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LIBRARY
Michigan State
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This is to certify that the

dissertation entitled

Functional Determinants of C/EBPbeta
In IL-6 and MCP-l Regulation

presented by

Chauncey J. Spooner

has been accepted towards fulﬁllment
of the requirements for

Ph.D. Microbiology &

Molecular Genetics

 

 

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6401 c:lCiFiC/DateDue.p65-p.15

 

FUNCTIONAL DETERMINANTS OF C/EBPB IN IL-6 AND MCP—l
REGULATION

By

Chauncey J. Spooner

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Molecular Genetics

2003

ABSTRACT

FUNCTIONAL DETERMINANTS or C/EBPB IN IL—6 AND MCP-l
REGULATION
By

Chauncey J. Spooner

C/EBP transcription factors, particularly C/EBPB and C/EBPS, are known to play an
important role in the regulation of numerous genes associated with the inﬂammatory
response and, in fact, are essential for the expression of many of these genes, including
IL-6 and MCP-l. Previous work in our lab demonstrated the importance of the bZIP
region of C/EBPB in the expression of IL-6 and MCP-l in a B lymphoblast cell line. In
addition to the bZIP domain, we have now found that serine 64 in the N-terminal
activation domain module 2 of C/EBPB is critical for the activation of MCP-l and, to a
lesser extent, IL-6 expression in response to lipopolysaccharide. Furthermore, internal
regulatory domains RDl and RD2 were found to impact C/EBPB activity in a promoter-
speciﬁc manner, as the presence of RD] and RD2 diminished C/EBPB activation of
MCP—l expression, while not blocking activation of IL-6 expression. In fact, the removal
of these regulatory domains actually reduced C/EBPB activity on the IL-6 promoter.
These results identify additional functional determinants for C/EBPB-mediated activation

of IL-6 and MCP-l expression, and suggest a model whereby C/EBPB may differentially .

regulate the expression of various proinﬂammatory cytokines through the differential
activity of the regulatory domains 1 and 2.

In addition to serine 64 and internal regulatory domains RBI and RD2, we have
identiﬁed a potential acetylation substrate in the bZIP of C/EBPB that is important for IL-
6 and MCP-l expression. Acetylation of has been shown to the function of many
transcription factors. We have found that the cooperation of the co-activators p300 and
GCN5 augments C/EBPB DNA-binding and transactivation, and that the enhancement of
C/EBPB DNA-binding was dependent on the acetyltransferase activity of GCN5, and not
p300. Moreover, p300-mediated augmentation of C/EBPB DNA-binding and
transactivation was dependent upon the motif KXKK, which has been identiﬁed as an
acetylation substrate in other transcription factors, such as p53 and GATA-l. We have
shown that the mutation of lysine residues that comprise this motif signiﬁcantly reduced
the expression of endogenous IL-6 in a B lymphoblast cell line in response to
lipopolysaccharide and IL-lB. We unexpectedly found that the GCN5 homolog P/CAF
actually reduced C/EBPB DNA-binding, which was also dependent on its
acetyltransferase activity. Furthermore, the mutation of lysines that block p300-mediated
augmentation of C/EBPB do not affect P/CAF inhibition of C/EBPB DNA-binding.
These results suggest that p300, presumably through its cooperation with GCN5, and

P/CAF differentially regulate C/EBPB activity through the use of alternative lysines.

ACKNOWLEDGEMENTS

Words cannot do justice to explain how truly appreciative I am of the guidance and
enormous support provided by mentor Dr. Richard C. Schwartz during my graduate
studies here at Michigan State University. His wisdom and involvement have played an
important role in the development, not only of my research, but me as a scientist as well.
I cannot thank him enough.

Furthermore, I would like to personally thank the members of my committee Dr.
Susan Conrad, Dr. Jerry Dodgson, Dr. Laura McCabe, and Dr. Ron Patterson for their
helpful input, guidance, and continued support throughout my graduate experience. In
addition, I would like to thank Peter F. Johnson at NCI-Frederick for generously
providing numerous reagents that have greatly assisted the progression of my research. I
would also like to thank Dr. Gao Hongwei, a former graduate student in the lab, for his
scientiﬁc advice and wonderful ﬁiendship.

Finally, I would like to thank my family, in particular my mother and father, for
always providing the unconditional love and support that has made all of this possible.
The sacriﬁces you have made for me have been an inspiration. None of this would be
possible without the love and support of my best friend and wonderful wife. Melissa,
you have been with me and supported me every step of the way through this entire

experience, and for that, I am eternally grateful.

iv

TABLE OF CONTENTS

List of Figures vii

List of Abbreviations x

Chapter 1 1
Literature Review 1
Introduction ..................................................................................................................... 2
l. C/EBP Family of Transcription Factors ................................................................ 2
1.1 C/EBPa ............................................................................................................. 7
1.2 C/EBPB ........................................................................................................... 10
1.3 C/EBPS ........................................................................................................... 13
1.4 C/EBPs ............................................................................................................ 14
1.5 C/EBPy ............................................................................................................ 15
1.6 C/EBPQ ........................................................................................................... 17
2. Inﬂammation ........................................................................................................ 18
2.1 Cytokine Overview ................................................................................... 18
2.1 . l Interleukin-6 ...................................................................................... 19
2.2 Chemokine Overview ............................................................................... 22
2.2.1 Chemokine and Chemokine Receptor Structure/Function ............... 23
2.2.2 Chemotaxis ....................................................................................... 26
2.2.3 Chemokine-mediated Pathology ....................................................... 29
3. Acetylation and Gene Regulation ........................................................................ 31
3.1 Chromatin Remodeling and Gene Expression .......................................... 31
3.2 Acetyltransferases ..................................................................................... 34
3.2.1 Gcn5 .................................................................................................. 34
3.2.2 P/CAF ............................................................................................... 36
3.2.3 p300 and CBP ................................................................................... 37
3.3 Factor Acetyltransferase Function ............................................................ 40
3.3. l DNA-Binding .................................................................................... 40
3.3.2 Protein-Protein Interactions .............................................................. 41
3.3.3 Protein Stability ................................................................................ 43
3.3.4 Signal Transduction Pathways .......................................................... 44
References ..................................................................................................................... 46

Chapter 2 69

Differential Roles of CIEBPﬂ Regulatory Domains in MCP-1 and
lL-6 Transcription 69

Abstract ..................................................................................................................... 70
Introduction ............................................................................................................... 71
Materials and Methods .............................................................................................. 75
Results ....................................................................................................................... 80

Discussion ................................................................................................................. 95
References ............................................................................................................... 101

Chapter 3 106
CIEBPB Activity is Differentially Regulated By Acetyltransferases

1 06
Abstract ................................................................................................................... 107
Introduction ............................................................................................................. 108
Materials and Methods ............................................................................................ 112
Results ..................................................................................................................... 1 18
Discussion ............................................................................................................... 154
References ............................................................................................................... 161

Summary and Future Directions 166

vi

List of Figures

CHAPTER 1

Figure 1-1 Hypothetical model of a dimeric bZIP protein interacting with its target
DNA. ..................................................................................................................... 5

Figure 1-2 Schematic representation of the C/EBP family members ........................... 6

CHAPTER 2

Figure 2-1 Figure 1. Diagram of the structures of C/EBPB and mutants used in these
studies. ................................................................................................................... 81

Figure 2-2 Ectopic expression of C/EBPB and its various amino-terminal deletions in
P388 lymphoblasts .................................................................................................... 83

Figure 2-3 Individual N-terminal functional domains of C/EBPB differentially
enhance and suppress MCP-l and IL-6 expression in stable transductants of P388
lymphoblasts ............................................................................................................. 85

Figure 2-4 ADM2 and/or 3 and the internal negative regulatory region of C/EBPB act
in opposition to each other in MCP-l expression ..................................................... 88

Figure 2-5 ADM2 and/or 3 and the internal negative regulatory region of C/EBPB are
important functional motifs in IL-6 expression ........................................................ 90

Figure 2-6 Serine 64 in C/EBPB ADM2 is important for MCP-l promoter-reporter
activity ................................................................................................................... 91

Figure 2-7 Serine 64 of C/EBPB is important for both MCP-l and IL-6 expression
from intact endogenous promoters ............................................................................ 93

Figure 2-8 The extent of hybridization was quantitated using a Storm Phosphimager
................................................................................................................... 94

CHAPTER3

Figure 3-1 C/EBP13213-297 is ineffective at supporting IL-1 [5 induction of IL-6 and
MCP-l ................................................................................................................. 119

vii

Figure 3-2 TSA Alleviates Reduced C/EBP8213-297 DNA-Binding in IL-1 B-treated

cells ................................................................................................................. 121
Figure 3-3 TSA and p300 Augment C/EBPB Activity ............................................. 122
Figure 3-4 C/EBPB DNA-binding is augmented by p300. ....................................... 124
Figure 3-5 C/EBPB DNA-binding is augmented by CBP. ....................................... 125

Figure 3-6 The association of C/EBP8213-297 with DNA is augmented by p300 ...... 126

Figure 3-7 C/EBPB acetylation is enhanced by p300 overexpression ...................... 128

Figure 3-8 p300 enhancement of DNA-binding is blocked by lysine to arginine
substitutions of C/EBPB residues 216 and 217 ....................................................... 130

Figure 3-9 p300 augmentation of transactivation is blocked by lysine to arginine
substitutions of C/EBPB residues 216 and 217 ....................................................... 131

Figure 3-10 Mutation of lysine residues 216 and 217 largely blocks C/EBPB
transactivation of the IL-6 and MCP-l promoter-reporters .................................... 132

Figure 3-11 p300 acetyltransferase activity is dispensable for augmentation of C/EBPB
binding ................................................................................................................. 134

Figure 3-12 p300 acetyltransferase activity is not required for augmentation of
C/EBPB transactivation by p300 ............................................................................. 135

Figure 3-13 ElA oncoprotein does not suppress p300-mediated augmentation of
C/EBPBZ13-297 DNA-binding ................................................................................... 137

Figure 3-14 P/CAF inhibits both p300 and CBP-mediated enhancement of C/EBPBZI 3-
297 DNA-bmdmg ..................................................................................................... 138

Figure 3-15 P/CAF HAT activity is required for its inhibition of C/EBPB DNA-
binding activity ....................................................................................................... 139

Figure 3-16 P/CAF suppression of CBP-mediated effects on C/EBPBZI 3-297 inﬂuences
protein stability in a HAT-dependent manner ........................................................ 142

Figure 3-17 GCN5 synergizes with p300 to enhance C/EBPB213-297 DNA-binding .. 144

Figure 3-18 GCN5 Cooperates with p300 to Enhance C/EBPB Transactivation ....... 145

viii

Figure 3-19 Differential Regulation of C/EBPB Binding Through Alternative Lysines
................................................................................................................. 147

Figure 3-20 Stable expression of C/EBPB lysine to arginine substitution mutants... 150

Figure 3-21 C/EBPBKZMR, and P388-CEBPBKKK214'216217RRR are reduced in their
ability to support induction of IL-6 expression in P388 stable transductants ........ 151

Figure 3-22 Quantitation of induced 11-6 expression in in P3 88 stable transductants 152

Figure 3-23 Hypothetical models of differential C/EBPB acetylation by GCN5 and
P/CAF ................................................................................................................. 157

ix

C/EBP

IL

MCP-l

LPS

bZIP

TNFa

GCSF

LAP

LIP

TPA

TGF-a

RDl and2

P/CAF

GCN5

HAT

List of Abbreviations

CCAAT/enhancer binding protein
Interleukin

Monocyte Chemoattractant protein-l
Lipopolysaccharide

basic region leucine zipper

Tumor Necrosis Factor on
Granulocyte colony stimulating factor
Liver activating protein

Liver inhibitory protein

Tumor promoter activators
Transforming growth factor-0t
Regulatory Domain 1 and 2
Activation domain module
p300/CBP associated factor

General control nonderepressible-S

Histone acetyltransferase

Chapter 1

Literature Review

Introduction

1. C/EBP Family of Transcription Factors

CCAAT/enhancer—binding proteins (C/EBPs) comprise a family of structurally and
functionally related transcription factors. To date, there are 6 known C/EBP isoforms,
C/EBPOL, B, y, 8, a, and Q (Cao, Umek et a1. 1991), which deﬁne a family of basic region-
leucine zipper (bZIP) transcription factors. These factors dimerize through their C-
terminal leucine zipper to form either homodimers or heterodimers with other family
members, a prerequisite for DNA-binding through the basic region (Landschulz, Johnson
et a1. 1988). Further diversity is generated by the use of alternative translation start sites
and promoters, differential splicing (Yamanaka, Kim et a1. 1997), and regulated
proteolysis (Descombes and Schibler 1991; Welm, Timchenko et a1. 1999), resulting in
the production of different sized polypeptides, post-transcriptional and post-translational
modiﬁcations of C/EBPs, and the ability of C/EBPs to interact with a myriad of other
proteins (McKnight 2001). Such mechanisms enable these proteins to perform numerous
functions in tissue and stage-speciﬁc transcriptional regulation in various biological
processes.

C/EBP family members are highly conserved (>90%) in the C-terminal bZIP
domain which precedes the leucine zipper region (Ramji and Foka 2002). The basic
region is the most conserved domain between C/EBP isoforms, which explains the
similarity in DNA-binding speciﬁcity within the C/EBP family (Williams, Cantwell et a1.

1991). In particular, an approximately 20 amino acid stretch, which is nearly identical

among every family member except C/EBPC, directly binds to the consensus binding site
for C/EBPs: 5’-T(T/G)NNGNAA(T/G)-3’ (Johnson 1993). The dimerization domain
contains a heptad repeat of 4-5 leucines that form a or-helical structure capable of
intercalating with other C/EBP monomers in parallel orientation (Agre, Johnson et a1.
1989; Hurst 1995). The highly conserved nature of the leucine zipper within the C/EBP
family permits the formation of heterodimers between any C/EBP isoform (Williams,
Cantwell et al. 1991). C/EBP-speciﬁc dimer formation as well as dimer formation with
other transcription factor families, such as the NF-kB and Fos/Jun families, is inﬂuenced
by electrostatic interactions between amino acids along the dimerization interface
(Vinson, Hai et al. 1993). Moreover, C/EBP dimerization speciﬁcity has been shown to
impact DNA-binding and transcriptional activation in a promoter-speciﬁc manner
(Catron, Brickwood et a1. 1998). Based on a model for C/EBP DNA—binding,
dimerization of individual polypeptides forms an “inverted Y” structure whereby each
arm of the “Y”, which is analogous to the basic region, interacts with one half of the
palindromic DNA-binding sequence in the major groove (Fig. 1) (Landschulz, Johnson et
al. 1988).

While C/EBPs share considerable amino acid sequence identity in the C—terminal
residues that make up the bZIP domain (Landschulz, Johnson et a1. 1988; Akira, Isshiki
et al. 1990; Chang, Chen et al. 1990; Poli, Mancini et al. 1990; Roman, Platero et a1.
1990; Ron and Habener 1992; Hurst 1995), they are quite divergent in structure and
function in the N-terminus (<20% sequence similarity) (Fig. 2) (Ramji and Foka 2002).
Family members vary from being strong activators of transcription, such as C/EBPOL, B,

5, and e (Cao, Umek et al. 1991; Nerlov and Ziff 1994; Williams, Baer et a1. 1995;

Yamanaka, Kim et al. 1997), to being dominant negative suppressors of C/EBP
transactivation, such as C/EBPy and C/EBPC (Ron and Habener 1992; Cooper,
Henderson et al. 1995). Presumably, at least part of this difference in activity is due to
the presence of three separate N-tenninal activation domains that are conserved in
C/EBPa, B, 8, and 8 (Williams, Baer et al. 1995; Williamson, Xu et al. 1998) and absent
in C/EBPy and C/EBPC; (Fig.2). In addition, C/EBPB and C/EBPa have been shown to
contain conserved negative regulatory domains that reduce their respective
transactivation potential on artiﬁcial C/EBP-dependent reporters (Williams, Baer et a1.
1995; Williamson, Xu et al. 1998). Our understanding of the function of these domains
has been extended by data in Chapter 2, which demonstrate the differential activity of the
internal negative regulatory domains of C/EBPB in MCP-l and IL-6 expression in a
murine B lymphoblast cell line.

Various ﬁmctions of C/EBP family members have been elucidated using a variety
of methods, including cell culture-based assays overexpressing or inhibiting C/EBP
expression, promoter analysis of C/EBP target genes, and/or transgenic and knock-out
studies in mice. Such studies have implied an important role for C/EBPs in many cellular
processes, including energy metabolism (McKnight, Lane et al. 1989), inﬂammation
(Poli 1998), and cellular proliferation and differentiation (Scott, Civin et al. 1992;
Darlington, Ross et al. 1998; Diehl 1998). Observations in mice lacking C/EBPa show a
reduction in glycogen synthase expression, which blocks glucose synthesis de novo
(Wang, F inegold et a1. 1995). Moreover, C/EBPs have been identiﬁed as direct targets of
inﬂammatory stimuli, such LPS and various cytokines (Tengku-Muhammad, Hughes et

al. 2000), which results in the expression of certain proinﬂammatory gene products

 

Figure 1-1 Hypothetical model of a dimeric bZIP protein interacting with its target
DNA. A “side-view” in which the leucine zipper is to the leﬁ, and the axis of the coiled-
coil is horizontal and parallel to the plane of the paper (Landschulz, Johnson et al. 1988).
One subunit of the C/EBP dimer is presented in red and the other is blue. The leucines
and basic residues with the zipper are displayed in yellow. (Images in this ﬁgure are
presented in color).

CIEBPa

CIEBPB

CIEBPy
CIEBPB
ClEBPs

ClEBPf

Figure 1-2 Schematic representation of the C/EBP family members. The position of

AD AD RD AD B ZIP

 

   
   

p42

p30

LAID" -. lmls- um.

LAP .mIﬁ-a- melt-I

LIP nil-I mm
P32 mum- “anal-ﬂ

p30

p27

p14

 

 

 

Modiﬁed From Ramji and Foka (Biochem J. 2002)

the activation domains (AD), negative regulatory domains (RD), basic region DNA-
binding domain (B), and the leucine zipper dimerization domain (ZIP). “?” indicates the
presence of an activation domain in the N-tenninus of C/EBPQ whose boundaries have
yet to be precisely determined. (Images in this ﬁgure are presented in color).

(Akira, Isshiki et al. 1990; Poli 1998). Finally, recent exciting discoveries have identiﬁed
critical roles for C/EBPs in cell cycle regulation, some surprisingly having nothing to do
with transcriptional regulation. Interestingly, C/EBPor was found to directly interact with
cdk2 and cdk4, independently of the C/EBPa DNA-binding domain (Wang, Iakova et al.
2001). This interaction prevented the association of cyclins with cyclin-dependent
kinases (cdk), thereby blocking cell cycle progression. The following sections discuss
the roles of C/EBP or, B, 5, a, y, and Q in the regulation of these, and many more cellular

processes.

1.1 C/EBPa

C/EBPa, the ﬁrst C/EBP family member to be cloned, was originally identiﬁed in
adult rat liver crude nuclear extracts as a DNA-binding factor capable of interacting with
retroviral LTR regulatory sequences, including the CCAAT box and enhancer core
elements (Johnson, Landschulz et al. 1987). The use of alternative translation initiation
sites allows for the production of two C/EBPa isoforrns (Ossipow, Descombes et al.
1993). The 42 kDa full-length form contains three N-terminal activation domain
modules (ADMs) that, among many other functions, were found to facilitate interactions
with elements of the basal transcription machinery, such as TBP and TFIIB, and promote
transcriptional activation of the serum albumin promoter (Nerlov and Ziff 1994; Nerlov
and Ziff 1995). In addition, the N-terminal activation domains can further modulate
C/EBPa activity by providing substrates for covalent modiﬁcations mediated by various
signaling pathways, as Ras-induced phosphorylation of C/EBPa within ADM3 promoted

granulocytic differentiation (Behre, Singh et al. 2002). A 30 kDa form, that lacks a

portion of the N-terminal transactivation domains, has a lower transactivation potential,
and does not block cell proliferation of 3T3-L1 preadipocytes, which is a normal ﬁmction
of C/EBPa (Lin, MacDougald et a1. 1993; Ossipow, Descombes et al. 1993).

C/EBPa mRNA expression is similar in mice and humans, and is observed in a
variety of cells, including intestine, lung, adrenal gland, and peripheral blood
mononuclear cells (Lekstrom-Himes and Xanthopoulos 1998), but is most abundant in
terminally differentiated liver and adipose tissue (Birkenmeier, Gwynn et al. 1989;
Williams, Cantwell et al. 1991; Antonson and Xanthopoulos 1995). This is supported by
various studies demonstrating the importance of C/EBPOL in the activation of liver- and
adipose-speciﬁc gene expression (Darlington, Ross et al. 1998; Lekstrom-Himes and
Xanthopoulos 1998). C/EBPoc"' mice have morphological derangements in hepatic
structure and die soon after birth of hypoglycemia. C/EBPa directly targets those genes,
such as glycogen synthase, phosphoenolpyruvate carboxykinase, and glucose-6-
phosphatase gene expression, which are necessary for proper glycogen production and
metabolism (Wang, Finegold et al. 1995; Lee, Sauer et al. 1997). In addition, C/EBPa
overexpression in 3T3-L1 cells induces adipocyte differentiation, while expression of
C/EBPa antisense in 3T3-L1 blocks lipid droplet formation, a hallmark of adipocyte
differentiation (Lin and Lane 1992; Lin and Lane 1994; Yeh, Cao et a1. 1995). The
murine C/EBPa proximal promoter contains a C/EBP binding site (Christy, Kaestner et
al. 1991; Legraverend, Antonson et a1. 1993). Therefore, C/EBPa expression is

maintained by auto-upregulation, presumably maintaining the differentiated state (Rosen,

Walkey et a1. 2000).

C/EBPa has been shown to play a critical role in the development of other cell types,
including hematopoietic cells (Scott, Civin et al. 1992; Radomska, Huettner et al. 1998).
C/EBPa-deﬁcient mice display a block during myeloid differentiation and lack
granulocytes (Zhang, Zhang et a1. 1997). Moreover, these mice are unresponsive to
Granulocyte Colony Stimulating Factor (G-CSF), presumably due to the absence of G-
CSF receptor (G-CFFR), a direct target of C/EBPa (Hohaus, Petrovick et al. 1995;
Smith, Hohaus et al. 1996). In the development of acute myeloid leukemia (AML), the
most obvious defect is a block in the differentiation of myeloid precursors to mature
granulocytes. Five heterozygous mutations of C/EBPa were identiﬁed in several patients
with acute myeloid leukemia (Pabst, Mueller et al. 2001). These mutations generated N-
terminal truncations of C/EBPa that inhibited the DNA-binding and transactivation of
granulocytic target genes in a dominant-negative manner, thus blocking granulocytic
differentiation. Interestingly, a recent study on C/EBPa-mediated granulocytic
differentiation suggests that the concentration of C/EBPa relative to PU. 1 , a macrophage-
speciﬁc differentiation factor that prevents neutrophil development in myeloid progenitor
cells, is critical for cell fate determination (Dahl, Walsh et al. 2003).

Recently, several studies have directly linked the anti-proliferative effect associated
with C/EBPa expression to cell cycle regulation in a transcription-independent manner.
Wang et al. observed a direct interaction between a N-terminal domain in C/EBPa and
cdk2 and cdk4, that prevents cdk2/cyclin complex formation and, thus, cell proliferation
(Wang, Iakova et al. 2001). In the case of cdk4, this interaction promotes proteasome-
dependent cdk4 degradation (Wang, Goode et al. 2002). This same region of C/EBPoc

that interacts with cdk2 and cdk4 was previously found to be critical for grth arrest

(Umek, Friedman et al. 1991). C/EBPa has also been shown to directly interact with and
stabilize p21, a cdk inhibitor, resulting in growth inhibition (Timchenko, Harris et al.
1997), which suggests the presence of multiple mechanisms in C/EBPa-mediated cell

cycle regulation.

1.2 C/EBPB

C/EBPB was initially identiﬁed as a cytokine-responsive nuclear factor (NF-1L6)
that bound to the promoter of various genes involved in the inﬂammatory response
(Akira, Isshiki et al. 1990; Isshiki, Akira et al. 1990; Poli, Mancini et a1. 1990). The use
of alternative translation start sites from C/EBPB mRNA may give rise to three isoforms,
38 kDa (LAP*), 35 kDa (LAP), and 20 kDa (LIP), with LAP and LIP as the most
prevalent variants of C/EBPB observed in most cells (Descombes and Schibler 1991).
The N-terminal extension present in LAP“ was shown to play a role in the recruitment of
the ATP-dependent chromatin remodeling complex SWI/SNF to activate the expression
of myeloid-speciﬁc genes (Kowenz-Leutz and Leutz 1999). LAP retains three highly
conserved N-terminal subdomains associated with transcriptional activation that are also
found in other C/EBPs (Williams, Baer et al. 1995). Unlike strong activators, such as
C/EBPa and C/EBPS, C/EBPB contains two regulatory domains (RDl and RD2) that
reduce its transactivation potential and DNA-binding activity, respectively (Williams,
Baer et al. 1995). In addition, RD2 inﬂuences C/EBPB activity in a cell-speciﬁc manner
(Williams, Baer et al. 1995). Data presented in Chapter 2 demonstrate promoter-speciﬁc
function for RD] and RD2-mediated inhibition of C/EBPB activity (CJS and RCS,

unpublished data). Several lines of evidence suggest that LIP, which lacks N-terminal

10

activation domains, primarily functions as a dominant-negative inhibitor of C/EBP-
mediated transcription by dimerizing and forming non-functional dimers with other
C/EBP members (Descombes and Schibler 1991). It can also inhibit transcriptional
activation by directly competing with activating forms of C/EBP for binding sites.
However, research in our lab and data presented in Chapter 2 demonstrates that LIP and
other C/EBPB variants lacking N-terminal activation domains can play a positive role in
IL-6 promoter activation in a B lymphoblastic cell line (Hu, Tian et a1. 2000).

The N-terminal functional domains of C/EBPB can participate in C/EBPB
activation by providing substrates for covalent modiﬁcation by various signaling
pathways. Activated Ras has been shown to promote human C/EBPB phosphorylation at
threonine 235, a MAPK substrate that resides in the N-terminal RD2, which resulted in
the activation of an IL-6-promoter/reporter (Nakajima, Kinoshita et al. 1993). Tumor
promoter activators (TPA)- or protein kinase C-induced phosphorylation of rat C/EBPB
at serine 105 was also found to enhance the transactivation potential of C/EBPB
(Trautwein, Caelles et al. 1993). Interestingly, this same residue is phosphorylated in
response to TGF-a-induced p90 ribosomal S kinase activation, which is associated with
hepatocyte proliferation (Buck, Poli et al. 1999). Data presented in Chapter 2
demonstrates the importance of C/EBPB serine 64 in MCP-l and IL-6 transcription in
P388 B lymphoblasts, implicating a kinase signaling pathway in their C/EBPB-mediated
expression. This is supported by data demonstrating serine 64 as a novel Ras-induced

phosphoacceptor site involved in the transformation of NIH 3T3 cells (J. D. Shuman and

P. F. Johnson, unpublished data).

11

In addition to phosphorylation, C/EBPB has recently been identiﬁed as a substrate
for sumoylation (Kim, Cantwell et al. 2002; Eaton and Sealy 2003) and acetylation (Xu,
Nie et al. 2003)[see Chapter 3]). C/EBPs with N-terminal activation motifs, such as
C/EBPa, B, 5, and 8, contain an evolutionarily conserved motif (I/V/L)KXEP. This motif
is also found in other transcription factors, including the AP-l family members c-Jun,
JunB, and JunD, and functions as a substrate for SUMO-1 modiﬁcation (Kim, Cantwell
et al. 2002). Interestingly, C/EBPB sumoylation enhanced promoter activation by
alleviating the inhibitory effects of C/EBP regulatory domains. Unlike sumoylation,
which enhances C/EBPB activity, acetylation by P/CAF was shown to reduce IL-3-
dependent C/EBPB transactivation of the Id-l promoter by diminishing its DNA-binding
activity (Xu, Nie et al. 2003). Data presented in Chapter 3 also demonstrate the
importance of P/CAF-mediated acetylation of C/EBPB and inhibition of DNA-binding.
However, additional data presented in Chapter 3 implies a role for the co-activators p300,
CBP, and GCN5 in enhanced DNA-binding and transactivation by C/EBPB, presumably
through the use of alternative lysine residues.

C/EBPB is believed to be a critical regulator of various aspects of inﬂammation
due to its ability to bind to the promoter region of many proinﬂammatory cytokines
(Shirakawa, Saito et a1. 1993; Pope, Leutz et a1. 1994; Wedel and Ziegler-Heitbrock
1995; Poli 1998; Pan, Hetherington et al. 1999), acute phase proteins (Ray and Ray
1994), and chemokines (Poli 1998; Sekine, Nishio et al. 2002). In addition, C/EBPB
transcription and transactivation are modulated by inﬂammatory stimuli, such as LPS, IL-
1, and IL-6 (Akira, Isshiki et al. 1990; Poli, Mancini et al. 1990; Alam, An et al. 1992;

Yin, Yang et al. 1996). A role for C/EBPB in inﬂammation is further supported by

12

studies in C/EBPB knockout mice, which have impaired TNF-OL induction (Poli 1998).
Moreover, C/EBPB"' mice have impaired tumor cytotoxicity of macrophages and are
highly susceptible to infection by Listeria monocytogenes (Tanaka, Akira et al. 1995),
presumably due to a defect in macrophage bactericidal function. IL-12 production is also
impaired in C/EBPB-deﬁcient mice, which results in altered T-helper cell function and
increased susceptibility to infection by Candida albicans (Screpanti, Romani et al. 1995).

Therefore, C/EBPB appears to play a critical role in both humoral and innate immunity.

1.3 C/EBP8

C/EBP8 was originally identiﬁed in rat as a C/EBP family member, like C/EBPB,
temporally expressed during the initial stages of adipocyte differentiation (Cao, Umek et
a1. 1991). C/EBP5 is primarily expressed in lung, intestine, and adipose tissue (Williams,
Cantwell et al. 1991; Alam, An et al. 1992), with the induction of high levels of
expression in various tissues, including myelomonocytic cells, in response to
inﬂammatory stimuli (Kinoshita, Akira et al. 1992; Scott, Civin et al. 1992). Unlike
C/EBPB and C/EBPs, C/EBP8 lacks internal regulatory domains that repress its
transactivation potential (Fig. 2). In addition, C/EBP5 seems to be largely induced at the
transcriptional level, while C/EBPB shows more post-transcriptional regulation
(Kinoshita, Akira et al. 1992; Ramji, Vitelli et al. 1993). However, C/EBP8 activity is
still comparable with that of C/EBPB (Cao, Umek et al. 1991; Hu, Tian et al. 2000).

C/EBP family members have been found to regulate the differentiation of hepatocytes
and adipocytes (Birkenmeier, Gwynn et al. 1989; Friedman, Landschulz et al. 1989; Cao,

Umek et al. 1991). In these studies, C/EBP6 and C/EBPB were observed during the

13

proliferative stages prior to differentiation, while high levels of C/EBPor were expressed
in terminally differentiated tissue. It is believed that C/EBP6 and C/EBPB initiate the
differentiation program of adipocytes and hepatocytes by binding to C/EBP regulatory
sites in the C/EBPor promoter, which blocks cell proliferation and promotes terminal
differentiation (Cao, Umek et al. 1991; Christy, Kaestner et al. 1991). This is supported
by observations in mice lacking both C/EBP6 and C/EBPB. These mice are unable to
support lipid droplet formation, a hallmark of adipocyte differentiation (Tanaka, Yoshida
et al. 1997). On the other hand, the transient pattern of C/EBP isoform expression is
reversed in the myeloid lineage, as C/EBPor expression is predominant in proliferating
myelomonocytic cells, while C/EBP8 and C/EBPB are signiﬁcantly higher during

granulopoiesis (Scott, Civin et al. 1992).

1.4 C/EBPs

Unlike other C/EBPs, which have a broad tissue distribution, C/EBPs mRNA and
protein is primarily expressed in cells of the hematopoietic lineage (Antonson, Stellan et
a1. 1996; Chumakov, Grillier et al. 1997). C/EBPs contains two introns and multiple
translation initiation codons in the context of a Kozak sequence, which allows the
generation of at least four subforms (32kDa, 30kDa, 27kDa, and 14kDa, respectively)
through differential splicing and the use of alternative promoters (Yamanaka, Kim et al.
1997). All four subforrns retain the highly conserved bZIP domain, but lack different
elements of the N-terminal activation and regulatory domains (Fig. 2). Like C/EBPB,
C/EBPs contains three N-terminal ADM3 and two conserved inhibitory domains

(Angerer, Du et al. 1999). The activation potential is progressively diminished by the N-

14

terminal truncation of additional activation domains, as the activation potential of the
30kDa C/EBPs variant is lower than that of the 32 kDa variant (Y arnanaka, Kim et al.
1997; Lekstrom-Himes 2001 ).

C/EBPe plays a critical role in myeloid differentiation, as C/EBPs-deﬁcient mice are
unable to form functional neutrophils and eosinophils (Morosetti, Park et al. 1997;
Yamanaka, Barlow et al. 1997). This phenotype is similar to one observed in patients
that have an uncommon neutrophil-speciﬁc granulocyte deﬁciency (Lekstrom-Himes
2001). Some of these patients have C/EBPe loss-of-function mutations (Lekstrom-Himes,
Dorman et a1. 1999; Gombart, Shiohara et al. 2001). Moreover, C/EBPs-deﬁcient mice
survive only several months aﬁer birth due to opportunistic infections by Pseudomonas
aeruginosa. While macrophages are present in both wildtype and C/EBPe'/' mice, the
absence of C/EBPs is associated with a decline in overall macrophage levels, impaired
macrophage function, down-regulation of cytokine production, and a signiﬁcant decrease
in phagocytic function (Tavor, Vuong et al. 2002). In addition, T-cell receptor-mediated
T cell proliferation is impaired in C/EBP8"' mice, which suggests a role for C/EBPs
activity in both the lymphoid and myeloid lineages. Therefore, these studies implicate
C/EBPa as an important transcription factor necessary for normal function and

development in hematopoiesis.

1.5 C/EBPy
C/EBPy, also referred to as Ig/EBP-l, was initially characterized as an ubiquitously
expressed transcription factor that bound to the immunoglobulin heavy chain enhancer

element (Roman, Platero et al. 1990). C/EBPy expression is highest in non-differentiated

1‘5

progenitor cells (Thomassin, Hamel et al. 1992). It is the principal C/EBP family
member present in early B cells, but decreases as B cells develop (Cooper, Berrier et al.
1994). In addition, C/EBPy is a critical transcription factor involved in the functional
maturation of NK cells, as NK cells derived from C/EBPy'/' mice display lower cytotoxic
activity and IFN-y production than those derived from control animals (Kaisho, Tsutsui et
al. 1999).

C/EBPy lacks N-terminal activation domains and has been shown to dimerize and,
consequently, attenuate the transcriptional activation of C/EBPor and C/EBPB target
genes, suggesting a dominant-negative regulation of C/EBP activity (Cooper, Henderson
et al. 1995). Although, recent work in our lab demonstrated that C/EBPy can play a
positive role in the expression of speciﬁc proinﬂammatory gene products in a cell type-
and promoter-dependent manner (Gao, Parkin et a1. 2002). In reported cases of
transcriptional activation in the presence of C/EBPy, the association with additional
transactivators seems to be a prerequisite for supporting promoter activity. For example,
C/EBPy cooperation with Stat6 and NF-KB p50/p65 heterodimer induced the gamma 3-
immunoglobulin promoter in B cells (Pan, Petit-Frere et al. 2000). In P388 murine B
lymphoblasts, ectopically expressed C/EBPa, B, and 5 were all observed predominantly
as heterodimers with C/EBPy (Hu, Baer et al. 1998; Hu, Tian et al. 2000; Gao, Parkin et
al. 2002). More signiﬁcantly, C/EBPy was shown to augment the activity of C/EBPB in
transactivating the IL-6 and IL—8 promoters. Furthermore, C/EBPy augmentation of

C/EBPB activity required co-expression of NF-kB p65 (Gao, Parkin et al. 2002).

16

1.6 C/EBPC

C/EBPQ, also referred to as CHOP (Ron and Habener 1992) or Gadd153 (Park,
Luethy et al. 1992), is ubiquitously expressed (Ron and Habener 1992). Unlike C/EBPor,
B, 8, and 7, which lack introns, C/EBPC contains three introns (Luethy, Fargnoli et al.
1990). Like C/EBPy, C/EBPQ lacks the N—terminal activation motifs observed in other
C/EBP isoforrns (Fig. 2). However, C/EBPQ undergoes inducible phosphorylation on
two serines at amino acids 78 and 81 in response to conditions of cellular stress (Wang
and Ron 1996). While C/EBPQ can dimerize with other C/EBPs due to the presence of
an intact leucine zipper domain, such heterodimers are unable to bind to consensus
C/EBP binding sites (Ron and Habener 1992). The presence of two proline residues in
the C/EBPC basic region alters its structure and inhibits binding to C/EBP regulatory
elements. Therefore, C/EBPI; is believed to function as a dominant-negative inhibitor of
C/EBP transcriptional activity by blocking the DNA-binding of C/EBP transactivators.
Although, certain subsets of genes activated under conditions of cellular stress contain
non-consensus C/EBP regulatory sites that permit C/EBPC heterodimers to bind and

activate gene expression (Ubeda, Wang et al. 1996; Sok, Wang et al. 1999; Ubeda,
Vallejo et al. 1999).

C/EBPQ was initially identiﬁed as a DNA-damage-inducible gene associated with
grth arrest and apoptosis, as C/EBPC expression correlated with apoptosis (Fornace,
Nebert et al. 1989; Barone, Crozat et al. 1994; Zhan, Lord et al. 1994). This is supported
by studies in mouse embryonic ﬁbroblasts (MEFs) derived from C/EBPQ-deﬁcient mice,

which exhibit a signiﬁcant reduction in programmed cell death when exposed to agents

that disrupt ER function compared to wildtype mice (Zinszner, Kuroda et al. 1998).

17

Abnormal nitric oxide production in activated pancreatic B cells has been implicated in
cell death in type-l diabetes, as B cells are susceptible to nitric oxide-induced apoptosis
(Eizirik, Flodstrom et al. 1996). Interestingly, pancreatic B cells in C/EBPQJ' mice are
unresponsive to nitric oxide-induced apoptosis (Oyadomari, Takeda et al. 2001), which

further supports the notion of C/EBPQ as a critical apoptotic regulator.

2. Inﬂammation
2.1 Cytokine Overview

Cytokines are soluble factors that are essential for communication between cells of
the immune system and play critical roles in various immune responses, including
inﬂammation, immunity, and hematopoiesis. Cytokines are low molecular weight
proteins (<30kDa) that are secreted by many cell types, including macrophages, B and T
lymphocytes, hepatocytes, endothelial cells, and ﬁbroblasts in response to various
stimuli. Altogether, there are believed to be well over 100 proteins with cytokine
activity. Many of these proteins can be grouped into one of four families: 1) the
interferon family, 2) the hematopoietin family, 3) the tumor necrosis factor family, and 4)
the Chemokine family. While the amino acid sequence may differ signiﬁcantly between
individual members of a given family, they retain a considerable degree of conservation
in secondary structure. For example, members of the hematopoietin family have
substantial a-helical structure, with little or no B-sheet structure. Cytokines, which
function as either autocrine or paracrine factors, facilitate communication between
various cells and cell types by directly binding to speciﬁc membrane receptors located on

target cells, which results in the activation of downstream kinase signaling cascades and

18

an eventual change in gene expression. Most cytokines are pleiotropic and can function
in either a positive or negative manner, depending on the target cell type.

To elicit a biological effect, cytokines must associate with speciﬁc receptors on a
given target cell. Moreover, the expression on the cell surface of speciﬁc cytokine
receptors determines the response of target cells to a particular cytokine. The
extracellular domain of most cytokine receptors has a common motif with sequences
found in the immunoglobulin superfamily (Miyajima, Kitamura et al. 1992). In addition,
the extracellular domain consists of highly conserved cysteine residues at speciﬁc
positions within each family. Cytokine receptors belong to one of ﬁve groups: 1) Class I
cytokine receptor family, which binds to hematopoietin cytokines, 2), Class II cytokine
receptor family, which binds the interferon cytokines, 3) immunoglobulin superfamily
receptors, 4) TNF receptor family, and 5) the Chemokine receptor family. One feature
common to class I and class II cytokine receptors is the presence of multiple subunits,
which are usually classiﬁed into those that interact with the ligand and those that mediate

signal transduction.

2.1.1 Interleukin-6

IL-6, a 26 kDa glycoprotein, was originally identiﬁed as a soluble factor produced by
T cells which is important for terminal B lymphocyte differentiation and induction of
immunoglobulin synthesis and secretion (Hirano, Yasukawa et al. 1986; Muraguchi,
Hirano et al. 1988). IL-6 is a pleiotropic cytokine that is produced by numerous cell
types, including macrophages, T and B lymphocytes, hepatocytes, and osteoblasts, in

response to diverse stimuli (Taga and Kishimoto 1997). In addition, IL-6 plays a critical

19

role in the regulation of various biological processes, such as proliferation,
differentiation, and cell survival (Muraguchi, Hirano et al. 1988; Uyttenhove, Coulie et
al. 1988).

IL-6 functions as an essential factor for B cell ﬁrnction, in particular, and as a
mediator of inﬂammation, in general. CD40 ligand-induced co-stimulation of B cells in
the presence IL-6 stimulates lg class switching, cell surface expression of CD38 (a
plasma cell differentiation marker), and Ig secretion (Urashima, Chauhan et al. 1996).
Moreover, an anti-IL-6 antibody prevented Ig secretion, which demonstrates the
importance of IL-6 for terminal B cell differentiation into plasma cells. In accordance
with a role for IL-6 in B lymphocyte proliferation, transgenic mice overexpressing IL-6

/ - .
+ + or IL-6+/ mice may

develop plasmacytomas (Hirano and Kishimoto 1989). While IL—6
develop plasma cell tumors, IL-6-deﬁcient mice are completely resistant to in vivo
plasmacytoma development (Hilbert, Kopf et al. 1995). In addition, the inﬂammatory
acute-phase response is severely impaired in IL—6 deﬁcient mice (Kopf, Baurnann et al.
1994). These same mice are unable to support a normal inﬂammatory response after
tissue damage or infection, and show a reduction in the expression of the acute phase
proteins: a2-macroglobulin (azM), serum amyloid P (SAP), Oil-acid glycoprotein,
(AGP), and haptoglobin (Hp) (Fattori, Cappelletti et al. 1994). IL-6 is also believed to
play a role in neutrophil ﬁrnction, as IL-6"' mice are unable to clear Listeria
monocytogenes infections (Dalrymple, Lucian et al. 1995). Finally, IL-6'l' mice were

shown to have a reduction in chemokine production, which resulted in impaired

leukocyte recruitment to local sites of infection (Romano, Sironi et al. 1997). These

20

studies demonstrate the importance of IL-6 as a mediator of terminal B cell
differentiation and of inﬂammation in response to localized infection.

IL-6 binds to a member of the Type I cytokine receptor family which is comprised of
the or chain (gp80 or IL-6R), which is unable to initiate downstream signaling events of
itself, presumably due to its short cytoplasmic domain (Y amasaki, Taga et al. 1988) To
initiate downstream signaling events, the IL—6/gp80 complex associates with gp130,
which is unable to bind IL—6 alone, but can form high-afﬁnity IL-6 binding sites when
interacting with the IL-6/gp80 complex (Hibi, Murakami et al. 1990). More importantly,
gpl30 contains signal transducing elements in its cytoplasmic tail and enables the IL-6
receptor to initiate a tyrosine kinase signaling pathway upon IL-6 binding (Murakami,
Hibi et a1. 1993). In addition to Janus kinase (JAK) activation, IL-6 can stimulate other
protein kinases, including the Src and Tec family tyrosine kinases (Matsuda, Takahashi-
Tezuka et al. 1995; Hallek, Neumann et al. 1997) and the MAPK pathway (Fukada, Hibi
et al. 1996). While gp130 lacks an enzymatic kinase domain, IL-6 receptor activation
results in the association of the protein tyrosine kinase JAK with the gpl30 membrane-
proximal cytoplasmic tail and its consequent activation (Lutticken, Wegenka et al. 1994;
Stahl, Boulton et al. 1994). This domain is highly conserved within other cytokine
receptor families (Murakami, Narazaki et al. 1991), which may explain the redundant
functions observed between different cytokines.

IL-6 has been shown to function as an autocrine factor and support its own production
by inducing the expression and enhancing the transactivation potential of the C/EBP
family of transcription factors, which are capable of binding to a cytokine-responsive

element in the IL-6 promoter (Isshiki, Akira et al. 1990). C/EBPB and C/EBP5

21

transcription is induced in response to cytokine stimulation, which results in the binding
and transactivation of various cytokine promoters, including IL-6 (Akira, Isshiki et al.
1990; Kinoshita, Akira et al. 1992). In addition, IL—6 was found to enhance the DNA-
binding activity and transactivation potential of C/EBP6 in the hepatocytic cell line
Hep3B, implying posttranslational modiﬁcations of C/EBPs induced by IL-6 (Poli,
Mancini et al. 1990). Work in our lab has shown that the ectopic expression of C/EBPor,
B, and 6 in a B lymphoblastic cell line can support IL-6 expression in response to LPS
and H.-1B (Hu, Baer et al. 1998). Interestingly, a truncated form of C/EBPB lacking all
of the known N-terminal activation and regulatory domains can also support IL-6
transcription, but is dramatically reduced in its ability to support IL-lB-induced IL-6
expression. We found that LPS and IL-lB differentially modulate C/EBPB DNA-binding
(Chapter 3), which is surprising since it is generally thought that the induction of the LPS
and IL-1 signaling pathways are similar following receptor activation. This difference in
inﬂammatory signaling suggests that LPS and IL-1 are not identical in their inﬂammatory

effects.

2.2 Chemokine Overview

The interaction of various cell types from the hematopoietic lineage in speciﬁc
microenvironments is required for an effective immune response in the presence of
antigen. Leukocytes constantly recirculate through secondary lymphoid organs in a
spatially and temporally regulated manner in order to maximize the probability of antigen
recognition, where antigen is displayed on antigen—presenting cells (APCs). Numerous

studies have demonstrated that the control of cell migration is regulated by the

22

differential expression of chemokines and chemokine receptors in varying cell types
under both homeostatic and inﬂammatory conditions. Chemokines primarily act on
neutrophil, monocyte, lymphocyte, and eosinophil migration and play a pivotal role in
host defense mechanisms. More recent evidence suggests that chemokines also play

fundamental roles in the development, homeostasis, and function of the immune system.

2.2.1 Chemokine and Chemokine Receptor Structure/Function

Currently, there are approximately 40 known human chemokines, also referred to as
chemotactic cytokines, and 20 chemokine receptors that have been identiﬁed based on
common genetic and structural features (Oppenheim, Zachariae et al. 1991). Like
cytokines, chemokines are produced and secreted by hematopoietic cells in response to
inﬂammatory stimuli and exert their effects locally in an autocrine and/or paracrine
manner (Baggiolini, Dewald et al. 1994). Chemokines are low molecular weight proteins
(~8 kDa to 14 kDa) that are subdivided into four separate families, CXC (or or-
chemokines), CC (or B-chemokines), C, and CX3C chemokine families, based on the
presence and context of highly conserved cysteine residues (Baggiolini, Dewald et al.
1994; Baggiolini, Dewald et al. 1997). CXC and CC are the two primary chemokine
families and are distinguished by the presence (CXC) or absence (CC) of a single amino
acid that separates the two conserved N-terminal cysteines (Baggiolini, Dewald et al.
1994). Disulphide bonds are formed between the ﬁrst and third cysteines (Cysl—CysB)
and the second and fourth cysteines (CysZ-Cys4), which is essential for the three-
dimensional structure of these proteins (Baggiolini 2001). Fraktalkine, which is the only

member of the CX3C chemokine family, is a membrane-bound chemokine that is

23

 

upregulated in brain inﬂammation. It contains three amino acids between the ﬁrst two
conserved cysteine residues (Bazan, Bacon et al. 1997; Pan, Lloyd et al. 1997). Finally,
lymphotactin and SCM-lB, are the only members of the C chemokine family, which
contain only two conserved cysteine residues (Kennedy, Kelner et al. 1995).

The divergent N-terminal and C-terminal regions are anchored to the conserved
backbone via disulﬁde bonds involving the conserved cysteine residues (Clore, Appella
et al. 1990; Rajarathnam, Clark-Lewis et al. 1995; Handel and Domaille 1996). C0-
precipitation and cross-linking studies suggests that chemokines function as dimers, as
mutations that prevent dimerization block ligand-receptor binding (Zhang and Rollins
1995). In addition, N-terminal deletions of chemokines, such as monocyte
Chemoattractant protein-l (MCP-l) and IL-8, inhibit dimerization and chemotaxis
(Moser, Dewald et al. 1993; Zhang, Rutledge et a1. 1994; Gong and Clark-Lewis 1995).
Interestingly, the presence of the amino acid motif ELR (glutamate-leucine-arginine) in
the N-terminus of CXC chemokines supports neutrophil-speciﬁc migration (Clark-Lewis,
Schumacher et al. 1991; Hebert, Vitangcol et al. 1991). Moreover, the introduction of
this motif in the N-terrninus of PF4, a CXC chemokine that normally attracts activated T
lymphocytes (Murphy, Tian et al. 1996) and lacks this moiety, changes PF4 into a
neutrophil-speciﬁc Chemoattractant (Clark-Lewis, Dewald et al. 1993). The basic amino
acids of the C-terminus mediate interactions between chemokines and acidic
macromolecules found in the surrounding extracellular matrix or on the cell surface,
where they remain in close proximity to their site of production (Chakravarty, Rogers et

al. 1998; Amara, Lorthioir et al. 1999).

24

To date, 6 CXC receptors and 10 CC receptors have been characterized (Baggiolini
2001). Considerable promiscuity is observed between CC and CXC chemokine-
chemokine receptor interactions, as certain chemokine receptors recognize multiple
chemokines, and vice versa. There are two primary contact sites within chemokines that
facilitate interactions with their respective receptors, including a N-terminal determinant
and a loop region within the highly conserved backbone between the second and third
cysteine (Clark-Lewis, Kim et al. 1995). It is believed that the loop region initially
engages the receptor. This restricts chemokine diffusion and facilitates the appropriate
orientation of the N-terminal domain, which is necessary for the N-terminus-mediated
binding and triggering of the chemokine receptor (Clark-Lewis, Kim et al. 1995). This is
supported by studies that demonstrate that a N-terminal deletion of MCP-l, which retains
its capacity to interact with a chemokine receptor, was unable to stimulate receptor
signaling (Zhang, Rutledge et al. 1994). Moreover, substitution of the ﬁrst 17 amino
acids from IP-lO and GROor, both ligands for CXC3 receptors, with corresponding
residues from SDF-l, a ligand for the CXC4 receptor, enables IP-lO and GROor-mediated
activation of CXC4 receptors (Loetscher, Gong et al. 1998).

Chemokine receptors are classiﬁed as members of the seven pass-transmembrane
spanning (7-TMS) family of Gi-protein-coupled receptors due to the inhibition of
chemokine signaling in the presence of Bordetella pertussis toxin (Thelen, Peveri et al.
1988). Chemokine receptor activation is associated with the activation of phospholipase
C (PLC)—B2 and PLC-B3 (Li, Jiang et al. 2000), which results in the induction of inositol
triphosphate (IP3)-induced intracellular Ca2+ release and diacylglycerol (DAG)-induced

protein kinase C (PKC) activation (Rhee and Bae 1997). While Gi-protein-coupled

25

receptor activation prevents CAMP production, chemokines such as MCP-l have been
shown to stimulate other signaling pathways, including the IP3 kinase and MAPK
pathways (Turner, Ward et al. 1995; Knall, Young et al. 1996). Mice that lack the
expression of pl 10, the catalytic subunit of P13Ky, had impaired neutrophil migration in
response to chemokine receptor stimulation (Sasaki, Irie-Sasaki et al. 2000). The re-
association of the G-protein By subunit with the Ga subunit blocks the signaling pathway
following GTP hydrolysis. More recent evidence suggests that the GTP-bound Gai-
subunit can bind and activate Src and Hck tyrosine kinase signaling pathways, suggesting
additional mechanisms for transducing signals mediated by chemokine receptor

activation (Ma, Huang et al. 2000).

2.2.2 Chemotaxis

Following chemokine receptor activation, sudden changes in cell morphology are
observed due to the polymerization and depolymerization of actin (Baggiolini 1998).
This results in the extension and retraction of lamellipodia, which function as the arms
and legs of a cell as it pushes the plasma membrane outward and pulls the plasma
membrane inward. In addition, chemokine stimulation induces integrin expression and
activation, which enhances cellular adherence to vascular endothelial cells prior to
extravasation (Springer 1994). Chemokine-induced activation also results in the
production of microbicidal oxygen radicals, secretion of proteolytic molecules from
cytoplasmic storage granules of neutrophils, CD8+ T cells, and monocytes, and the
release of histamine from basophils, and the release of cytotoxic proteins from

eosinophils (Baggiolini, Dewald et al. 1997). The fact that higher concentrations of

26

 

chemokines enhance receptor activation and downstream signaling events suggests that
these events occur in the proximity of infection and inﬂammation, where higher levels of
chemokine expression are observed.

The control of the immune response is dependent on the regulated expression of
chemokines and chemokine receptors, which are essential in directing leukocyte
migration to speciﬁc locations under homeostatic and inﬂammatory conditions. The
expression of speciﬁc homeostatic chemokines is required for various processes in
leukocyte function. This includes the targeted recirculation of lymphocytes from the
bloodstream to specialized microenvironments in lymphatic organs that regulate
differentiation, the control of cell survival, and to direct activated effector cells to speciﬁc
sites of infection. Lymphocytes enter peripheral lymph nodes through high endothelial
venules (HEVs), which exclude the entrance of other cell types, such as monocytes and
neutrophils. Several studies suggest that the passage of certain cell types through HEVs
is regulated by the speciﬁc expression of the chemokine receptor CCR7, whose
expression is restricted primarily to B and T lymphocytes (Campbell, Bowman et al.
1998). SLC, a ligand for CCR7, is expressed by HEVs and enhances adhesion and
lymphocyte recruitment into secondary lymphoid organs (Gunn, Tangemann et al. 1998).
In addition, T cells are unable to enter the lymph nodes in SLC-deﬁcient mice (Gunn,
Kyuwa et al. 1999). This can be reversed by intraderrnal injection of SLC (Stein, Rot et
al. 2000). Furthermore, CCR7"' mice have severe defects in T and B lymphocyte
migration (Forster, Schubel et al. 1999). Mice lacking BLRl, a chemokine receptor
expressed on mature B lymphocytes, were also found to have abnormal Peyer’s patches

and inguinal lymph nodes (Forster, Mattis et al. 1996). Moreover, lymphocyte

27

chemotaxis into splenic follicles is severely impaired, migration of activated B cells to
the T cell—rich zone into B cell follicles of the spleen is blocked, and functional germinal
centers are absent in the spleens of mice deﬁcient in BLRl expression.

As B and T lymphocytes enter the lymph node, they migrate to distinct
microenvironments due to the expression of particular chemokines and chemokine
receptors that permit separate stimulatory events by antigen displayed on MHC receptors
of antigen presenting cells (APCs). Following antigen stimulation, a change in
chemokine and chemokine receptor expression induces the migration of T and B cells
toward one another, whereby the T cell receptor recognizes antigen displayed by MHC
class II molecules on the B cell surface. This interaction results in T cell-dependent B cell
activation, proliferation, and germinal center formation (Garside, Ingulli et al. 1998). T
cell migration in response to antigen stimulation results in the upregulation of CXCR5
and CCR4, which are responsive to the chemokine BLC/BCA-l and are expressed in the
B cell zone of secondary lymphoid tissues (Flynn, Toellner et al. 1998). In addition,
CCR7, which is responsive to the T cell zone-speciﬁc ELC chemokine, is upregulated in
antigen-stimulated B cells and promotes the migration of activated B cells near T cells in
the lymph node (Ngo, Tang et a1. 1998). Such studies suggest that chemokines are
essential for lymphocyte migration into speciﬁc microenvironments in the immune
system, which is a prerequisite for an effective immune response.

Unlike homeostatic chemokines, which regulate lymphocyte homing in secondary
lymphoid organs, inﬂammatory chemokines play a pivotal role in the chemotaxis of other
cells involved in innate immunity, including neutrophils and monocytes (Schall and

Bacon 1994). Inﬂammatory chemokines, such as IL-8 (Bazzoni, Cassatella et al. 1991),

28

RANT ES (Schall, Bacon et al. 1990), IP-10 (Luster and Ravetch 1987), and MCP-l
(Leonard and Yoshimura 1990) are produced by endothelial cells, epithelial cells, and
leukocytes in response to various inﬂammatory signals, including bacterial products,
viruses, and various cytokines. The recognition of an infection or physical damage to
tissues by sentinel cells, such as macrophages, is normally responsible for the initiation of
the inﬂammatory response, which results in the production and secretion of cytokines and
chemokines at the site of infection. The release of cytokines and chemokines induces the
upregulation and activation of selectins and integrins on the surfaces of endothelial cells.
It is thought that neutrophils and monocytes ‘role’ along the endothelium through
reversible interactions with selectins until they encounter a high local concentration of
chemokines (Butcher 1991). Chemokines enhance integrin-ligand afﬁnity, resulting in
stable adhesion between leukocytes and the vascular endothelium followed by leukocyte
extravasation. These inﬁltrating leukocytes then migrate along a chemokine gradient
until they encounter the site of infection. This is supported by the fact that cytokine
induction and monocyte recruitment to sites of inﬂammation are severely impaired in

MCP-l'l' mice (Lu, Rutledge et al. 1998).

2.2.3 Chemokine-mediated Pathology

Chemokines are believed to play an important role in the progression of several
diseases. Most notably, certain chemokines were identiﬁed as suppressive factors for
HIV infection in viva (Cocchi, DeVico et al. 1995). This is supported by ﬁndings that
demonstrate a correlation between the increased production of speciﬁc chemokines and a

reduction in HIV infection of CD4+ T cells (Paxton, Martin et al. 1996). In 1996, the

29

chemokine coreceptor CXCR4 was identiﬁed as a cofactor, along with CD4, necessary
for HIV-1 entry into cells, as CXCR4 enabled nonhuman cell types expressing CD4 to
support HIV -1 Env-mediated cell fusion and HIV —1 infection (Feng, Broder et al. 1996).
Moreover, chemokine receptor-speciﬁc antibodies blocked cell fusion and infection with
normal CD4+ human target cells. SDF-l, a CXC chemokine, was identiﬁed as a ligand
for CXCR4. Overexpression of SDF-l prevented infection of CD4+ HeLa cells by T-cell-
line-tropic (T tropic) HIV-1, but did not block CCRS-mediated macrophage tropic ﬂyi-
tropic) HIV-1 infection (Bleul, Farzan et al. 1996). However, CC chemokines, such as
RANTES, macrophage inhibitory protein-la (MIP-lor), and MIP-IB, which block M-
tropic viral infection (F eng, Broder et a1. 1996), were unable to inhibit CXCR4-mediated
HIV-l entry (Oberlin, Amara et al. 1996). Moreover, individuals that contain an internal
32 base pair deletion in CCR5, which encodes a truncated protein that cannot be detected
at the cell surface, are resistant to sexually transmitted HIV-1 infection of macrophages,
but can be infected with T-tropic viruses (Liu, Paxton et al. 1996; Samson, Libert et al.
1996). These and other studies demonstrate the importance of chemokines and
chemokine receptors in HIV infection.

The essential role of chemokines in normal leukocyte trafﬁcking suggests a role
for aberrant chemokine expression and/or function in the progression of various
inﬂammatory pathologies, including atherosclerosis. A critical early event in the
development of atherosclerotic lesions is the adherence and migration of monocytes
through the endothelium of the arterial cell wall and their differentiation into specialized
macrophage foam cells that accumulate excess low-density lipoprotein (LDL) (Jonasson,

Holm et a1. 1986). This results in the eventual occlusion of the vessel due to

30

atherosclerotic plaque buildup or by plaque rupture-mediated thrombus formation (Ross
1995). It is believed that arterial endothelial cells secrete chemoattractants in response to
injuries associated with atherosclerosis, such as high blood pressure and hyperlipidemia
(Taubman, Rollins et al. 1992), as MCP-l overexpression is observed in macrophage-rich
areas in the presence of high levels of oxidized LDL in human and rabbit atherosclerotic
lesions (Yla-Herttuala, Lipton et al. 1991). Moreover, LDL-receptor and MCP-l double
knockout mice fed a high cholesterol diet were found to have reduced levels of
macrophage inﬁltration in their arterial cell walls and less lipid deposition in the aorta,
similar to the case with LDL-receptor’l' mice (Gu, Okada et al. 1998). These studies

suggest a critical role for chemokine function in the progression of atherosclerosis.

3. Acetylation and Gene Regulation
3.1 Chromatin Remodeling and Gene Expression

Transcriptional activation in eukaryotes is a highly regulated process involving the
binding of sequence-speciﬁc transcriptional activators and non-DNA-binding
transcriptional co-activators, the reorganization of chromatin structure, and the
association of general transcription factors and RNA polymerase II at the promoter of a
gene. One of the major barriers to transcriptional initiation is the presence of
histonezDNA interactions in which 146 base pairs of DNA are wrapped around a histone
octarner consisting of two polypeptides each of H2A, H2B, H3, and H4 to form a
nucleosome (Wolffe 1992). Various studies have shown that the nucleosomal structure
represses activation of gene expression, as the transcriptional machinery has limited

access to the regulatory regions in the promoter of a gene, (Grunstein 1990; Owen-

31

Hughes and Workman 1994; Paranjape, Kamakaka et al. 1994; Felsenfeld 1996). In
order to overcome this initial obstacle to activating transcription, the acetylation of
histones has been found to disrupt interactions between both adjacent nucleosomes and
histone octamers with DNA (Wolffe and Pruss 1996; Grunstein 1997; Kouzarides 1999;
Cheung, Briggs et al. 2000).

Histone acetylation is a post-translational, reversible modiﬁcation that occurs by
transferring an acetyl moiety from acetyl-coenzyme A (acetyl-CoA) to the s-amino
groups of often highly conserved lysine residues within the N-terrninal basic region of
histones (Loidl 1994). Several studies have shown that the acetylation of histones is
linked with transcriptional activation (Hebbes, Thome et a1. 1988; Turner 1993; Brownell
and Allis 1996; Grunstein 1997). It is believed that the positively charged histone N-
terminal tail region associates with negatively charged DNA or facilitates interactions
between adjacent nucleosomes (Fletcher and Hansen 1995). Therefore, the acetylation of
lysines, which neutralizes the positively charged residue and increases hydrophobicity
(Kuo and Allis 1998), is responsible for promoting the dissociation of histonezDNA
interactions (Norton, Imai et al. 1989; Hong, Schroth et a1. 1993; Steger and Workman
1996) and causes a reduction in the compaction of nucleosomal arrays by disrupting
intemucleosomal contacts (Bauer, Hayes et al. 1994; Garcia-Ramirez, Rocchini et al.
1995; Fletcher and Hansen 1996; Luger and Richmond 1998; Tse, Sera et al. 1998).
Destabilization of the nucleosomal structure leads to the exposure of sequence-speciﬁc
DNA regulatory sites, which permits the subsequent binding of the transcriptional
apparatus. This is supported by the fact that hyperacetylated histones are associated with

a decondensed chromatin structure, whereas hypoacetylated histones have been observed

32

to be enriched in a more condensed chromatin structure oﬂen associated with
heterochromatic DNA (O'Neill and Turner 1995). In vitro studies using chromatin
templates reconstituted with either hypoacetylated or hyperacetylated histones found that
the acetylation of histones promotes the access of elements of the basal transcription
machinery to nucleosomal DNA templates, which leads to the enhancement of
transcription (Lee, Hayes et al. 1993; Anderson, Lowary et al. 2001; Sewack, Ellis et al.
2001). Chromatin immunoprecipitation (ChIP) assays using antibodies speciﬁc for
acetylated histones have demonstrated a correlation between the recruitment of histone
acetyltransferase (HAT) complexes, increased histone acetylation in the proximal
promoter of certain genes, and increased promoter activity (Kuo, vom Baur et al. 2000).
In contrast, hypoacetylation at particular promoters is associated with the recruitment of
histone deacetylases (HDACs) and transcriptional repression (Khochbin, Verdel et al.
2001). Moreover, amino acid substitution of N-terminal residues of histone H4
encompassing highly conserved, acetylated lysines signiﬁcantly reduced activation of the
GAL] promoter in vivo in S. cerevisiae (Durrin, Mann et al. 1991 ).

Interestingly, recent evidence suggests that the acetylation of histones on speciﬁc
residues may also promote transcriptional activation by serving as a substrate for the
recruitment of speciﬁc regulatory factors to a genetic locus (Strahl and Allis 2000).
Histones can be modiﬁed by phosphorylation, ubiquitination, ADP-ribosylation,
acetylation, and methylation (Bradbury 1992). Strahl and Allis hypothesize that
alternative combinations of these covalent modiﬁcations either sequentially or
concomitantly produce a “histone code” that directs the recruitment of speciﬁc factors of

the transcription apparatus to promote gene expression.

33

3.2 Acetyltransferases

Acetylation is catalyzed by histone acetyltransferases (HATS) that are classiﬁed
into one of two categories (Brownell and Allis 1996): 1) type A HATS are localized in
the nucleus and are believed to acetylate histones and other nuclear factors associated
with transcriptional regulation, and 2) type B HATS are found in the cytoplasm and are
thought to acetylate newly synthesized histones prior to chromatin assembly during S
phase (Ruiz-Carrillo, Wangh et al. 1975; Allis, Chicoine et al. 1985). Acetyltransferases
are also grouped into two families on the basis of sequence similarity in homologous
functional domains: 1) Gcn5-related N-acetyltransferase, and 2) MYST family (Sterner

and Berger 2000).

3.2.1 Gcn5

A 55 kDa polypeptide (p55) was the ﬁrst A-type acetyltransferase to be identiﬁed
and was discovered in the ciliate T etrahymena thermophila (Brownell, Zhou et al. 1996).
p55 is a homolog of GCN5 (General control nonderepressible-S) (Georgakopoulos and
Thireos 1992), a known transcriptional co-activator in S. cerevisiae (Berger, Pina et al.
1992; Marcus, Silverman et al. 1994; Silverrnan, Agapite et al. 1994) and was shown to
have acetyltransferase activity in vitro by its ability to acetylate free histones (Brownell
and Allis 1995; Kuo, Brownell et al. 1996). GCN5 homologs have been identiﬁed in
several organisms, including mouse (Xu, Edmondson et al. 1998), human (Candau,
Moore et al. 1996), Drosophila melanogaster (Smith, Belote et al. 1998), and
T oxoplasma gondii (Hettmann and Soldati 1999), which suggests an important role for

GCN5 activity that may be conserved throughout eukaryotes.

34

To date, there are several known functional domains in S. cerevisiae GCN5
(Sterner and Berger 2000), including a C-terminal bromodomain (Haynes, Dollard et al.
1992) that recognizes acetylated residues. Interestingly, the bromodomain can repress
human GCN5 activity in viva and in vitra through its interaction and subsequent
phosphorylation by the DNA-dependent protein kinase (DNA—PK) holoenzyme, (Barlev,
Poltoratsky et al. 1998). An ADA2 interaction domain, adjacent to the bromodomain,
plays an important role in the recruitment of speciﬁc transcriptional regulators, such as
DNA-binding transactivators (Silverman, Agapite et al. 1994), TATA-binding protein
(Barlev, Candau et a1. 1995) and the core transcription factor TFIIB (Chiang,
Komarnitsky et al. 1996). In addition, a HAT domain is located immediately to the N-
terminus of the ADA2 binding domain and supports GCN5 acetyltransferase activity
(Brownell, Zhou et al. 1996) and adaptor-mediated transcriptional activation in viva
(Candau, Zhou et al. 1997). Several highly conserved residues in the HAT domain are
critical for GCN5 catalytic activity and have been demonstrated to play an important role
in histone acetylation, chromatin remodeling, and transcriptional activation in viva
(Gregory, Schmid et al. 1998; Kuo, Zhou et al. 1998; Wang, Liu et al. 1998).
Interestingly, mutational analyses have demonstrated that the removal of sequences N-
terrninal to the HAT domain were dispensable for GCN5-mediated acetylation and robust
transcriptional activation (Candau, Zhou et a1. 1997). These data support a model
whereby GCN5 functions as an activator of transcription through its ability to promote
remodeling of the chromatin structure by directly modifying histones via recruitment of
multisubunit complexes containing ADA2, which was shown to bridge DNA-binding

transcriptional activators with the basal transcription machinery.

35

Unlike yeast GCN5 (yGCNS), some higher eukaryotic GCN5 proteins, including
those of mice (mGCNS), humans (hGCN5) (Xu, Edmondson et al. 1998) and Drasaphila
(Smith, Belote et al. 1998), contain an additional 400-amino acid domain at the N-
terminus. While hGCN5 has similar substrate speciﬁcity to yGCN5, it is unable to
complement yGCNS activity in gcn5’ yeast cells (Wang, Mizzen et a1. 1997). However,
an chnS chimera containing the HAT domain of hGCN5 was capable of supporting
transcriptional activation in gcn5' cells. These ﬁndings suggest both the evolutionary
conservation of GCN5 HAT activity and the divergence of GCN5 function, presumably
reﬂected by the additional N-tenninal sequences in higher eukaryotes. In addition, there
are multiple hGCN5 variants as a result of alternative splicing, adding an additional level
of complexity to GCN5 activity. While the hGCN5 N-terminal extension is dispensable
for acetylation of free histones (Yang, Ogryzko et al. 1996; Wang, Mizzen et al. 1997),
only full-length hGCN5 and mGCNS can acetylate nucleosomal histones (Xu,
Edmondson et al. 1998), suggesting a role for the GCN5 N-terminus in chromatin-
speciﬁc acetylation. In higher eukaryotes, GCN5 is able to physically interact with
additional co-activators, such as p300 and CBP, to facilitate the acetylation of various

substrates (Sterner and Berger 2000).

3.2.2 P/CAF

P/CAF (p300/CBP Associated Factor) was initially identiﬁed on the basis of its
homology to GCN5 (~70% sequence identity) with the majority of conservation residing
in the C-terminal HAT domain (Sterner and Berger 2000). Like GCN5, full-length
P/CAF can acetylate both free and nucleosomal histones (Kuo, Brownell et al. 1996;

Yang, Ogryzko et al. 1996), with substrate speciﬁcity similar to GCN5 (Xu, Edmondson

36

et al. 1998; Schiltz, Mizzen et al. 1999). While P/CAF and GCN5 are ubiquitously
expressed in mouse, their relative mRNA expression levels vary in different tissues. The
ratio of mouse GCN5 to P/CAF expression is higher in thymus, spleen, testis, brain, and
embryonic tissue, while being reduced in heart, liver, kidney, and skeletal muscle (Xu,
Edmondson et al. 1998). In vitra and in viva studies have demonstrated the ability of
P/CAF to associate with p300 and CBP (Yang, Ogryzko et al. 1996). The adenoviral
oncoprotein ElA disrupts P/CAF association with p300 and CBP due to its ability to
compete for the same interaction domain. While P/CAF and GCN5 N«terminal domains
mediate p300 and CBP interactions, these co-activators bind to separate regions within
p300 and CBP. The co-activators p300 and CBP interact with numerous DNA-binding
factors, including CREB (Chrivia, Kwok et al. 1993; Kwok, Lundblad et al. 1994), c-Jun
(Arias, Alberts et al. 1994), F05, (Bannister and Kouzarides 1995), C/EBPB (Mink,
Haenig et al. 1997), and nuclear receptors (Kamei, Xu et al. 1996). Therefore, P/CAF
could be targeted to speciﬁc promoters through its interaction with p300/CBP to
selectively acetylate speciﬁc histone residues and/or chromatin remodeling complexes to

facilitate promoter activation.

3.2.3 p300 and CBP

p300 and CBP (CREB binding protein) are large (~300kDa) nuclear proteins that
have various functional roles in transcriptional regulation, including the linkage of
various transcription factors with the basal transcriptional apparatus (Sterner and Berger
2000), acetyltransferase activity and chromatin remodeling (Bannister and Kouzarides

1996; Ogryzko, Schiltz et al. 1996), integration of both positive and negative signal

37

transduction pathways on gene expression (Arany, Newsome et al. 1995), and cell cycle
regulation, differentiation, and apoptosis (Lundblad, Kwok et al. 1995; Eckner, Yao et al.
1996; Yuan, Condorelli et al. 1996; Blobel, Nakajima et al. 1998; Giordano and
Avantaggiati 1999). Several lines of evidence suggest that aberrant p300/CBP activity
may play an important role in tumor progression in certain human cancers, as inactivating
somatic mutations of p300 are observed in gastric and colon cancers (Barbeau,
Charbonneau et al. 1994; Miller and Rubinstein 1995; Muraoka, Konishi et a1. 1996).

p300 and CBP are highly homologous (~63% amino acid identity) transcriptional co-
activators that are ubiquitously expressed (Giles, Peters et al. 1998). Like P/CAF and
GCN5, p300 and CBP contain intrinsic acetyltransferase activity (Bannister and
Kouzarides 1996; Ogryzko, Schiltz et al. 1996) and a bromodomain (Jeanmougin, Wurtz
et al. 1997). Unlike GCN5 and P/CAF, while p300 and CBP can acetylate all four
histones within nucleosomes, they preferentially acetylate H3 and H4. Therefore, the
ability of p300/CBP to recruit speciﬁc subsets of additional co-activators with
acetyltransferase activity, such as GCN5, P/CAF (Xu, Edmondson et al. 1998), SRC-l
and ACTR (Smith, Onate et al. 1996; Yao, Ku et al. 1996; Chen, Lin et al. 1997; J enster,
Spencer et al. 1997; Chen, Lin et al. 1999), may determine the speciﬁc function for p300
and CBP on various promoters.

p300 and CBP contain multiple interaction domains for numerous factors, including
nuclear hormone receptors (RAR), estrogen receptor (ER), and thyroid-hormone receptor
(TR) (Chakravarti, LaMorte et a1. 1996), acetyltransferases P/CAF and GCN5 (Yang,

Ogryzko et al. 1996), numerous transcription factors, such as CREB, p53, C/EBPB, and

NF-KB (Lundblad, Kwok et al. 1995; Dai, Akimaru et al. 1996; Gu and Roeder 1997;

38

Mink, Haenig et al. 1997; Vanden Berghe, De Bosscher et al. 1999), general transcription
factors like TFIIB and TBP (Kwok, Lundblad et al. 1994; Yuan, Condorelli et al. 1996),
and nuclear receptor co-activators, such as SRC-l and ACTR (Smith, Onate et al. 1996;
Yao, Ku et al. 1996; Chen, Lin et al. 1997; Jenster, Spencer et al. 1997; Chen, Lin et al.
1999). The presence of multiple interaction domains for numerous factors in a large
protein such as p300 and CBP supports a model whereby p300 and CBP interact with
multiple factors Simultaneously to facilitate speciﬁc biological responses. This notion is
supported by the fact that SRC-l can associate with the C-terminus of p300/CBP, while
nuclear receptors interact with N-terminal determinants, and that these interactions are
important for PR activation (Smith, Onate et al. 1996).

In addition to connecting various transcriptional modulators to elements of the basal
transcription machinery, p300 and CBP function as a critical juncture, integrating diverse
Signaling pathways to modulate gene expression. For example, IFN-or was Shown to
induce Stat2 and p300/CBP cooperation to stimulate promoter activation (Bhattacharya,
Eckner et al. 1996), while ElA overexpression, which competes for the Stat2 interaction
domain of p300/CBP, represses IFN-or-induced Stat2 transactivation. Interestingly,
insulin-induced Ras activation was shown to stimulate S6 kinase pp90R5k interaction with
the BIA-binding domain of p300/CBP, which is associated with the repression of CAMP-
responsive promoters through CREB (N akaj ima, Fukamizu et al. 1996). Moreover, p300
association with various transcription factors, including C/EBPB, inﬂuences the
phosphorylation state and consequent modulation of p300 activity (Schwartz, Beck et a1.
2003). Furthermore, the level of p300/CBP phosphorylation is altered during the cell

cycle, where maximal phosphorylation is observed during mitosis (Yaciuk and Moran

39

1991). These data demonstrate the importance of kinase signaling cascades in
modulating p300/CBP activity. A recent study identiﬁed a novel catalytic domain in the
N-terminus of p300 that is responsible for protein turnover. p300, which interacts with
numerous transcription factors, including p53 (Gu and Roeder 1997), was Shown to
catalyze the polyubiquitination and subsequent proteasome-mediated degradation of p53

in the absence of EIA in vitra and in viva (Grossman, Deato et al. 2003).

3.3 Factor Acetyltransferase Function

Histones are by far the best-characterized substrates for protein acetylation.
Acetylation of histones has traditionally been thought to correlate with an active
transcriptional state, due to its association with the remodeling of the chromatin structure
and exposure of regulatory sites for the binding of the transcriptional machinery to the
promoter of a gene. More recent evidence suggests that histone acetyltransferases may
further regulate various aspects of transcription by directly modifying elements of the
transcriptional machinery to enhance or repress their respective activity. This regulation
can alter DNA-binding, protein-protein interactions, protein stability, and the integration

of signaling pathways.

3.3.1 DNA-Binding

p53, a tumor suppressor/transcription factor that enhances the expression of apoptotic
genes in response to DNA damage (Chen, Ko et al. 1996; Levine 1997) was one of the
ﬁrst transcription factors identiﬁed as an acetylation substrate (Gu and Roeder 1997). In
vitra and in viva studies identiﬁed two adjacent lysine residues within the highly

conserved motif KXKK as a substrate for acetylation by p300. This acetylation motif is

40

located near the DNA-binding domain of p53. Unexpectedly, acetylation was found to
increase p53 DNA-binding and enhance its transactivation potential (Gu and Roeder
1997). Like p53, KXKK motifs in GATA-l and E2F-l located at the N-terminal
boundary of their respective DNA binding domains were identiﬁed as acetylation
substrates. In both cases, acetylation in the presence of p300 and P/CAF, respectively,
augmented their transactivation potential (Boyes, Byﬁeld et al. 1998; Martinez-Balbas,
Bauer et al. 2000). Moreover, the co-expression of GATA-l and E2F-1 with
acetyltransferase-deﬁcient mutants of p300 and P/CAF was unable to augment DNA-
binding and promoter activity. It is worth noting that, while acetylation increases the
overall negative charge of these proteins near the DNA-binding domain, this modiﬁcation
leads to an increase in DNA-binding. This suggests a mechanism whereby acetylation
may induce a conformational change in protein structure that enhances the DNA-binding
activity of certain transcription factors.

However, acetylation may also lead to the inhibition of DNA-binding of certain
transcription factors. P/CAF and GCN5-mediated acetylation at a highly conserved lysine
residue in the DNA-binding domain of lRF-7 reduces its DNA-binding activity and
transactivation potential (Caillaud, Prakash et al. 2002). In another instance, P/CAF
acetylation of C/EBPB was found to diminish its DNA-binding activity in IL-3-dependent
expression of the Id-l gene (Xu, Nie et al. 2003). Research presented in Chapter 3 shows
that, while P/CAF reduces C/EBPB DNA-binding, the co-expression of p300 with the
P/CAF homolog GCN5 dramatically enhances C/EBPB DNA-binding through the use of

alternative lysines.

3.3.2 Protein-Protein Interactions

41

Acetylation of sequence-speciﬁc transactivators has also been shown to regulate
transcription by impacting protein-protein interactions. P/CAF-mediated acetylation of
TALl, a critical regulator of hematopoietic development, was shown to increase DNA-
binding in viva and in vitra and to promote TALl-dependent promoter activation. The
augmentation of TALl activity by P/CAF acetylation was associated with the disruption
of TALl interactions with the transcriptional co-repressor mSin3A (Huang, Qiu et al.
2000). EKLF is another hematopoietic transcription factor (Miller and Bieker 1993)
shown to be a substrate for acetylation by p300/CBP (Zhang and Bieker 1998). While
EKLF DNA-binding is unaffected by acetylation, p300/CBP-mediated acetylation of
EKLF stimulated transactivation of the B-globin promoter in viva by increasing EKLF
cooperativity with the SWI-SNF chromatin remodeling complex (Zhang, Kadam et al.
2001). Tat, a viral transactivator of human immunodeﬁciency virus (HIV) (Cullen 1998),
is another transcription factor whose function is impacted by acetylation (Kieman,
Vanhulle et al. 1999), as in viva and in vitra binding of Tat to the P/CAF bromodomain
and synergistic activation of the HIV promoter was dependent on P/CAF-mediated Tat
acetylation (Dorr, Kierrner et al. 2002).

Acetylation may also regulate gene expression by disrupting protein-protein
interactions. The proto-oncogene BCL6 represses transcription of genes involved in
lymphocyte activation and differentiation in B cell germinal centers (Reljic, Wagner et al.
2000; Shaffer, Yu et al. 2000). p300 was recently shown to acetylate and consequently
inhibit BCL6 activity in viva by disrupting its ability to recruit HDACs (Bereshchenko,
Gu et al. 2002). The inability to recruit deacetylase activity, which is normally associated

with transcriptional repression (Pazin and Kadonaga 1997), blocks the inhibitory function

42

of BCL6, as well as its ability to induce cellular transformation. In addition, the
acetylation of the ERor hinge/ligand—binding domain by p300 suppresses ERa sensitivity

to estrogen on an ER-dependent luciferase reporter (Wang, F u et al. 2001).

3.3.3 Protein Stability

Ito et al. demonstrated that the acetylation motif KXKK in p53 may also function as a
substrate for MDM2-mediated HDAC recruitment and ubiquitination resulting in protein
degradation (Ito, Kawaguchi et al. 2002). This data suggests that one possible ﬁrnction of
HAT-dependent acetylation is to prevent protein degradation by blocking MDM2-
dependent ubiquitination. Like p53, E2F is acetylated by P/CAF and p300/CBP at the
highly conserved KXKK acetylation motif adjacent to its DNA binding domain
(Martinez-Balbas, Bauer et al. 2000; Marzio, Wagener et al. 2000). As observed in
numerous transcription factors including p53, E2F acetylation enhances its DNA-binding
activity and transactivation potential. Furthermore, P/CAF acetylation was Shown to
facilitate the stabilization of activated (dissociated from Rb) E2F (Martinez-Balbas,
Bauer et al. 2000). Interestingly, Rb was shown to promote the deacetylation of E2F by
forming a complex with E2F and a HDAC. In another instance, acetylation of SREBPl,
a critical regulator of adipocyte differentiation (Osborne 2000; Rosen, Walkey et al.
2000), by p300/CBP was shown to increase the stability of SREBPla and SREBP2 by
blocking ubiquitination of the same residues that are acetylated (Giandomenico,
Simonsson et al. 2003). Data presented in Chapter 3 demonstrate the importance of the

KXKK motif of C/EBPB in co-activator-mediated protein stabilization.

43

3.2.4 Signal Transduction Pathways

The identiﬁcation of numerous non-histone proteins that are substrates for acetylation
has revealed the involvement of protein acetylation in many biological processes,
including signal transduction. POP-l, the Caenarhabditis elegans homolog of lymphoid
enhancer factor/T-cell factor (LEF/T CF), is a transcription factor that plays an important
role in the Wnt signaling pathway during embryonic development (van Noort and Clevers
2002). A recent study demonstrated that p300/CBP-mediated in viva acetylation
promoted the nuclear import of POP-1 and was critical for its appropriate nuclear
localization and function during C. elegans development (Gay, Calvo et al. 2003).
Interestingly, the acetylation of POP-1 is similar to that of NF-KB, where p65 acetylation
was also shown to regulate NF-kB nuclear retention (Chen, Fischle et al. 2001). p65
acetylation was shown to block the formation of NF-kB-IkBor complexes, a prerequisite
for nuclear export (Arenzana-Seisdedos, Turpin et al. 1997).

Recent studies have also demonstrated an important role for acetylation in cell cycle
regulation. The Rb protein can block the G1 to S phase transition via its interaction and
sequestration of E2F, a transcription factor which promotes cell cycle progression
(Weinberg 1995; Dyson 1998). The phosphorylation of Rb by Cdks disrupts the Rb-E2F
complex, allowing E2F to initiate transcription of genes required for entry into S phase
(Nevins 1992). Interestingly, acetylation of Rb in a cell-cycle-dependent manner by
p300/CBP inhibits the phosphorylation of Rb by Cdks (Chan, Krstic-Demonacos et al.
2001), thereby blocking entry into S phase. In addition, acetylation can regulate the cell
cycle in response to DNA damage. p73, a tumor suppressor protein related to p53

(Levrero, De Laurenzi et al. 2000), is acetylated by p300 in response to DNA damage-

44

induced c-abl activation (Costanzo, Merlo et al. 2002). Moreover, lysine to arginine
substitutions that prevent acetylation block the ability of p73 to support the induction of

apoptosis in response to the DNA-damage-inducing agent doxorubicin.

45

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leucine zipper exchange between GCN4 and C/EBP." Science 246(4932): 922-6.

Akira, S., H. Isshiki, et al. (1990). "A nuclear factor for IL-6 expression (NF-1L6) is a
member of a C/EBP family." Embo J 9(6): 1897-906.

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70

Chapter 2

Differential Roles of C/EBPB Regulatory Domains in MCP-l and IL-6

Transcription

71

Abstract

C/EBPB is a member of the CCAAT/enhancer binding protein family of transcription
factors and has been shown to be a critical transcriptional regulator of various
proinﬂammatory gene products, including interleukin-6 (IL-6) and monocyte
chemoattractant protein-1 (MCP-l). To examine the comparative roles of the C/EBPB N-
terrninal activation and regulatory domains in lipopolysaccharide (LPS)-induced MCP-l
and IL-6 expression, various N-terminal truncations and deletions of C/EBPB were
ectopically expressed in P388 murine B lymphoblasts using both retroviral transduction
and transient transfection. P388 cells lack endogenous C/EBPB expression and are
normally unresponsive to LPS for expression of IL-6 and MCP-l. Unexpectedly,
regulatory domains 1 and 2 between amino acids 153-212 of C/EBPB Signiﬁcantly
inhibited C/EBPB activation of MCP-l expression, while not affecting activation of IL-6
expression, demonstrating the importance of this region in conferring promoter-speciﬁc
effects. Moreover, serine 64 of C/EBPB in activation domain module 2 is critical for
both IL-6 and MCP-l expression. These data suggest a model whereby C/EBPB
differentially regulates proinﬂammatory cytokine transcription through the activity of
multiple functional determinants. These data identify serine 64 as an additional
functional determinant for C/EBPB-mediated activation of IL-6 and MCP-l expression,
and suggest a model whereby CfEBPB regulates proinﬂammatory cytokine transcription

through the differential activity of the regulatory domains 1 and 2.

72

Introduction

CCAAT/enhancer-binding proteins (C/EBPS) comprise a family of basic region-
leucine zipper (bZIP) transcription factors that, as a prerequisite for DNA-binding
through their basic region, dimerize through their adjacent C-terrninal leucine zipper to
form either homodimers or heterodimers with other C/EBP family members (Ramji and
Foka 2002). While C/EBP’S are well conserved in their bZIP regions, they exhibit more
limited conservation in their N-terminal activation and regulatory domains (Ramji and
Foka 2002). C/EBPa, B, 5, and e commonly function as strong transcriptional activators,
while C/EBPy and Q generally act as dominate negative repressors of C/EBP
transcriptional activators (Lekstrom-Himes and Xanthopoulos 1998). C/EBPQ-C/EBP
heterodimers can also activate transcription through a non-consensus binding site (Wang

et a1 1998; Sok et al 1999), and recent work has Shown that C/EBPy as a heterodimer

with C/EBPB can augment transcription in a promoter and cell-speciﬁc manner (Gao,
Parkin et al. 2002). C/EBP binding sites have been identiﬁed in the promoter regions of
numerous cytokine and other proinﬂammatory gene products, including interleukin-6
(IL-6) (Akira, Isshiki et al. 1990), IL-12 (Plevy, Gemberling et al. 1997), tumor necrosis
factor or (TNFor) (Pope, Leutz et al. 1994), IL-l-B (Shirakawa, Saito et al. 1993), IL-8
(Matsusaka, Fujikawa et al. 1993), monocyte chemoattractant protein-1 (MCP-l)
(Sekine, Nishio et al. 2002), granulocyte-colony stimulating factor (G-CSF) (Dunn,
Coles et al. 1994), myeloperoxidase (Ford, Bennett et al. 1996), inducible nitric oxide
synthase (Lowenstein, Alley et al. 1993), and lysozyme (Goethe and Loc 1994).
Moreover, C/EBPB and C/EBPB activity is inﬂuenced by various inﬂammatory stimuli,

including LPS (Alam, An et al. 1992; An, Hsieh et al. 1996), IL-6 (Poli, Mancini et al.

73

1990), IL-l (Akira, Isshiki et al. 1990), and TNFor (Yin, Yang et al. 1996), suggesting an
important role for C/EBPS in mediating the inﬂammatory response. We previously
demonstrated that the stable expression of C/EBPor, B, 8, or s was capable of conferring
lipopolysaccharide (LPS)-induced IL-6 and MCP-l expression to a murine B
lymphoblast cell line (Bretz, Williams et al. 1994).

While C/EBPs are quite divergent in their N-terminus, three separate activation
domain modules (ADMl, 2, and 3) are shared by C/EBPa, B, 5, and 3 (Williams, Baer et
al. 1995). Unlike C/EBPor and 5, C/EBPB and s also have internal regulatory regions
(RBI and RD2), which conditionally repress their transactivation potential (Williams,
Baer et al. 1995). The functional importance of N-terrninal determinants in C/EBP-
mediated transcriptional regulation is supported by the fact that C/EBPB153-297 (LIP), a
truncated form of C/EBPB that initiates at Met 153 and lacks N-terrninal activation
domains, can function as a repressor of C/EBP transcriptional activity by either directly
competing with activating forms for C/EBP binding sites or as a dominant negative by
heterodimerizing with activating C/EBP family members (Descombes and Schibler
1991). Moreover, C/EBPy, which also lacks N-terminal activation motifs, was reported
to inhibit C/EBP transcriptional activity by either forming inactive heterodimers with
C/EBPa and B, or by competing with activating C/EBP isoforms (Cooper, Henderson et
al. 1995).

The N-terminal region modulates C/EBPB activity by providing domains for
protein-protein interactions and post-translational modiﬁcations. N-terrninal activation
domains in C/EBPB are reported to associate with the coactivator p300 (Mink, Haenig et

al. 1997) and the chromatin remodeling complex SWI/SNF (Kowenz-Leutz and Leutz

74

1999), inﬂuencing the regulation of C/EBP-dependent promoters. In addition, various
signal transduction pathways impact C/EBPB activity at N-terminal substrates. Ras-
mediated phosphorylation of rat C/EBPB at a mitogen-activated protein kinase (MAPK)
Site located at threonine 189 enhances transactivation of a target promoter, and mutation
of the residues that comprise this site reduce ras-mediated C/EBPB activation (Nakajima,
Kinoshita et al. 1993). This MAPK substrate is also responsive to growth hormone
(GH)-induced phosphorylation, as an alanine substitution at threonine 189 inhibits c-fos
promoter activation in response to GH (Piwien-Pilipuk, MacDougald et al. 2002). TPA-
induced activation of the protein kinase C pathway results in phosphorylation of rat
C/EBPB at serine 105 and enhanced C/EBPB transactivation (Trautwein, Caelles et al.
1993). This site has also been Shown to function as a substrate for TGF-or-induced
phosphorylation by p90 ribosomal S kinase, which increases C/EBPB activity and
hepatocyte proliferation (Buck, Poli et al. 1999). A more recent study demonstrated that
sumoylation of C/EBPB at a N-terrninal motif alleviated the inhibitory effects mediated
by Regulatory Domain 1 (RDl) (Kim, Cantwell et al. 2002). These ﬁndings demonstrate
the functional signiﬁcance of N-terrninal motifs in response to various signaling events in
C/EBPB-mediated transcriptional regulation.

Contrary to the notion of the N-terminal domains being essential for C/EBP
transactivation, at least two studies have demonstrated the dispensability of the N-
terminus in C/EBPB-mediated transcriptional regulation. C/EBPB153-297 (LIP) and
progesterone receptor B cooperate to activate progesterone response element-driven
promoters (Christian, Pohnke et al. 2002). Previous work in our laboratory showed that

LIP, as well as the C/EBPB bZIP domain (C/EBPB213-297), retain the capacity to support

75

LPS-inducible activation of a proximal IL-6 promoter-luciferase reporter in P388 murine
B lymphoblasts (Hu, Tian et al. 2000). The C/EBPB bZIP domain is capable of
supporting LPS-induced expression of both IL—6 and MCP-l in stable transfections (Hu,
Tian et al. 2000). However, the phosphorylation of the MAPK site at threonine 189 of
C/EBPB was Shown to impact IL-6 expression (Nakajima, Kinoshita et al. 1993), and
thus, notwithstanding the activity of LIP and the C/EBPB bZIP domain, the N-terrninal
region of C/EBPB does play a role in IL-6 transcription. This led us to examine the role
of the N-terminal region of C/EBPB in IL-6 and MCP-l expression. We expected the N-
terrninal region to have a similar function in the activation of these two proinﬂammatory
gene products, as our previous studies found their regulation to parallel each other. In
this report, we unexpectedly found that a region encompassing the RD] and RD2 internal
regulatory domains of C/EBPB signiﬁcantly inhibited its activation of both a MCP-l
proximal promoter-reporter and the endogenous intact MCP-l promoter, while having
little effect on activation of the IL-6 promoter. We also found that serine 64 in the N-
terrninal ADM2, a residue that is highly conserved among C/EBP family members, is
critical for both optimal IL-6 and MCP-l expression. Our current ﬁndings suggest a role
for RDl and RD2 in conferring promoter speciﬁcity to C/EBPB-mediated transcriptional
activation, and identify serine 64 as an additional ﬁrnctional determinant for C/EBPB-

mediated activation of both IL-6 and MCP-l expression.

76

Materials and Methods

Cell Culture. P388 cells are murine B lymphoblasts (Bauer, Holmes et al. 1986)
(American Type CultureCollection; CCL 46). P388-Neo, P388-CB, and P388—CB213-297
cells have been described previously by Hu et al (Hu, Tian et al. 2000). P388 cells were
cultured in RPMI 1640 medium supplemented with 5% fetal calf serum and 50 M B-
mercaptoethanol. All LPS inductions of IL-6 and MCP-l were conducted with LPS
derived from Escherichia cali serotype 055:B5 (Sigma) added to 10 ug/ml.

Retroviral Transductions. C/EBPB mutants were expressed from pZIP-Neo
SV(X)1, a retroviral vector derived from Moloney murine leukemia virus (Cepko,
Roberts et al. 1984). Retroviral stocks were prepared by transient transfection of 293T
cells (Pear, Nolan et al. 1993) that were grown in Dulbecco’s modiﬁed Eagle’s medium
supplemented with 10% FCS. Retroviral expression vectors (3 pg) were cotransfected
with a packaging construct (3 pg), pMOV-w (Mann, Mulligan et a1. 1983). These
transfections were performed using DMRIE-C reagent according to the manufacturer’s
instructions (Invitrogen) upon 70% conﬂuent 293T cells on 60 mm plates. Retrovirus-
containing supematants were harvested 48 hours post-transfection by centrifugation at
1500 rpm for 5 min followed by ﬁltration through a 0.45-uM-pore-size ﬁlter. Retroviral
infections were performed on 60 mm plates by the addition of 3 ml virus stock to 2 x 106
P388 cells in standard grth medium with the total volume supplemented to 8 ug/ml
polybrene (Sigma). The cells and retrovirus were incubated at 37°C for 3 hours during
which time the plates were swirled every 30 min. The infection medium was then

replaced with standard growth medium. After 24 hours, the cells were split to four 60

77

mm plates and G418 (Invitrogen) was added to a ﬁnal concentration of 1 mg/ml for
selection of resistant cells.

Transient Transfections. Transient transfections were conducted with 2 x 106 P388
cells, 4 pg of DNA, and 8 pl of DMRIE-C reagent in 1.2 ml of OPTI-MEM medium.
The DNA was comprised of either 1 pg of MCP-l promoter-reporter or IL-6 promoter-
reporter, 0.1 pg of SV40 early promoter-reporter, 2 pg of wildtype or truncated C/EBPB
expression vector, and pMEX plasmid to total 4 pg. Cells were incubated in the
transfection mixture for 5 hours followed by addition of RPMI 1640 medium
supplemented to 15% with FCS. After 20 hours, the medium of certain transfections was
supplemented with 10 pg/ml LPS. After 4 hours in the absence of presence of LPS,
transfected cells were harvested, lysed and analyzed for luciferase activity by using the
Luciferase Reporter Gene Assay Kit (Roche Molecular Biochemicals) and for B-
galactosidase activity by using the Luminescent B-Galactosidase Genetic Reporter
System 11 (Clontech).

Expression Vectors and Promoter Reporters. All forms of C/EBPB expressed in
these experiments are derived from rat C/EBPB22-297 (LAP). For retroviral transductions,
rat C/EBPB deletions were constructed by the transfer of C/EBPB37-297, C/EBPB64-297,
C/EBPB153-297, and C/EBPB187-297 BamHI/HindIII fragments from pMEX (Williams,
Cantwell et al. 1991) into the BamHI site of pZIP-NEO SV(X)1 with BamHI linkers.
The C/EBPB(S64A) mutant was generated by site-directed mutagenesis and the C/EBPB-
encoding EcaRI/HindIII fragment from pCDNA3.1 was transferred into the BamHI site

of pZIP-NEO SV(X)1 with BamHI linkers. Inserted sequences were transcribed from the

Moloney murine leukemia virus promoter and the gene conferring G418-resistance was

78

expressed from a subgenomic splicing product from the same promoter. For transient
transfections, C/EBPS were expressed from pMEX, which utilizes the Moloney murine
sarcoma virus promoter.

The IL-6 promoter reporter consists of the murine IL—6 promoter (Tanabe, Akira et al.
1988) (-250 to + I) inserted into the luciferase vector pXP2 (Nordeen 1988). The MCP-l
promoter-reporter consists of the murine MCP-l promoter (U eno, Sonoda et a1. 2000) (-
322 to +59) inserted into the luciferase vector pGV-B2 (Toyo Ink Mfg. Co., Ltd.). The
SV40 early promoter-reporter is a commercial product, p-Bgal control (BD Biosciences
Clontech), where the SV40 early promoter and enhancer sequences are cloned upstream
and downstream, respectively, of the lacZ gene.

RNA Isolation and Analysis. Total RNA was isolated using TRIzol reagent
(Invitrogen) according to the manufacturer’s directions. RNAs were electrophoresed
through 1% agarose/formaldehyde gels and transferred to membranes that were
hybridized and washed to a stringency of 0.1% x SSPE at 65°C. Hybridization probes
were prepared with a random priming kit (Invitrogen) with the incorporation of 5’-[or-
32P]dATP (3000 Ci/mmol; NEN Life Science Products Inc.) The IL-6 probe was a 0.65
kilobase murine cDNA (from N. Jenkins and N. Copeland, National Cancer Institute-
Frederick Cancer Research and Development Center). The MCP-l probe was a 0.58
kilobase mmine cDNA (Rollins, Morrison et al. 1988). The glyceraldehye-3-phosphate
dehyrdogenase (GAPDH) probe was a 1.3 kilobase rat cDNA (Fort, Marty et al. 1985).

Western Analysis. Nuclear extracts were prepared as described below. The extracts
(50 pg) were adjusted to 1X Laemmli sample buffer (Laemmli 1970) and analyzed by

sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis. Gels were transferred

79

to a Protran membrane (Schleicher and Schuell) and antigen-antibody complexes were
visualized with the Enhanced Chemiluminescence kit (Amersham Pharmacia Biotech).

Electrophoretic Mobility Shift Assay (EMSA). Nuclear extracts were prepared as
follows. Cells were washed in phosphate-buffered saline and lysed in 15 mM KCl, 10
mM HEPES (pH 7.6), 2mM MgClz, 0.1 mM EDTA, lmM dithiothreitol, 0.1% (v/v)
Nonidet P-40, 0.5mM phenyhnethylsulfonyl ﬂuoride, 2.5 pg/ml Leupeptin, 5 pg/ml
antipain, and 5 pg/ml aprotinin for 10 min. on ice. Nuclei were pelleted by centrifugation
at 14,000 x g for 1 min. at 4°C. Proteins were extracted from nuclei by incubation at 4°C
with vigorous vortexing in Buffer C (420 mM NaCl, 20 mM HEPES, (pH 7.9), 0.2 mM
EDTA, 25% (v/v) glycerol, 1 mM dithiothreitol, 0.5mM phenyhnethylsulfonyl ﬂuoride,
2.5pg/ml Leupeptin, 5 pg/ml antipain, and 5 pg/ml aprotinin). Nuclear debris was
pelleted by centrifugation at 14,000 x g for 30 min. at 4°C and the supernatant extract
was collected and stored at -70°C.

The EMSA probe was a double-stranded oligonucleotide containing an optimal
C/EBP binding site (5’-
GATCCTAGATATCCCTGATTGCGCAATAGGCTCAAAGCTG-3’ annealed with 5’-
AATTCAGCTTTGAGCCTATTGCGCAATCAGGGATATCTAG-3’) labeled with the
incorporation of 5’-[0L-32P]dATP (3000 Ci/mmol; NEN Life Science Products Inc.) and
Klenow DNA polymerase. Underlined sequences correspond to the binding motif of
C/EBPB.

DNA binding reactions were performed for 20 min at room temperature in a 25-pl
reaction mixture containing 6 pl of nuclear extract (1 mg/ml in Buffer C) and 5 pl of 5x

binding buffer (20% (w/v) Ficoll, 50 mM HEPES (pH 7.9), 5 mM EDTA, 5 mM

80

dithiothreitol). The remainder of the reaction mixture contained 1 pg of poly(dI-dC), 300
pg of probe, bromophenol blue to a ﬁnal concentration of 0.06% (w/v), and water to
volume. The remainder of the reaction mixture contained KCl to a ﬁnal concentration of
50 mM, Nonidet P-40 to a ﬁnal concentration of 0.1%, 1 pg of poly(dI-dC), 300 pg of
probe, bromophenol blue to a ﬁnal concentration of 0.06% (w/v), and water to volume.
For supershiﬁs, nuclear extracts were preincubated with antibodies for 20 min. at 4°C
prior to the binding reaction. Samples were electrophoresed through 5.5%
polyacrylamide gels in 1x TBE (90 mM Tris base, 90 mM boric acid, 0.5 mM EDTA) at
160V.

Antibodies. Rabbit anti-C/EBPB speciﬁc to the carboxyl terminus (product C-19)
and normal rabbit IgG were purchased from Santa Cruz Biotechnology. Rabbit anti-
C/EBPB speciﬁc to the amino terminus has been described (Williams, Cantwell et al.
1991). Rabbit anti-C/EBPy (Ig/EBP) was prepared against a synthetic peptide

corresponding to the carboxyl terminus of C/EBPy (Parkin, Baer et al. 2002).

81

Results

Individual N-Terminal Functional Domains in C/EBPﬂ Promote and Repress
MCP-I Expression—We previously demonstrated that stable expression of C/EBPor, B, 8,
and 8 in P388 murine B lymphoblasts, which lack expression of all C/EBP family
members except C/EBPy, all supported LPS-inducible IL-6 and MCP-l mRNA
expression (Bretz, Williams et al. 1994; Hu, Baer et al. 1998). We found that this
redundancy among the C/EBPs is based on the ability of the well-conserved bZIP region
by itself to support LPS induced IL-6 and MCP-l expression (Hu, Tian et al. 2000). The
C/EBPB bZIP region (C/EBPB213-297), lacking all known conventional activation and
regulatory domains, behaved similarly to intact C/EBPB in its ability to support both IL-6
and MCP-l transcription in response to LPS (Hu, Tian et al. 2000), suggesting a similar
role for C/EBPB in both IL-6 and MCP-l transcription. While C/EBPB213_297 and LIP
activated an IL-6 promoter-reporter in response to LPS, the removal of N-terrninal
activation and regulatory motifs reduced its transactivation potential relative to intact
C/EBPB (Hu, Tian et al. 2000). To investigate more directly the role of C/EBPB N-
terrninal activation and regulatory domains in the regulation of MCP-l and IL-6
expression, we generated stable retroviral transductants of P388 cells ectopically
expressing various N-terrninal truncations of C/EBPB (Fig. 1). Stable expression of
C/EBPB and its various deleted forms was initially characterized by EMSA of nuclear
extracts from the transduced populations. Progressive deletion of the N-terminal
functional domains did not prevent binding to a consensus C/EBP binding motif, as

protein-DNA complexes supershiﬁed

82

Activation and Basic Lcucine

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

came 22 1 11 Riiimm Dom am im- Region 297
cram... 37 297
cmpsm 64 297
mp3,,“ 102 297
c/EBpe,,,_,,, (up) 153 W 297

 

 

 

 

cansmmm 187 297

 

CIEBP5,,,_,,, (mp) 213 -. 7////////////////n 297

Figure 2-1 Diagram of the structures of C/EBPB and mutants used in these
studies.

83

upon incubation with a C/EBPB-speciﬁc antibody were observed in nuclear extracts from
P388-CB (LAP), P388-CB37-297, P388-CB64497, P388-CB153-297 (LIP), P388-CB137-297, and
P388-CB213-297 (Fig. 2A). EMSA species of C/EBPB N-terminal deletions (P388-CB37-297,
P388-CB64497, P388-CB153-297 (LIP), P388-CB137-297, and P388-CB213-297) were only
supershiﬂed by antibody speciﬁc to the C-terminus of C/EBPB, while antibodies speciﬁc
to the N- and C-terminus of C/EBPB both supershifted EMSA species of intact C/EBPB.
Antiserum speciﬁc for C/EBPy was also added to binding reactions to differentiate
between C/EBPB homodimers and C/EBPBzC/EBPy heterodimers. Interestingly,
homodimers (supershiﬁed by antibody speciﬁc to the C-terminus of C/EBPB, but not
antibody speciﬁc to C/EBPy) are only observed as a major binding species with
expression of P388-CB153-297 (LIP), P388-CB137-297, and P388-CB213-297. Western analysis
of nuclear extracts from transductants conﬁrmed expression of the various N-terminal
deletions (Fig. 2B). Interestingly, C/EBPB137-297 and C/EBPB213-297 are under expressed

in comparison to other forms, while producing robust EMSA Species, suggesting greater
binding activity for these deleted forms.

P388 cells stably transduced for C/EBPB (LAP), C/EBPB37-297, C/EBPB64-297,
C/EBPB153-297, C/EBPB187-297, and C/EBPB213-297 expression were treated with LPS over a
time course of 0, 4, 8, and 24 hrs, during which RNA was isolated. Northern analyses
showed that amino acids 22 through 63 of C/EBPB (C/EBPB37-297 and C/EBPB64-297),
which encompass ADMl and a portion of ADM2 (Williams, Baer et al. 1995), are
dispensable for LPS-induced MCP-l transcription in P388 cells (left panel, Fig. 3).

Deletion of an additional 88 amino acids (C/EBPB153-297 [LIP]), which removed all of the

84

 

Noe CIEBPﬂ 37476 64-276 163-278 187276 213-297

Neo
187-297

creeps
37-297
64-297
1 53297
213-297

i\\\\\\\\\\l \{t‘nm‘ \\\l ‘l ”“1“ “\U
' . l- \\ l
‘1“. \ Mm ,l“\"‘“_\‘\“r\‘

 

 

Figure 2-2 Ectopic expression of C/EBPB and its various amino-terminal
deletions in P388 lymphoblasts. (A). An EMSA was performed using nuclear extracts
Isolated from P388-NCO, P388-CB, P388-CB37497, P388-(3364-297, P388-CB153-297, P388-
CB137-297, and P388-CB213-297. Binding reactions were carried out with a C/EBP
consensus binding site and included normal rabbit IgG (N), N-terrninus-speciﬁc C/EBPB
antibody (B-N), C-terminus-speciﬁc C/EBPB antibody (B-C), or C-terminus-speciﬁc anti-
C/EBPy (y). The positions of C/EBPzDNA complexes are indicated by open arrows and
C/EBP-speciﬁc antibody supershiﬁ species are indicated by solid arrows. (B). A western
analysis was performed using nuclear extracts from P388 stable transfectants of C/EBPB
and its deletion mutants. A C-terminus-speciﬁc C/EBPB primary antibody was used to
detect C/EBPB. B-tubulin was detected as a control for protein loading.

85

   

Hours LPS
Tretmant , 0 4.. .8

P388—N00
P388—C/EBPB
P388-37—297
P388-64297
P388-153497
P388— 187-297

P388—213—297

Figure 2-3 Individual N-terminal functional domains of C/EBPB differentially
enhance and suppress MCP-l and IL-6 expression in stable transductants of P388
lymphoblasts. Northern analyses of MCP-l (left panel) and IL-6 (middle panel)
expression in P388 transductants. RNA was isolated over time courses of LPS treatment
as shown. Thirty micrograms of RNA were analyzed on Northern blots that were
successively hybridized to probes for MCP-l , IL-6, and GAPDH.

86

known activation domains in C/EBPB, completely blocked the ability of C/EBPB to
support LPS-induced MCP-l expression, suggesting the presence of a critical activation
motif(s) for MCP-l transcription located between amino acids 64 and 152 of C/EBPB.
Since western blot analysis of nuclear extracts showed expression of C/EBPB153-297 to be
comparable to C/EBPB (LAP), the lack of activity associated with this deletion was not
caused by inefﬁcient expression (Fig. ZB). This lack of activity was surprising, since we
had previously found C/EBPB153.297 to be active in supporting LPS-induced IL-6
expression (see middle panel, Fig. 3). Successive deletion of additional residues between
amino acids 153 and 212 (C/EBPB137-297 and C/EBPB213-297), which encompasses RDl
and RD2 of C/EBPB, alleviates the loss in activity observed with C/EBPB153-297 in
stepwise fashion. This is consistent with both RBI and RD2 exerting an inhibitory effect
on C/EBPB in LPS-induced MCP-l transcription. Since C/EBPB213_297 expression is
much lower than that of C/EBPB (LAP) (Fig. 2B), its activity is not the result of over
expression. Collectively, these data suggest the presence of critical activation and
inhibition motifs located between amino acids 64 and 152 and amino acids 153 and 212
of C/EBPB, respectively, that are important for MCP-l regulation.

In order to conﬁrm the above results and further localize the regions of C/EBPB
mediating positive and negative regulation of MCP-l expression, the same deletion series
used in the previous experiment with the addition of C/EBPB102-297, as well as three
internal deletion mutants, C/EBPBA105-212, C/EBPBA137-212, and C/EBPBA134-212, were
examined in transient transfections of P388 cells using a proximal MCP-l-promoter-

luciferase reporter (Fig. 4). C/EBPBAIBHIZ lacks RD2, C/EBPBAI 37-212, lacks RDl and

87

RD2, and C/EBPBA105-212 lacks RDl and RD2, as well as an “extended” RDl region that
has also been reported to exert minor inhibitory effects (Williams, Baer et al. 1995). The
latter region contains a well-conserved sumoylation site at lysine 134 (Kim, Cantwell et
al. 2002). Similar to the stable transductants of P388 cells, C/EBPBbAm retained its
capacity to support MCP-l transcription, while C/EBPB153-297 activity was reduced
Signiﬁcantly reduced in comparison to C/EBPB. C/EBPBIOMW was also inactive, further
localizing the activation region to amino acids 64 through 101, encompassing a portion of
ADM2 and ADM3. Consistent with region containing RDl and RD2 serving an
inhibitory function, internally deleted C/EBPBAmsm and C/EBPBA137-212 Showed
enhanced activity relative to intact C/EBPB (LAP), while C/EBPBA134-212 and C/EBPB
(LAP) were similar in activity.

C/EBPﬁ RD 1 and 2 Function in a Promoter-Speciﬁc Manner—Northern analysis
of IL-6 expression in the P388 cells stably transduced for the C/EBPB deletion series
found that C/EBPB (LAP), C/EBPB37-297, C/EBPB64-297, C/EBPB137-297, and C/EBPB213-297
displayed levels of activity in stimulating IL-6 expression that were similar to those
observed for MCP-l expression, but C/EBPB153-297 (LIP) was robust in its ability to
support LPS-induced IL-6 expression (middle panel, Fig. 3). Thus, RDl and RD2 appear
to behave in a promoter-speciﬁc manner. In order to conﬁrm the differential activity of
RD] and RD2 in MCP-l and IL—6 expression, a vector expressing internally deleted
C/EBPBAIOHIZ was transiently co-transfected with the IL-6 promoter-reporter into P388
cells. This internal deletion, which completely removes RD] and RD2, showed a strong

enhancement of activity in MCP-l expression relative to intact C/EBPB

88

MCP-l (-322 to +59)-Lac

604

   

p<(l.05 p50.05

 

943-05 p<0.05 p<o.05

Relative Luciferase Expresslon

         

 

Vector CIEBPB 64-297 102-297 153-297 187-297 213-297 1-297 1-297 1-297
A104-212 A137-212A184-212

Figure 2-4 ADM2 and/or 3 and the internal negative regulatory region of
C/EBPB act in opposition to each other in MCP-l expression. Transient transfections
of P388 lymphoblasts were canied out in duplicate with the MCP-l promoter-reporter.
Luminometer values were normalized for expression from a co-transfected SV40 early
promoter-B-galactosidase reporter. These values were then normalized to a relative value
of 1 for the cells not receiving a C/EBP expression vector. The data presented are the
mean of three experiments with their standard error. ("‘ Signiﬁcantly different from intact
C/EBPB).

89

(LAP) (Fig. 4). In contrast, C/EBPBA105-212 was reduced in its ability to support IL-6
expression in comparison to intact C/EBPB (LAP) (Fig. 5). This result is consistent with
the notion that RD] and RD2 suppress C/EBPB activity on the MCP—l promoter, but do
not function in a suppressive manner in IL-6 expression.

Serine 64 in C/EBPﬂ ADM2 is Important for both MCP-I and IL-6 Promoter
Activation—Our deletion analysis localized a region from amino acid 64 to 101 that was
necessary for C/EBPB stimulation of MCP-l expression (Figs. 3 and 4). One candidate
activation motif in this region is serine 64, which is conserved among C/EBPor, B, 6, and
a (Johnson, P. F. and S. C. Williams, 1994. CAAT/enhancer binding (C/EBP) proteins. In
Liver Gene Expression, Yaniv, M. and Tronche, F. (eds.) Raven Press, New York, pp.
231-258). This site has been identiﬁed as a Ros-induced phosphoacceptor Site (J.
Shuman and PF. Johnson, unpublished data). In order to evaluate the role of serine 64 in
C/EBPB activation of MCP-l transcription, we examined the ability of a mutant C/EBPB
with a serine to alanine substitution at amino acid 64 (C/EBPBSMA) to transactivate the
MCP-l proximal promoter. While C/EBPB induced MCP-l promoter activity ~40-fold
in transient transfections of P388 cells, C/EBPBSW, resulted in ~10-fold induction, or
~75% loss of activity (Fig. 6A). The magnitude of the defect in C/EBPBSMA suggests
that loss of serine 64 may be the critical lesion in deletion of amino acids 64 to 101 (Fig.
4). C/EBPBSMA was also defective in its ability to transactivate the IL-6 promoter with
LPS stimulation (Fig. 5). C/EBPB (LAP) induced IL-6 promoter activity in the absence
and presence of LPS ~13-fold and 50-fold, respectively, while C/EBPBSMA induced IL-6

promoter activity in the absence and presence of LPS ~4-fold and 16-fold, respectively,

90

lL-6 (-250 to +1)-Luc

 

 

 

 

 

 

 

 

70-
60—
r: 50-
.2
H
2 40+
‘6
< 30- p<o. 05 P<0 05
E
‘2 20
p<0 05 p<0. 05
0 _. . i ,
Control llClEBF’lti CIEBPB ClEiBPB CIEBPB CIEBPB CIEBPB
LPS 864A 864A A105—212 A105-212
LPS LPS
Figure 2-5 ADM2 and/or 3 and the internal negative regulatory region of

C/EBPB are important functional motifs in IL-6 expression. Transient transfections
using a proximal IL-6 promoter-reporter with and without 4 hours of LPS treatment.
Transient transfections of P388 lymphoblasts were carried out in duplicate with the IL-6
promoter—reporter (-250 to +1). Luminometer values were normalized for expression
from a co-transfected SV40 early promoter-B-galactosidase reporter. These values were
then normalized to a relative value of l for the cells not receiving a C/EBP expression
vector. The data presented are the mean of three experiments with their standard error.
(* signiﬁcantly different from wildtype C/EBPB; ** signiﬁcantly different from wildtype

C/EBPB plus LPS).

91

MCP-1 (-322 to +59)-Luc
60 -

50—
404

30i

p<0.05

20-

Fold Activatlon

10-

 

 

0___—

Control CIEBPB

 

 

 

Figure 2-6 Serine 64 in C/EBPB ADM2 is important for MCP-l promoter-
reporter activity. (A). Transient transfections using a proximal MCP-l promoter-
reporter (-322 to +59). All transfections were carried out in duplicate. Luminometer
values were normalized for expression from a co-transfected SV40 early promoter-B-
galactosidase reporter. These values were then normalized to a relative value of l for the
cells not receiving a C/EBP expression vector. The data presented are the mean of four
experiments with their standard error. (* signiﬁcantly different from wildtype C/EBPB).

92

or a 70% reduction in IL—6 promoter activation under either condition. An EMSA of
nuclear extracts from transient transfections of P388 cells demonstrates that C/EBPBSMA
is expressed and capable of binding DNA (Fig. 6B).

To conﬁrm the reduced activity of C/EBPBSMA on the endogenous MCP-l and IL-6
promoters, we generated stable transductants in P388 cells. Similar to the results
obtained in transient transfections, serine 64 was shown to be critical for MCP-l
expression. Wildtype C/EBPB induced endogenous MCP-l expression ~33-fold more
than C/EBPBSMA transductants after 8 hours of LPS treatment (Fig. 7A and 8A). In the
case of IL-6, the level of maximal expression was observed after 24 hours of LPS
stimulation in C/EBPB (LAP) transductants and at 8 hours of LPS induction in
C/EBPBSMA transductants in P388 cells. We found that serine 64 also impacts C/EBPB-
mediated IL-6 expression, as LPS-induced IL-6 expression in P388-C/EBPB
transductants was 6.3 fold more than that observed in P388 cells ectopically expressing
C/EBPBSMA (Fig. 7A and 8B). Therefore, mutation of serine 64 of C/EBPB appears to
have a lesser impact on IL-6 than MCP-l expression, as P388-CB364A transductants retain
~16% of its activity on the IL-6 promoter in comparison to wildtype C/EBPB (Fig. 8B),
as opposed to being virtually inactive on the MCP-l promoter (Fig. 8A). Expression of
P388-CB364A protein was conﬁrmed and found to be higher than intact C/EBPB (LAP) in

P3 88 transductants making its reduced activity all the more dramatic (Fig. 7B).

93

Neo creeps sm (3)

"NEWS 04 8240 4 824 04824
Tr.mnt qgil"““,‘.‘,li““',lv 4 .v u . '

lL-6 “

 

 

    
 
     

«1‘1 111‘"

ill 51"" ll “1.. .. . will
“11.11 11111 W‘jllll“ “11““ . _ . 1‘ 11

.“h I‘ 1I . ‘ ‘ _ lj“".\
MCP-1 “‘11“; ym":“nullllhmw11mm“ “111‘ “i“

“111“
llul“ lll‘“ . Milli“; “v““iii “gill“ M
{iii MW in 1““Wul“111W“[illl‘l“\:illll)m 'l “ 1‘ M W11“

“twill

  
    

 

“W
1111“ i

”ii i
. " . “ 11
m ”1“" all“ lllli‘wullll“ . ." “iii“ “will“‘m‘iilllllwnlll‘l‘?

 

       

 

GAPDH
B
Neo CIEBPB SMA
f: Wag-«T111111 ' “111" 1.411‘ ll'.‘..‘.llll‘.l‘rlilh :1
\ WB: creeps
WB: B-tubulin
Figure 2-7 Serine 64 of C/EBPB is important for both MCP-l and IL-6

expression from intact endogenous promoters. (A). Northern analyses of MCP-l and
IL-6 expression in P388 transductants. RNA was isolated over the time course of LPS
treatment as shown. Thirty micrograms of RNA were analyzed through successive
hybridization to probes for MCP-l, IL-6, and GAPDH. (B). A western analysis was
performed using nuclear extracts from P388 lymphoblasts stably transduced for C/EBPB
and C/EBPBSMA expression. A C-terminus-speciﬁc C/EBPB primary antibody was used
to detect C/EBPB. B-tubulin was detected as a control for protein loading.

94

ID

MOP-1 rrRNA expression

 

 

 

 

 

 

 

 

 

 

 

 

 

7 0.15 -
n_ C
9 3 005 —I—DlC2-2
:3 3 ' ‘ +864A(3)
a; 0 -——¢—.-—a—-,—¢-—r:A—.
0 4 8 24
Hours LPS Treatment
B
IL-6 n'RNA Expression
‘3 § +Neo
_ to
g :3 —I—DIC2-2
% 3 +864A(3)
m l
Hours lL-1 Treatment
Figure 2-8 The extent of hybridization was quantitated using a Storm

Phosphimager (Molecular Dynamics). The quantities for (A), MCP-l expression and
(B), IL-6 expression were analyzed through successive hybridization to probes for MCP-
1, IL-6, and GAPDH. Neo is representative of P388-neo samples. DIC2—2 refers to a

P388 stable transductant cell line ectopically expressing wildtype C/EBPB.

95

Discussion

These studies demonstrate promoter speciﬁcity in the ability of regulatory domains 1
and 2 (RBI and RD2) to inhibit C/EBPB activity. RDl and RD2 suppressed C/EBPB
activity in MCP-l expression, but not IL-6 expression in a B lymphoblast cell line. The
ability of C/EBP8153-297, also known as LIP, to selectively inhibit C/EBPB-regulated
genes may lie with the differential activity of RBI and RD2 rather than differential
requirements for C/EBPB activation domains. Additionally, serine 64 of C/EBPB was
found to be critical for the robust activity of C/EBPB in the activation of both MCP-l and
IL-6 expression. In the case of MCP-l expression, substitution of serine 64 with alanine
was as effective as deletion of amino acids 22 through 101 in reducing C/EBPB activity.
This suggests that serine 64 is the critical activation motif for C/EBPB-mediated MCP-l
expression.

RDl and RD2, components of the internal negative regulatory region of C/EBPB,
were initially characterized, respectively, as modulating C/EBPB activation domains and
cell type speciﬁcity (Williams, Baer et a1. 1995). RDl functions to inhibit the
transactivation potential of C/EBPB, presumably by causing a closed conformation that
interferes with access to the activation domain by signaling elements (Williams, Baer et
al. 1995). RD2 inhibits DNA binding in a cell-speciﬁc manner, perhaps by inducing a
conformation that interferes with basic regionzDNA interactions (Williams, Baer et al.
1995). While C/EBPB is a critical activator of various proinflammatory gene products
(Lowenstein, Alley et al. 1993; Matsusaka, Fujikawa et a1. 1993; Shirakawa, Saito et al.

1993; Dunn, Coles et al. 1994; Pope, Leutz et al. 1994; Ford, Bennett et al. 1996; Akira

96

and Kishimoto 1997; Plevy, Gemberling et al. 1997; Sekine, Nishio et al. 2002), its
negative regulatory region has neither been previously shown to inhibit the expression of
these genes, nor to operate in a promoter specific manner. In fact, a MAPK site at
threonine 189 within RD2 was shown to play a role in enhancing IL-6 promoter activity
(Nakajima, Kinoshita et al. 1993). The decrease in C/EBPB activity in IL-6 transcription
observed upon deletion of the regulatory region (C/EBPBA105-212, Fig. 5) is certainly both
consistent with threonine 189 having a positive role and RBI and RD2 having no

negative effect in IL-6 expression.

It was surprising that the negative regulatory region reduced C/EBPB activity in
MCP-l, but not IL-6 expression (Figs. 3 and 4). In a previous report, we demonstrated
the dispensability of C/EBPB N-terminal motifs in both LPS-induced MCP-l and IL-6
expression in P388 lymphoblasts (Hu, Tian et al. 2000), suggesting a common role for
C/EBPB in the regulation of both MCP-l and IL-6. C/EBPB and NF-KB synergistically
activate the IL-6 promoter (Matsusaka, Fujikawa et a1. 1993) and, consistent with this,
direct interaction between the bZIP of C/EBPB and the NF-KB Rel homology domain has
been observed (Stein and Baldwin 1993; Stein, Cogswell et a1. 1993). We found that the
activity of the C/EBPB bZIP in the absence of N-terminal motifs required the NF-KB site
of the IL-6 promoter (Hu, Tian et al. 2000), and that augmentation of C/EBPB activity by
C/EBPy required formation of a heterodimeric leucine zipper and co-expression of NF-
KB (Gao, Parkin et al. 2002). A mechanism for IL—6 activation whose essential feature is
the requirement for the bZIP region of C/EPBB to synergize with NF-KB is suggested by

all of these studies. The current studies conﬁrm the dispensability of C/EBPB N—terminal

97

regions for this function and point to an auxiliary role for activation sites such as serine
64.

The role of C/EBPB in MCP-l expression is less well understood than for IL-6
expression. While C/EBPB can play an essential role in MCP-l expression (Bretz,
Williams et al. 1994) and this function requires only the bZIP domain (Hu, Tian et al.
2000), the association of C/EBPB with NF-KB is not as clear as it is with IL-6. Two NF-
KB binding sites have been identiﬁed at —2280 and —2261 bp from the starting point of
MCP-l transcription in the rat (Wang et al., Kidney Int. 57: 2011-2022 [2000]) and
C/EBPIS binding sites have been identiﬁed at —2579 and ~3107 bp (Sekine, Nishio et al.
2002). Working in a murine system, we found that the behavior of the intact MCP-l
gene and a —322 bp MCP-l proximal promoter in response to C/EBPB was virtually
identical (see Figs. 3, 4, 6, and 7), yet an examination of the proximal promoter ﬁnds no
likely NF-KB sites and the mutation of potential C/EBP binding sites did not affect the
proximal promoter’s response to C/EBPB (MatInspector V2.2 at http:
//transfac.gbf.de/TRANSFAC/ and data not shown). Perhaps C/EBPB regulates MCP-l
expression through a highly degenerate site or through an indirect mechanism.
Nonetheless, the negative regulatory region including RDl and RD2 has a profound
inhibitory effect on the activity of C/EBPB in MCP-l expression, while not affecting IL-6
expression. The incremental enhancement of C/EBPB activity with progressive deletion
of the negative regulatory region (see C/EBPB]37-297 and C/EBPBZI3-297, Fig. 4) suggests
that both RD] and RD2 contribute to inhibition, although the failure of C/EBPA134-212
(deleting RD2 by itself) to augment activity suggests a greater role for the extended RDl

region, if not RDl itself. These inhibitory motifs are conserved in C/EBPe and were

98

found to suppress C/EBPs transactivation (Angerer, Du et al. 1999). It will be
illuminating to see if these motifs display differential ﬁinction among C/EBPe-regulated
genes.

We observed that removal of highly conserved serine 64 in ADM2 of C/EBPB was as
debilitating to MCP-l transcription as removal of all amino acids N-terminal to the
negative regulatory region (Figs. 3, 4, and 7). The region of C/EBPB encompassing
serine 64 is known to associate with the co-activator, p300 (Mink, Haenig et al. 1997).
The co-expression of p300 with C/EBPBSW, in transient transfections stimulates the
MCP-l promoter as much as does p300 with intact C/EBPB (data not shown), suggesting
a role for this site other than co-activator recruitment. Although serine 64 may have
some function in a mechanism that purely serves to enhance C/EBPB activity on the IL-6
promoter, it is likely that it functions to alleviate the negative regulatory region in MCP-l
expression. These data suggest a mechanism where C/EBPB exists in a form inactive for
MCP-l transcription. Following an extracellular signal, the induction of a kinase cascade
would lead to the phosphorylation of C/EBPB at serine 64, which would in turn reduce
the inhibition mediated by the internal negative regulatory domains of C/EBPB through a
conformational change allowing more effective DNA binding. We have, indeed,
observed that the progressive removal of the internal negative regulatory domains, which
resulted in a progressive increase in MCP-l promoter activity (left panel, Fig. 3), is also
associated with a progressive increase in C/EBPB DNA-binding (Fig. 2A). RD2 has
previously been implicated in the inhibition of DNA-binding (Williams, Baer et al.
1995). Interestingly, a motif in C/EBPB that encompasses serine 64 is nearly identical to

a site in the N-terminal transactivation domain of c-jun that is phosphorylated by Jun

99

Kinase to become a substrate for the prolyl isomerase, Pinl (Wulf, Ryo et al. 2001). Pinl
alters protein function through conformational change (Zhou, Kops et al. 2000). Future
investigations should assess the ability of Pinl to augment C/EBPB activity and a
possible dependence on serine 64 for that activity, as well as the ability of compensatory
mutations in the negative regulatory region of C/EBPB to suppress mutation of serine 64.

Interestingly, homodimers are only observed as a major binding species with
expression of P388-C0534” (LIP), P388-C0137-297, and P388-C0234” (Fig. 2B). This
was observed in multiple independent cell lines (data not shown) and may imply an
important role for N-terminal sequences in promoting heterodimerization, as well as
suppressing the internal negative regulatory region. While heterodimerization of C/EBPB
with C/EBPy promotes IL-6 expression (Gao, Parkin et al. 2002) and has been reported to
inhibit gene expression in other contexts (Cooper, Henderson et al. 1995), C/EBPB
homodimerization is reported to be essential for its activity on the ICAM-l promoter
(Catron, Brickwood et al. 1998). Thus, the modulation of dimerization may have
important consequences for gene expression.

The promoter speciﬁcity of the C/EBPB internal negative regulatory region has
important implications for the function of LIP, a form of C/EBPB that initiates at amino
acid 153 and lacks activation domains (Descombes and Schibler 1991). LIP has been
reported to be a transdominant inhibitor of C/EBP activity (Descombes and Schibler
1991). Because LIP retains RDl and RD2 of the internal negative regulatory region, our
ﬁndings suggest that LIP inhibits C/EBP-dependent gene expression in a promoter-
speciﬁc fashion. The ability of otherwise inhibitory LIP to play a positive role in LPS

stimulation of IL-6 transcription could provide a mechanism for IL-6 production to drive

100

the maturation of B cells, while suppressing other proinﬂammatory cytokines such as
MCP-l. Similarly, the negative regulatory region could modulate expression in intact
C/EBPB. In the absence of signaling to C/EBPB activation domains, C/EBPB would lose

the ability to participate in the transactivation of MCP-l and other proinﬂammatory

genes, but would retain activity in IL-6 expression.

101

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106

Chapter 3

C/EBPB Activity is Differentially Regulated By Acetyltransferases

107

Abstract

C/EBPB is a critical transcriptional regulator of numerous genes associated with
inﬂammation, including the proinﬂammatory cytokine IL-6. We previously found that a
truncated form of CfEBPB (C/EBPBZI3497) lacking all known activation and regulatory
domains was still capable of supporting LPS-induced IL-6 expression in a B lymphoblast
cell line. Interestingly, this truncated form of C/EBPB retains a KXKK motif adjacent to
the basic region that is conserved among several C/EBP family members and has been

demonstrated to be an acetylation substrate in several other transcription factors. This
suggested to us that C/EBPB might be a target for acetylation. We report here that p300
and GCN5 synergistically co-activate C/EBPB, with the acetyltransferase activity of
GCN5 being necessary for stimulating the DNA-binding activity of C/EBPB. Mutation
of lysines in the KXKK motif adjacent to the C/EBPB DNA-binding domain blocked
p300-enhancement of C/EBPB DNA-binding and promoter activation. Moreover, the
integrity of the KXKK motif was critical for LPS and IL-lB-induced IL-6 expression in
P388 B lymphoblasts stably transduced for C/EBPB expression. In contrast to the
aforementioned ﬁndings, the overexpression of the GCN5 homolog P/CAF reduces
C/EBPB DNA-binding activity. This effect is dependent upon the acetyltransferase
activity of P/CAF. The differential effects of GCN5 and P/CAF are likely mediated by
the acetylation of alternative lysines in C/EBPB. These results suggest a novel regulatory
mechanism whereby the co-activators GCN5 and P/CAF differentially regulate C/EBPB

activity in an acetyltransferase-dependent manner.

108

Introduction

C/EBPs comprise a family of bZIP transcription factors that are effectors in the
transcriptional regulation of numerous proinﬂammatory gene products and acute phase
proteins, including IL—6, IL-loc, IL-lB, IL-8, TNF-or, G-CSF, inducible NO synthase,
lysozyme, haptoglobin, ou-acid glycoprotein, C-reactive protein, and serum amyloids
(Poli 1998). In particular, both C/EBPB and C/EBP8 are able to transactivate an IL-6
promoter/reporter in transient transfection assays (Akira, Isshiki et al. 1990; Kinoshita,
Akira et a1. 1992). Moreover, the stable transfection of C/EBPa, B, 5, and e in a B
lymphoblast cell line that lacks the expression of these C/EBP isoforms supports LPS-
induced IL-6 and MCP-l expression (Bretz, Williams et al. 1994; Hu, Baer et al. 1998;
Williams, Du et al. 1998). The primary mechanism of C/EBPS regulation during the
inﬂammatory response is transcriptional (Kinoshita, Akira et al. 1992; Ramji, Vitelli et
al. 1993), as LPS, IL-1, and IL-6 induce its mRNA expression (Zhang and Rom 1993).
While C/EBPB mRNA levels can also be induced in response to inﬂammatory stimuli
(Akira, Isshiki et al. 1990; Natsuka, Akira et al. 1992), the transactivation potential of
C/EBPB is primarily regulated by post-transcriptional mechanisms (Akira, Isshiki et al.
1990; Poli, Mancini et al. 1990; Ramji, Vitelli et al. 1993).

Several lines of evidence suggest that post-translational modiﬁcations are important
modulators of C/EBPB DNA-binding activity and transactivation. The in vitro
phosphorylation of serine 240 in the basic region of rat C/EBPB by protein kinase A
(PKA) and protein kinase C (PKC) attenuates its DNA-binding activity (Trautwein, van

der Geer et al. 1994), as does Akt and glycogen synthase kinase-3 phosphorylation of

109

serine 185 in rat C/EBPB (Piwien-Pilipuk, Van Mater et al. 2001). In addition to
modulation of DNA-binding activity, ras oncogene-induced phosphorylation of human
C/EBPB at a MAPK substrate site corresponding to threonine 235 was associated with
enhanced transactivation of an IL-6 promoter-reporter (N akajima, Kinoshita et a1. 1993).
This same site has been implicated in IFN-y- and growth hormone-induced
transactivation by C/EBPB (Hu, Roy et al. 2001; Piwien-Pilipuk, MacDougald et al.
2002). Tumor promoting activator (TPA)-mediated PKC activation also enhances rat
C/EBPB transactivation through the phosphorylation of serine 105 (Trautwein, Caelles et
al. 1993), in addition to the PKC modulation of C/EBPB DNA-binding already mentioned
(Trautwein, van der Geer et al. 1994). Phosphorylation of this identical site by p90
ribosomal S kinase in response to TGF-a is critical for hepatocyte proliferation (Buck,
Poli et al. 1999). Interestingly, phosphorylation of serine 277 in the leucine zipper
domain of rat C/EBPB by calcium/calmodulin—dependent kinase enhanced C/EBPB
activity (Wegner, Cao et al. 1992). This ﬁnding demonstrated the presence of an
activation motif outside of the N-terminal Activation Domain Modules (ADMs)
(Williams, Baer et al. 1995) that are critical for C/EBPB activity. More recently, motifs
conserved between C/EBPa, B, 8, and 8 were found to be substrates for sumoylation and
acetylation (Kim, Cantwell et al. 2002; Xu, Nie et al. 2003). Sumoylation of a lysine
residue in Regulatory Domain 1 (RDl) of C/EBPe reduced the inhibitory effects
mediated by this regulatory domain (Kim, Cantwell et al. 2002). This conserved
sumoylation motif is also found in c-Fos and F038, and functions as an inhibitory domain
in these transcription factors (Brown, Sutherland et al. 1995). Studies from Xu et al.

suggest that the acetylation of C/EBPB by P/CAF and CBP at evolutionarily conserved

110

lysine residues located adjacent to the basic region diminishes the DNA-binding activity
of C/EBPB concomitant with a reduction in IL-3-dependent expression of Id-l (Xu, Nie
et al. 2003).

A number of transcription factors have been identiﬁed as substrates for acetylation by
the histone acetyltransferases (HATS) p300, CBP, and P/CAF, such as p53 and p73 (Gu
and Roeder 1997; Costanzo, Merlo et al. 2002), GATA-l (Boyes, Byﬁeld et a1. 1998),
E2F-1 (Martinez-Balbas, Bauer et a1. 2000), NF-KB (Chen, Fischle et a1. 2001), EKLF
(Zhang and Bieker 1998), Rb (Chan, Krstic-Demonacos et al. 2001), BCL6
(Bereshchenko, Gu et a1. 2002), POP-1 (van Noort and Clevers 2002), TALl/SCL
(Huang, Qiu et al. 2000), androgen receptor (Fu, Wang et al. 2002), and MyoD
(Sartorelli, Puri et al. 1999). In many of these transcription factors, including p53,
GATA-l, E2F, NF-KB, TALl/SCL, and MyoD, the acetylation substrate is located
adjacent to the DNA-binding domain and results in enhanced afﬁnity of the protein for
DNA. In addition to modulating DNA-binding activity, acetylation of some transcription
factors has been shown to impact protein-protein interactions (Zhang and Bieker 1998;
Chen, Fischle et al. 2001; Wang, Fu et al. 2001; Bereshchenko, Gu et al. 2002), protein
stability (Martinez-Balbas, Bauer et al. 2000; Ito, Kawaguchi et al. 2002; Giandomenico,
Simonsson et al. 2003), and to mediate various signal transduction pathways (Gay, Calvo
et al. 2003). HATS are believed to modify a small number of lysines within a given
protein (Kouzarides 2000). Comparison of known acetylation substrates in numerous
transcription factors suggests a clustering of lysine residues as part of the recognition
motif for acetylation. Consistent with this, p53, GATA-l, E2F family members, POP-1,

BCL-6, and NF-KB contain a more speciﬁc motif, KXKK, or in the case of TALl,

lll

KXXKK, as the major substrate for acetylation. Like these transcription factors,
C/EBPa, [3, and 8 contain the KXKK motif directly adjacent to the DNA-binding
domain; in the case of C/EBPB, the motif is located at amino acids 214-217.

Previous work in our lab demonstrated that a truncated form of C/EBPB (C/EBPBm-
297), composed solely of the bZIP domain, supports LPS-induced IL-6 expression (Hu,
Tian et al. 2000). While C/EBPB;13_297 retains the capacity to support LPS-induced IL-6
expression, we subsequently found that this mutant is ineffective in stimulating IL-6
expression in response to IL-10, and that this distinction in inﬂammatory signaling
reﬂects a difference in C/EBPB DNA-binding activity. The presence of a putative
acetylation motif and the aforementioned modulation of DNA-binding led us to explore
the role of acetylation in modulating C/EBPB activity. Here we show that p300 augments
the DNA-binding activity and transactivation potential of C/EBPB independently of its
acetyltransferase activity. GCN5 cooperates with p300 to co-activate C/EBPB with the
acetyltransferase activity of GCN5 necessary to enhance the DNA-binding of C/EBPB.
In contrast, P/CAF, an acetyltransferase closely related to GCN5, reduces C/EBPB DNA-
binding. This activity of P/CAF is dependent upon its acetyltransferase activity. The
opposing activities of GCN5 and P/CAF in modulating C/EBPB are likely to be
dependent on the acetylation of alternative lysines. The importance of this regulatory
mechanism is underscored by the fact that the integrity of the C/EBPB KXKK acetylation

motif is critical for IL-6 expression in a B lymphoblast cell line.

112

Materials and Methods

Cell Culture. P388 cells are murine B lymphoblasts (Bauer, Holmes et al. 1986)
(American Type CultureCollection; CCL 46). P388-Neo, P388-CB, and P388-C0213-297
cells have been described previously by Hu et a1 (Hu, Tian et al. 2000). P388 cells and
their derivatives were cultured in RPMI 1640 medium supplemented with 5% fetal calf
serum and 50 uM B-mercaptoethanol. All IL-6 and MCP-l inductions were conducted
with LPS derived from Escherichia coli serotype 055:B5 (Sigma-Aldrich) added to 10
jig/ml.

Expression Vectors and Promoter-Reporters. All forms of C/EBPB expressed in
these experiments are derived from rat C/EBPBzz-297 (LAP). For retroviral transductions,
C/EBPB and mutant forms were expressed from pZIP-NEO SV(X)1, a retroviral vector
derived from Moloney murine leukemia virus (Cepko, Roberts et a1. 1984). C/EBPB
sequences were inserted into the BamHI site of the vector. Inserted sequences were
transcribed from the Moloney murine leukemia virus promoter and the gene conferring
G418-resistance was expressed from a subgenomic splicing product from the same
promoter.

For transient transfections, C/EBPB and mutant forms were expressed from pMEX,
which utilizes the Moloney murine sarcoma virus promoter (Williams, Cantwell et al.,
1991). Expression vectors for the following coactivators have been described: murine
p300 and p300AHAT (Boyes, Byﬁeld et al., 1998); murine HA-tag CBP (from P. F.
J Ohnson, NCI-Frederick) murine P/CAF and P/CAFAHAT (Martinez-Balbas, Bauer et al.

2000); murine pCMV-sport2 FLAG-tag GCN5 was (from T. Kouzarides, Cambridge

113

University). The GCNSAHAT mutant was generated ﬁom the murine GCN5 expression
vector by site-directed mutagenesis using the QuikChange Kit (Stratagene) duplicating a
previously described HAT-defective mutant (Paulson, Press et al. 2002).

The IL-6 promoter-reporter consists of the murine IL-6 promoter (Tanabe, Akira et al.
1988) (-250 to + 1) inserted into the luciferase vector pXP2 (Nordeen 1988). The MCP-l
promoter-reporter consists of the murine MCP-l promoter (U eno, Sonoda et al. 2000) (-
322 to +59) inserted into the luciferase vector pGV-BZ (Toyo Ink Mfg. Co., Ltd.). The
2XC/EBP promoter-reporter consists of two tandemly arrayed C/EBP consensus binding
sites inserted into a minimal thymidine kinase promoter-luciferase reporter (Miller,
Shuman et a1. 2003). The SV40 early promoter-reporter is a commercial product, p-Bgal
control (BD Biosciences Clontech), where the SV40 early promoter and enhancer
sequences are cloned upstream and downstream, respectively, of the lacZ gene.

Reporter Assays. Transient transfections were conducted with 2 x 106 cells, 4 pg of
DNA, and 8 pl of DMRIE-C reagent (Invitrogen) in 1.2 ml of OPTI-MEM medium
(Invitrogen). Unless otherwise speciﬁed, the transfected DNA was comprised of 1.0 pg
of promoter-reporter, 0.1 pg of SV40 early promoter-reporter, 0.5 pg of
C/EBPBexpression vector, 1.0 pg of coactivator expression vector, and pMEX plasmid to
total 4 pg. Cells were incubated in the transfection mixture for 5 hours followed by
addition of RPMI 1640 medium supplemented to 15% with FCS. Aﬂer 24 hours, the
medium of certain transfections was supplemented with 10 pg/ml LPS. After 4 hours in
the absence or presence of LPS, transfected cells were harvested, lysed and analyzed for

luciferase activity by using the Luciferase Reporter Gene Assay Kit (Roche Molecular

114

Biochemicals) and for B-galactosidase activity by using the Luminescent B-Galactosidase
Genetic Reporter System 11 (BD Biosciences Clontech).

Retroviral Transductions. The generation of stable transductants of C/EBPB lysine
to arginine mutations was accomplished by retroviral infection. Retroviral stocks were
prepared by transient transfection of 293T cells (Pear, Nolan et al. 1993) that were grown
in Dulbecco’s modiﬁed Eagle’s medium supplemented with 10% FCS. Retroviral
expression vectors (3 pg) were cotransfected with a packaging construct (3 pg), pMOV-
w (Mann, Mulligan et al. 1983). These transfections were performed using DMRIE-C
reagent (Invitrogen) upon 70% conﬂuent 293T cells on 60 mm plates. Virus stocks were
harvested 48 hours post-transfection by centrifuging the supematants at 1500 rpm for 5
min followed by ﬁltration through 0.45-pM-pore-size ﬁlters. Retroviral infections were
performed on 60 mm plates by the addition of 3 ml virus stock to 2 x 106 cells in the
presence of 8 pg/ml polybrene (Sigma-Aldrich). The cells were then incubated at 37°C
for 3 hours during which time the plates were swirled every 30 min. The infection
medium was then replaced with standard growth medium. Aﬁer 24 hours, the cells were
split to four 60 mm plates and G418 (Invitrogen) was added to a ﬁnal concentration of 1
mg/ml for selection of resistant cells.

Transient Transfections for Nuclear Extracts. Transient transfections of P388
cells were conducted with 16 pl of DMRIE-C reagent (Invitrogen), 4 pg of C/EBPB,
C/EBPB213-297, or C/EBPB(KK216,217RR), 2 pg of p300, p300AHAT, CBP, GCN5,
GCNSAHAT, P/CAF, or P/CAFAHAT, and pMEX plasmid to total 8 pg. Cells were
incubated in the transfection mixture for 5 hours followed by addition of RPMI 1640

medium supplemented to 15% with FCS. Aﬁer 24 hours, nuclear extracts were prepared.

115

Cells were washed in phosphate-buffered saline and lysed in 15 mM KCl, 10 mM
HEPES (pH 7.6), 2 mM MgC12, 0.1 mM EDTA, 1mM dithiothreitol, 0.1% (v/v) Nonidet
P-40, 0.5 mM phenylmethylsulfonyl ﬂuoride, 2.5 pg/ml Leupeptin, 5 pg/ml antipain, and
5 pg/ml aprotinin for 10 min. on ice. Nuclei were pelleted by centrifugation at 14,000 x
g for 1 min. at 4°C. Proteins were extracted from nuclei by incubation at 4°C with
vigorous vortexing in Buffer C (420 mM NaCl, 20 mM HEPES, (pH 7.9), 0.2 mM
EDTA, 25% (v/v) glycerol, 500 nm trichostatin A (TSA), 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl ﬂuoride, 2.5 pg/ml leupeptin, 5 pg/ml antipain, and 5 pg/ml
aprotinin). Nuclear debris was pelleted by centrifugation at 14,000 x g for 30 min. at 4°C
and the supernatant extract was collected and stored at -70°C.

Electrophoretic Mobility Shift Assay (EMSA). The EMSA probe was a double-
stranded oligonucleotide containing an optimal C/EBP binding site (5’-
GATCCTAGATATCCCTGATTGCGCAATAGGCTCAAAGCTG-3’ annealed with 5’-
AATTCAGCTTTGAGCCTATTGCGCAATCAGGGATATCTAG-3’) labeled with the
incorporation of 5’-[a-32P]dATP (3000 Ci/mmol; PerkinElmer Life Sciences) and
Klenow DNA polymerase. Underlined sequences correspond to the C/EBP binding
motif.

Unless otherwise speciﬁed, DNA binding reactions were performed for 20 min at
room temperature in a 25-pl reaction mixture containing 6 pl of nuclear extract (1 mg/ml
in Buffer C) and 5 pl of 5x binding buffer (20% w/v) Ficoll, 50 mM HEPES (pH 7.9), 5
mM EDTA, 5 mM dithiothreitol). The remainder of the reaction mixture contained 1 pg
of poly(dI-dC), 300 pg of probe, bromophenol blue to a ﬁnal concentration of 0.06%

(w/v), and water to volume. The remainder of the reaction mixture contained KCl to a

116

ﬁnal concentration of 50 mM, Nonidet P-40 to a ﬁnal concentration of 0.1%, 1 pg of
poly(dI-dC), 300 pg of probe, bromophenol blue to a ﬁnal concentration of 0.06% (w/v),
and water to volume. For supershiﬁs, nuclear extracts were preincubated with antibodies
for 20 min. at 4°C prior to the binding reaction. Samples were electrophoresed through
5.5% polyacrylamide gels in 1x TBE (90 mM Tris base, 90 mM boric acid, 0.5 mM
EDTA) at 160V.

Western Analysis. Nuclear extracts were prepared as described above. The extracts
(50 pg) were adjusted to 1X Laemmli sample buffer (Laemmli 1970) and processed by
sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis. Gels were transferred
to a Protran membrane (Schleicher and Schuell) and antigen-antibody complexes were
visualized with the Enhanced Chemiluminescence kit (Amersham Pharmacia Biotech).

Immunoprecipitation. For detection of acetylation of ectopically expressed Flag-
tagged C/EBPB, equal amounts of nuclear extracts from transiently transfected P388 cells
were diluted to 150 pl in Buffer C and 300 pl of Buffer C lacking NaCl and glycerol, and
then immunoprecipitated with a mouse monoclonal antibody against a FLAG epitope
overnight at 4°C. Immune complexes were immobilized on Protein A beads and washed
3 times with cell lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1mM EDTA, 1mM
EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM DTT,
0.5mM phenylmethylsulfonyl ﬂuoride, 2.5pg/ml leupeptin, 5 pg/ml antipain, and 5 pg/ml
aprotinin). Immunoprecipitated proteins were resolved by 15% SDS-PAGE and analyzed
by western blotting with antibodies against acetyl-lysine and C/EBPB.

RNA Isolation and Analysis. Total RNA was isolated using TRIzol reagent

(Invitrogen) according to the manufacturer’s directions. RNAs (30 pg) were

117

electrophoresed through 1% agarose/formaldehyde gels. Transfers to membranes were
hybridized and washed to a stringency of 0.1% x SSPE at 65°C. Hybridization probes
were prepared with a random priming kit (Invitrogen) with the incorporation of 5’-[a-
32P]dATP (3000 Ci/mmol; PerkinElmer Life Sciences). The IL-6 probe was a 0.65
kilobase murine cDNA (from N. Jenkins and N. Copeland, National Cancer Institute-
Frederick Cancer Research and Development Center). The MCP-l probe was a 0.58
kilobase murine cDNA (Rollins, Morrison et al. 1988). The glyceraldehye-3-phosphate
dehyrdogenase (GAPDH) probe was a 1.3 kilobase rat cDNA (Fort, Marty et al. 1985).
Antibodies. Rabbit anti-C/EBPB speciﬁc to the C-terminus (product C-19), normal
rabbit IgG, and rabbit anti-B-tubulin (II-235) were purchased from Santa Cruz
Biotechnology. Rabbit anti-C/EBPy (Ig/EBP) was prepared against a synthetic peptide
corresponding to the C-terminus of C/EBPy (Parkin, Baer et al. 2002). Anti-FLAG M2
monoclonal antibody was purchased from Si gma-Aldrich. Anti-acetyl-lysine monoclonal

antibody was purchased from Upstate Biotechnology.

118

Results

C/EBPﬂnMw is dramatically reduced in its ability to support IL-Iﬂ-induced IL-6
and M CP-I expression in comparison to LPS-induced expression—We previously
demonstrated that the bZIP region of C/EBPB was by itself capable of supporting IL-6
expression in response to LPS in P388 lymphoblasts (Hu, Tian et a1. 2000). We extended
our studies to include IL-IB, another proinﬂammatory stimulus known to induce IL-6
expression. RNA was isolated over time courses of LPS and IL-IB treatment from P388
cells stably transduced for C/EBPB (LAP) and C/EBPBZI3-297 expression and examined
for IL-6 and MCP-l expression by Northern analyses (Fig. 1). Surprisingly, we found
that the C/EBPB bZIP region was ineffective at supporting IL-l B-induced IL-6 and MCP-
1 expression.

T SA enhances C/EBPﬂ DNA-binding and transactivation—We explored whether
some structural component of the C/EBPB bZIP region other than the DNA-binding
domain and leucine zipper might play a role in the differential modulation of activity
observed between LPS and IL-lB treatments. Examination of the amino acid sequence of
C/EBPB revealed the motif KXKK juxtaposed to the DNA-binding domain at residues
214-217, which is identical to the acetylation motif found in several transcription factors,
including p53 (Gu and Roeder 1997), E2F (Martinez-Balbas, Bauer et al. 2000), and
GATA-l (Boyes, Byﬁeld et al. 1998). Therefore, we investigated whether this
acetylation motif is an important regulatory feature for C/EBPB activity.

To investigate whether LPS and IL-lB did, in fact, differentially affect C/EBP[321 3-297

activity in P388 cells, we compared the DNA-binding activity ofC/EBP13213_297 in stable

119

A

Hours of
Treatment

P388-Neo

P388-
CIEBPB

P388-

CIEBPB 213 297

(I!
Q
U"

Q
A

0.3 +
02 1

lL-6 mRNA

8

 

IL-6 MCP-l GAPDH

LPS IL-lﬁ LPS IL-lé LPS IL-lﬁ
024 82424 824 ..024824_2 4824 02.4 82424824

'u.‘Fh’b—'¢r’.l.""rﬂ'. . eh- - .11.!le -¢&i:§
O I. , .
t

 

 

 

    

I'.I»oua

 

Control + LPS

  

+
+ C/EBP6 + LPS
+ C/EBPJ321M97 + LPS
+ Control + lL-18
-B— CIEBPfS + IL-16
-A— C/EBP 213,297 + IL-18

 

0 2 4 6 8101214161823224

Figure 3-1

Hours of Treatment

C/EBP13213-297 is ineffective at supporting IL-lB induction of IL-

6 and MCP-l. RNA was isolated over time courses of LPS and IL-18 treatment as
indicated. (A). Northern blots were successively hybridized to probes for IL-6, MCP-l,
and GAPDH. (B). The extent of hybridization was quantitated using a phosphorimager.
The quantities for IL-6 and MCP-l hybridizations were normalized to the quantities for
GAPDH hybridizations in the same samples.

120

transductants treated with LPS and IL-IB. An electrophoretic mobility shift assay
(EMSA) revealed less C/EBPBZIMW bound to a C/EBP consensus-binding site under IL-

IB stimulation than under LPS stimulation (Fig. 2A). Pretreatment of C/EBPBQO
expressing cells with 500 nM trichostatin A (TSA), an inhibitor of deacetylases that often
results in increased protein acetylation, augmented C/EBPBZI3-297 DNA-binding under
both LPS and IL-lB treatment (Fig. 2A). Since expression of C/EBP8213-297 in nuclear
extracts was equivalent under these different conditions (Fig. 2B), the changes in DNA
binding activity are not due to alteration in levels of expression or nuclear translocation.
Furthermore, TSA largely reversed the deﬁcit in DNA binding observed in nuclear
extracts from IL-l B-treated cells (Fig. 2A).

To evaluate whether the TSA-mediated alterations in DNA binding corresponded to
enhanced transactivation activity, a C/EBPB expression vector was transiently transfected
with a 2X-C/EBP promoter-reporter in the presence and absence of TSA (Fig. 3). Since
the acetyltransferase p300 had previously been shown to interact with C/EBPB (Mink,
Haenig et a1. 1997), we also examined its activity in these transient transfections. TSA
enhanced the activity of C/EBPB in a dose-dependent manner and, furthermore, co-
transfection with a p300 expression vector increased C/EBPB activity to a level
comparable to that seen upon treatment with 500 nM TSA (Fig. 3). A C/EBP- dependent
reporter was used instead of an IL-6 or MCP-l promoter-reporter in order to isolate the
effects of TSA and p300 upon C/EBPB from effects upon other transcription factors that
would be observed on more complex promoters. The ability of TSA treatment to
alleviate the weak binding observed for C/EBPB213-297 under IL-lB treatment, coupled

with the ability of TSA to enhance both the DNA binding and

121

 

 

 

P388-C/EBPB213297
LPS IL.”
-TSA | #311 -TSA l +1311
norm: 0124801145 012801246
I. in , ; ‘ . .

    

CIEBPﬁ213297/
C/EBP5213297

Unbound
Probe

 

 

 

 

WB: B-tubulln

 

Figure 3-2 TSA Alleviates Reduced C/EBP13213-297 DNA-Binding in IL-lﬂ-
treated cells. P388 cells ectopically expressing C/EBPBm-297 were treated with 10
pg/ml LPS or 5 ng/ml IL-lB for 4 hours. Some cells were additionally treated with
500pM TSA for 4 hours prior to and during LPS or IL-18 stimulation. (A). EMSA was
performed using the indicated quantities of nuclear extract. Arrows indicate the positions
of C/EBP:DNA complexes. (B). C/EBPBZI3497 expression was detected in nuclear
extracts by western blotting with C-terminus-speciﬁc anti-C/EBPB.

122

1000
900
800
700
600
500

400
300
200
100

Relatlvo Luclferase Expression

 

pMEX 250nM 500nM p300 C/EBPB C/EBPB CIEBPB CIEBPB
TSA TSA 250 500 p300
TSA TSA

Figure 3-3 TSA and p300 Augment C/EBPB Activity. Transient transfections
of P388 cells reporter were carried out in duplicate using a 2X-C/EBP promoter-reporter.
Expression vectors for C/EBPB and p300 were used as indicated. Some cells were
additionally treated with TSA for 8 hours. Luminometer values were normalized for
expression from a co-transfected SV40 early promoter B-galactosidase reporter. These
values were then normalized to a relative value of 1 for cells receiving “empty”
expression vector (pMEX). The data presented are the means of four independent
transfections with their standard error.

123

transactivation activities of C/EBPB suggests an important role for acetylation in
regulating C/EBPB activity. It also suggests that the inability of C/EBP02.3_297 to
support robust IL-6 and MCP-l transcription in response to IL-1l3 may reﬂect inadequate
acetylation of C/EBP0213-297. Furthermore, the ability of p300 to enhance C/EBPB
transactivation as effectively as TSA suggested a candidate acetyltransferase for the
modulation of C/EBPB activity.

p300 and CBP augment C/EBPﬁ DNA-binding—Having observed that p300 was as
effective as TSA at enhancing C/EBPB activity, we examined the effects of p300
overexpression on C/EBPB DNA-binding activity by transiently transfecting P388 cells
with a vector expressing either intact C/EBPB or C/EBP13213,297 in the absence or presence
of a vector expressing p300. An EMSA revealed that p300 enhanced the DNA-binding
activity of C/EBPB and C/EBP[32,3_297 homodimers, as well as C/EBPB:y and C/EBPBZW
297:7 heterodimers (Fig 4A). Western blot analysis of nuclear extracts revealed equivalent
amounts of C/EBPB and C/EBP0213-297 expression in transient transfections of P388 cells
with and without a p300 expression vector (Fig. 43). Moreover, intact C/EBPB and
C/EBP0213-297 DNA-binding was also enhanced in the presence of CBP, a p300 homolog
(Fig. 5).

To determine whether p300 augmentation of DNA-binding inﬂuenced association
rates of C/EBP0213-297, nuclear extracts from transiently transfected P388 cells were
incubated with radiolabeled C/EBP consensus oligonucleotide and binding reactions were
loaded onto an EMSA gel at various time points during the incubation. Figure 6 shows

that the co-expression of C/EBPBZI 3-297 with p300 led to more rapid DNA-binding than

124

   
 

Vector CIEBPB CIEBPB 21: :97
-p300 +p300 -p300 |+p300 -p300 l+paoo
AbNﬂNB NBNB Nays
." 4- . " t 4-
“ ¢ Supershﬂts . 4. Supershifts
I a II “4‘ . ‘
M n
I.
CEBPﬂzn-wﬂ’ ‘1...
u a, ~ -‘ ., CIEBPamm» -' .
”‘4‘ ﬁ . CIEBPﬁZU-ZW 5“,.
. . EIP'A’

      

Unbound
Probe
Lane 1 2 34 5 6 78 9101112
CIEBPﬂ — — + + _ _
CIEBPﬁﬁ3.291 — — _ _' + ‘l'
p300 — + — + _ +

WB: ﬂ-tubulln

Figure 3-4 C/EBPB DNA-binding is augmented by p300. (A). EMSA of nuclear
extracts from P388 cells transiently transfected with expression vectors for C/EBPB,
C/EBP0213-297, and/or p300. Binding reactions included normal rabbit IgG (N) or C-
terrninus-speciﬁc anti-C/EBPB ([3). Arrows indicate the positions of C/EBPzDNA
complexes and supershifts. (B). C/EBPB and C/EBPBZMW expression were detected in
nuclear extracts by western blotting with C-terminus-speciﬁc anti-C/EBPB.

125

Vector CIEBPB CIEBPBmQW
-CBP +CBP -CBP |+CBP -CBP +CBP

Ab N N N N N
645 ->
M 'V
CJEBPﬁm_miy'>
CIEBPBZMWI ->
CIEBPBm_,,.,
Unbound
Probe "

Figure 3-5 C/EBPB DNA-binding is augmented by CBP. EMSA of nuclear
extracts from P388 cells transiently transfected with expression vectors for C/EBPB,
C/EBPBQO, and/or CBP. Binding reactions included either normal rabbit IgG (N) or
C-terminus-speciﬁc anti-C/EBPB (B). Arrows indicate the positions of C/EBPB:DNA
complexes.

 

126

Relative DNA Binding

 

  
   
 

 

 

 

20 + CiEBPBmm-ﬂr
15 ~
+ CiEBPﬂmmﬁCIEBPBM

10 ~

5 _ + CIEBPﬁamﬂy'i- p300

0 I

CIEBPB i
0 10 20 30 4o 50 60 7o +ceaps:$:.psoo
Minutes
Figure 3-6 The association of C/EBPBnMn with DNA is augmented by p300.

EMSA was performed using nuclear extracts of P388 cells transiently transfected for
C/EBP0213-297 expression in the presence and absence of p300 expression. Binding
reactions were sampled over a 60-minute time course at room temperature. The
abundance of C/EBPBZIHW homodimers and C/EBPle3.297/y heterodimers was measured
by a phosphorimager and is presented in relative units. “Images in this figure are
presented in color.”

127

that observed with C/EBPBZI3-297 alone. This was found to be the case for both
C/EBPBZI3497 homodimers and heterodimers with C/EBPy, with C/EBPl3213-297
homodimers being more dramatically affected.

p300 enhances C/EBPﬂ acetﬂation—The co-activator p300 was previously shown
to physically interact with and enhance C/EBPB transactivation (Mink, Haenig et al.
1997). More recently, p300 binding to C/EBPB was found to trigger phosphorylation,
and subsequent activation of p300 as a coactivator of C/EBPB (Schwartz, Beck et al.
2003). To verify that p300 inﬂuences C/EBPB acetylation, we immunoprecipitated
ectopically expressed FLAG-tagged C/EBPB in the absence or presence of p300
overexpression with a FLAG-tag-speciﬁc antibody. An anti-acetyl lysine antibody and a
C-terminus-speciﬁc anti-C/EBPB- antibody were used in a western blot analysis. Figure
7 shows that p300 overexpression enhances C/EBPB acetylation approximately 2.7-fold
over basal levels.

The conserved 10(KK motif is critical for p3 00-mediated augmentation of
C/EBPﬂ activity—To establish the functional signiﬁcance of the KXKK motif on p300
augmentation of C/EBPB activity, we substituted the two lysines in C/EBPB at positions
216 and 217 with arginines, which block potential acetylation, while minimizing
alterations to protein structure. These same residues have been identiﬁed as an
acetylation substrate in other transcription factors (Gu and Roeder 1997; Boyes, Byﬁeld
et al. 1998) and, therefore, were predicted to be a substrate for p300-mediated acetylation
of C/EBPB. The transient co-transfection of C/EBPB and C/EBPB(KK—)RR) (K216R
and K217R) with and without p300 in P388 cells demonstrated that loss of the two

lysines largely blocked the ability of p300 to augment DNA-binding (Fig. 8A).

128

FLAG-cream — + +

p300 — — +

 

'
I
.‘

 

, _ IP: «an-FLAG
, , 1"“ ,4». . E waza-Acetyl- LYS

I'n'n (HI, “I"! O ‘ 'II"? ‘I. "I I‘-."'. 'w' 'l 4' "-‘. ‘J'I‘TIV l.“'_"'w"" ‘m “'\I*3
ﬂIl‘é.h!':l".. "LII'!'1"'..l'sal;ﬂgm-I.x’l at“!

 

 

7‘5
l
. - W E W33 “'C’EBPB
‘ .. __ J. 1
Relative
Acetylation 1 2'7
Figure 3-7 C/EBPB acetylation is enhanced by p300 overexpression. P388

cells were transiently transfected with vectors expressing FLAG-tagged C/EBPB with and
without a p300 expression vector. Nuclear extracts of these cells were then analyzed by
immunoprecipitation using an anti-F LAG antibody, separated by PAGE, and then
subjected to western detection using either anti-acetyl lysine or C-terminus—speciﬁc anti-
C/EBPB. The relative acetylation of C/EBPB was determined by densitometry. Levels of
acetylation were normalized to the amount of C/EBPB detected and the level of
acetylation in the cells transfected with C/EBPB alone was set to a value of 1.

129

Western blot analysis of the nuclear extracts showed that neither the p300—mediated
enhancement of C/EBPB DNA-binding activity nor the lack of enhancement for
C/EBPB(KK—>RR) was the result of altered protein levels (Fig. 8B). Interestingly,
however, the expression of C/EBPB(KK—>RR), both in the absence and presence of p300,
was reduced in comparison to wildtype C/EBPB. At the same time, an increase in
C/EBPB(KK—>RR) expression was observed in co-transfections with p300 (Fig. 8B).
These results suggest a relationship between acetylation status of C/EBPB and protein
stability, in addition to modulation of activity.

Since the DNA-binding activity of C/EBPB(KK—>RR) could not be augmented by
p300, we examined whether this mutation inﬂuences the transactivation potential of
C/EBPB. Figure 9 shows that p300 overexpression stimulates wildtype C/EBPB activity
on a C/EBP-dependent reporter. The ability of p300 to co-activate a C/EBP-dependent
promoter-reporter was dependent on lysines 216 and 217 of C/EBPB, as p300 did not
appreciably enhance transactivation by C/EBPB(KK—)RR). In addition, substitution of
lysines 216 and 217 with arginine reduced the basal transactivation potential of C/EBPB.
Highlighting the functional signiﬁcance of these lysine residues, C/EBPl3(KK—+RR) was
also much less effective than wildtype C/EBPB in supporting transcription from the IL-6
and MCP-l promoter-reporters (Figure 10).

p3 00 acetyltransferase activity is dispensable for augmentation of C/EBPﬂ DNA-

binding and transactivation—To determine whether acetylation was a direct

consequence of p300 enzymatic activity or recruitment of an additional acetylase, we

130

CBPNKK—rRR)

 

   

B
ClEBPB ClEBPﬂ ClEBPﬂ ClEBPﬂ
+paoo (KK—>RR) (KK—>RR)
+psoo
we: cream
till
Figure 3-8 p300 enhancement of DNA-binding is blocked by lysine to arginine

substitutions of C/EBPB residues 216 and 217. (A). EMSA was performed using
nuclear extracts from P388 cells transiently transfected for C/EBPB or
C/EBPB(KK—>RR) expression with and without p300 expression. Binding reactions
included either normal rabbit IgG (N) or C-terminus-speciﬁc anti-C/EBPB (B). (B).
C/EBPB or C/EBPB(KK—>RR) expression were detected by Western blot of nuclear
extracts (50 pg) using C-terminus-speciﬁc anti-C/EBPB.

131

2X-ClEBP-Luc

 

 

 

2507
200 d
r:
.2
*5 150 ‘
.2
‘6
d
g 100 --
o
u.
504
0
Vector pm CIEBPB ClEBPn CIEBPB ClEBPp
p300 (KK—rRR) (KK-rRR)
p300
Figure 3-9 p300 augmentation of transactivation is blocked by lysine to

arginine substitutions of C/EBPB residues 216 and 217. Transient transfection of
P388 cells with expression vectors for C/EBPB, C/EBPB(KK-—)RR), and p300 were
carried out in duplicate with a 2X-C/EBP promoter-reporter as indicated. Luminometer
values were normalized for expression from a co-transfected SV40 early promoter-[3-
galactosidase reporter. These values were then normalized to a relative value of 1 for
cells receiving “empty” expression vector (Vector). The data presented are the means of
three experiments with standard error. (* signiﬁcantly different from wildtype C/EBPB;
** signiﬁcantly different from wildtype C/EBPB with p300 overexpression).

132

 

           

 

161
3...
E12-
:11
1o-
3
.2 OJ
3 so
.1
2 4«
'3
'6 2‘
C 0‘
LPS LPS ms Control ClEBPﬂ CIEBPB
Control + + (Kit-RR)
cam cream
(KK-‘RRJ
lL-6 MCP-l

Figure 3-10 Mutation of lysine residues 216 and 217 largely blocks C/EBPB
transactivation of the IL-6 and MCP—l promoter-reporters. Transient transfections
of P388 cells were carried out in duplicate with IL-6 and MCP-l promoter-reporters.
Luminometer values were normalized for expression from a co-transfected SV40 early
promoter-B-galactosidase reporter. These values were then normalized to a relative value
of 1 for cells receiving “empty” expression vector (Control). Cells transfected with the
IL-6 promoter-reporter were treated with LPS for 4 hours prior to assay. The data
presented are from one experiment.

133

compared the activity of an expression vector for p300 to one for p300AHAT that
encodes a protein lacking acetyltransferase activity. In transient co-transfections of P388
cells with expression vectors encoding C/EBP13213-297 and/or p300 and p300AHAT, both

p300 and p300AHAT similarly enhanced C/EBP13213-297 DNA-binding activity (Fig.11),

indicating that augmentation of C/EBPl3213-297 DNA-binding activity was not dependent
upon the acetyltransferase activity of p300. To determine if there are ﬁmctional
consequences to a lack of p300 acetyltransferase activity, we examined the effects of
p300 and p300AHAT on C/EBPB-dependent promoter activation in transient
transfections. Consistent with the dispensability of p300 acetyltransferase activity for
augmentation of C/EBPB DNA-binding, p300AHAT was able to enhance C/EBPB
transactivation (Fig. 12).

The ElA oncoprotein, which binds in close proximity to the acetyltransferase domain
of p300/CBP, has been shown to inhibit the HAT activity of p300/CBP and block p300-
mediated acetylation of p53 (Chakravarti, Ogryzko et al. 1999). Therefore, we examined
whether ElA blocks p300-mediated augmentation of C/EBPBZIMW DNA-binding by
transiently co-transfecting P388 cells with C/EBPBZIHW and p300 expression vectors in
the absence or presence of EIA expression vector. Consistent with the observation that
the activity of p300 was not dependent on its acetyltransferase function, we found that the
co-transfection of EIA expression vector with p300 failed to inhibit p300-mediated
enhancement of C/EBP8213-297 DNA-binding (Fig. 13, Left Panel). A western blot
analysis showed equivalent C/EBPB213-297 expression levels in the absence or presence of

p300 and ElA expression vectors (Fig. 13B).

134

emits-z! + + +
p300 " + ‘
p300 AHA‘I’

 

 

"4- CIEBPBNMWIy

‘C/EBPBQBQWI
' C/EBPB213-297

 

 

 

 

Figure 3-11 p300 acetyltransferase activity is dispensable for augmentation of
C/EBPB binding. EMSA was performed using nuclear extracts of P388 cells transiently
transfected for C/EBP13213-297, p300, and p300AHAT expression as indicated.

135

1201

100-4

Relative Luciferase Expreslon
8
1

 

 

 

m A
20 —
Control p31!) p3“) CIEBPﬁ ClEBPﬂ CiEBPﬂ
AHAT +paoo +peoo
AHAT
Figure 3-12 p300 acetyltransferase activity is not required for augmentation of

C/EBPB transactivation by p300. Transient transfection of P388 cells were carried out
in duplicate with a 2XC/EBP promoter-reporter. All values were normalized to a value
of 1 for the control transfection with reporter and “empty” expression vectors. Values
presented are the means from one experiment.

136

P/CAF Attenuates C/EBPﬁ DNA-Binding activity—The data demonstrating
enhanced acetylation of C/EBPB with p300 overexpression (Fig. 7) and a dependence
upon C/EBPB lysines 216 and 217 for p300-mediated enhancement of DNA-binding
activity and transactivation (Fig. 8A, 9, and 10) implicate acetylation of C/EBPB as being
critical to p300-mediated enhancement of its activity. Since the activity of p300 was not
dependent on its acetyltransferase activity, we examined whether additional
acetyltransferases, such as P/CAF and GCN5 (Brownell, Zhou et al. 1996; Yang,
Ogryzko et a1. 1996), that are known to associate with p300, might be active in
augmenting C/EBPB activity. We ﬁrst examined whether P/CAF might be capable of
augmenting C/EBPB213297 DNA-binding activity. Unexpectedly, we found that the co-
expression of the acetyltransferase P/CAF with either p300 or CBP in P388 cells actually
reduced C/EBPB213-297 DNA-binding in comparison to that observed with p300 or CBP
alone (Fig. 14A). Western blot analysis of nuclear extracts from transiently transfected
P388 cells revealed equivalent C/EBPB213-297 expression levels in the absence or presence
of p300, CBP, and/or P/CAF expression vectors (Fig. 14B). Interestingly, we found that
increasing amounts of EIA expression in P388 cells co-transfected with a constant
amount of P/CAF expression vector reversed P/CAF-mediated inhibition of C/EBPB213-
297 DNA-binding activity (Fig. 13, Right Panel). Western blot analysis of nuclear
extracts showed that C/EBPB213-297 expression levels were slightly enhanced in the
presence of CBP expression vector, while co-transfection of P/CAF expression vector
reduced C/EBPB273-297 expression levels (Fig. 13B). Interestingly, co-transfection with

ElA expression vector reversed the reduction in C/EBPB213-297 expression levels (Fig.

137

    
 

t" C/EBPﬂzrsv'I/Y
*‘i‘ CIEBPp,,,_,,,/
C/EBPBmasn

 

7 8 9101112

   

B
ClEBPﬂnm + + + + + + +
p300 — + — — — - —
E1A - § - — .. 9- H
CBP - - _ + + + +
”c” _' ' .‘ .-..‘ T.-. T __-T.._
Left Panel Right Panel
Figure 3-13 ElA oncoprotein does not suppress p300-mediated augmentation

of C/EBPB273.297 DNA-binding. (A) Left Panel: EMSA was performed using nuclear
extracts of P388 cells transiently transfected for C/EBPB213-297, p300, and ElA expression
as indicated. The binding reactions for cells transfected with vector for C/EBPB213.297
included either normal rabbit IgG (N) or C-terminus-speciﬁc anti-C/EBPB (B). Right
Panel: EMSA was performed using nuclear extracts of P388 cells transiently transfected
for C/EBPB213_297, CBP, P/CAF, and ElA expression as indicated. BIA expression
vector was transfected in increasing quantities of 0.5 pg (Lane 11) and 1.0 pg (Lane 12).
The binding reactions for cells transfected with vector for C/EBPB213-297 included either
normal rabbit IgG (N) or C-terminus-speciﬁc anti-C/EBPB (B). (B). C/EBPB2.3_297
expression was detected in a western blot of nuclear extracts (50 pg) from P388 cells
transiently transfected for C/EBPB213-297 expression with and without p300, ElA, CBP,
and P/CAF and expression vectors using C-terrninus-speciﬁc anti-C/EBPB.

138

     

P300 '— ' T " '
CBP - - + + +
PICAF " ’ ' + T"
P/CAF ' “WP/CA1:
WW“ ' \ll
l \ l l l l l l
l l l o
‘ ‘T‘ll‘i‘El‘E‘llE
CIEBPBanI "’
C/EBPBnmv
3
p300 - + - - -
CBP — - — - + + +
PICAF — — + H — — + H
— _ ..
- — ...........

Left Panel Right Pane!

Figure 3-14 P/CAF inhibits both p300 and CBP-mediated enhancement of
C/EBPB213.297 DNA-binding. (A) Left Panel: EMSA was performed using nuclear
extracts of P388 cells transiently transfected for C/EBPB213.297, p300, and P/CAF
expression as indicated. P/CAF expression vector was transfected in increasing
quantities of 0.5 pg and 1.0 pg. Right Panel: EMSA was performed using nuclear
extracts of P388 cells transiently transfected for C/EBP0213-297, CBP, and P/CAF
expression as indicated. P/CAF expression vector was transfected in increasing
quantities of 0.5 pg and 1.0 pg. One binding reaction for cells transfected for C/EBPBm-
297 expression alone included C-terminus-speciﬁc anti-C/EBPB (B). (B). C/EBPB213-297
expression was detected in a western blot of nuclear extracts (50 pg) from P388 cells
transiently transfected for C/EBP0213.297 expression with and without p300, CBP and
P/CAF expression vectors using C-terminus-speciﬁc anti-C/EBPB.

creams — + + + +
PICAF - - - r -
PICAF Al-IAT

 

1345
M
Lane
CIEBPB -— + + +
PICAF — — + —
PICAFAHAT

WBI CICEBP‘

 

Figure 3-15 P/CAF HAT activity is required for its inhibition of C/EBPB DNA-
binding activity. (A). EMSA was performed using nuclear extracts of P388 cells
transiently transfected for C/EBPB, P/CAF, and P/CAFAHAT expression as indicated.
One binding reaction derived from cells transfected for C/EBPB expression by itself
included C-terminus-speciﬁc anti-C/EBPB (B). (B). C/EBPB expression was detected in
a western blot of nuclear extracts (50 pg) from P388 cells transiently transfected for
C/EBPB expression with and without P/CAF and P/CAFAHAT expression vector using
C-terminus-speciﬁc anti-C/EBPB.

140

13B). In addition to its ability to inhibit p300 HAT activity, ElA also inhibits P/CAF
HAT activity by either displacing P/CAF from p300/CBP (Yang, Ogryzko et al. 1996)
and/or by directly binding to the P/CAF HAT domain (Chakravarti, Ogryzko et a1. 1999).
In light of this, ElA abrogation of P/CAF-mediated attenuation of DNA-binding suggests
that P/CAF acetyltransferase activity may play an important role in its inhibition of
C/EBPB activity.

P/CAF inhibition of C/EBPﬂ DNA-binding is dependent on its HA T activity—We
investigated whether P/CAF inhibition was dependent upon its HAT activity by co-
transfecting C/EBPB with vectors for P/CAF and P/CAFAHAT, which lacks
acetyltransferase activity. While P/CAF expression alone reduced C/EBPB DNA-
binding, the P/CAF acetyltransferase-deﬁcient mutant was unable to appreciably inhibit
C/EBPB DNA-binding (Fig. 15A). A western blot analysis showed fairly equivalent
C/EBPB expression levels under the various conditions (Fig. 15B). This suggests that
P/CAF inhibition of C/EBPB DNA-binding is dependent on its acetyltransferase activity
and is consistent with our previous experiment showing that the ectopic expression of
ElA, an inhibitor of P/CAF HAT activity, reverses P/CAF suppression of C/EBPB213-297
DNA-binding (Fig. 13, Right Panel). We also found that P/CAF suppression of CBP-
mediated enhancement of C/EBPB213-297 DNA-binding was dependent on P/CAF HAT
ﬁmction (Fig. 16A). Interestingly, a western blot analysis revealed a modulation of
C/EBPB273-297 expression levels in the presence of the co-activators CBP and P/CAF.
This is consistent with previous data demonstrating that alteration of the KXKK motif
affects steady state protein levels (Fig. 8B). In transient co-transfections of P388 cells,

CBP expression elevated C/EBPB213-297 expression 1.6-fold (Fig. 16B). We unexpectedly

141

found that P/CAF diminished C/EBPB273-297 expression in comparison to C/EBPB213-297
alone (Fig. 16B). This reduction was dependent on the HAT activity of P/CAF, as
C/EBPB213-297 expression levels were nearly equivalent in extracts with C/EBPB213-297
alone and extracts with C/EBPB213.297 and P/CAFAHAT (Fig. 16B). In addition, the co-
expression of C/EBPB213_297 and CBP yielded similar elevation of C/EBP213-297 protein
levels as co-expression of C/EBPB213-297, CBP, and P/CAFAHAT (Fig. 163, 1.6-fold
versus 1.7-fold). These data imply distinct roles for p300/CBP and P/CAF in co-
activator-dependent modulation of protein stability, as p300/CBP activity seems to
promote protein stability, while P/CAF activity supports protein degradation.
C/EBP,62,3_297 DNA-binding and transactivation is augmented by GCN5 in a HA T -
dependent manner—-The fact that p300 acetyltransferase activity is dispensable for
augmentation of C/EBPB activity, while P/CAF not only lacks co-activation activity, but
actually reduces C/EBPB DNA-binding suggested that some other acetyltransferase was
involved in augmentation of C/EBPB function. Therefore, we examined whether GCN5,
a homolog of P/CAF that is known to associate with p300 (Sterner and Berger 2000),
might augment C/EBPB activity. To test this notion, we transiently transfected P388
cells with various combinations of C/EBPB213_297, p300, GCN5, and GCNSAHAT
expression vectors; GCN5 was expressed over a titration of increasing quantities of
expression vector (Fig. 17, Left and Right Panels). p300 expression enhanced
C/EBPB273-297/y and C/EBPB213-297/C/EBPB213-297 DNA-binding 1.4-fold and 2.6-fold,
respectively (Fig. 17A, Lane 1 vs. 4), while GCN5 expression had no measurable impact
on C/EBPB213-297/y DNA-binding and enhanced C/EBPB213-297/C/EBPB273297 DNA-

binding 1.7-fold (Fig. 17A, Lane 1 vs. 5). In contrast to these relatively modest effects,

142

CBP — — + - — + +
PIC AF — - — + — + -
P/C AFAHAT — — — — + — +

eleeesmmh -> i'
creeesmmi +,

 

 

Lane
CIEBPBmQ97 + + + + + +
CBP — + — — + +
P/CAF — — + — + —
P/CAFAHAT — — — + — +
WB: C/EBPB

Lane 1 2 3 4 5 6
Protein levels 1.0 1.6 0.9 1.0 1.4 1.7
Figure 3-16 P/CAF suppression of CBP-mediated effects on C/EBPB213297

inﬂuences protein stability in a HAT-dependent manner. (A). EMSA was performed
using nuclear extracts of P388 cells transiently transfected for C/EBPB277297, CBP,
PxCAF. and P/C‘AFAHAT expression as indicated. The binding reaction for cells
transfected with vector for C/EBPB213297 alone included C—terminus speciﬁc anti-C/EBPB
(B). (B). C'EBPB expression was detected in a western blot of nuclear extracts (50 pg)
from P388 cells transiently transfected for C/EBPB273297 expression with and without
CBP. PI’CAF. and P/CAFAHAT expression vector were examined using C-tenninus-
speciﬁc anti—(T. EBPB.

143

we found that GCN5 synergized with p300 to augment C/EBPB213_297 DNA-binding (Fig.
17A, Lanes 1 vs. 6-8). C/EBPB213-297/y and C/EBPB213-297/C/EBPB273-297 DNA-binding
was augmented 3.2-fold and 16.1-fold, respectively, at the largest quantity of GCN5
expression vector (2 pg) (Fig. 17, Lane 8). Moreover, GCN5 augmentation of
C/EBPB273-297 DNA-binding is dependent on its acetyltransferase activity, as C/EBPB213-
297 DNA-binding was reduced in nuclear extracts from P388 cells transiently co-
transfected with a vector for GCN5 lacking acetyltransferase activity (GCNSAHAT) in
comparison to co-transfection with a vector for wildtype GCN5 (Fig. 17A, Lane 10 vs.
11). In addition, GCNSAHAT did not synergize with p300 to enhance C/EBPB213,297
DNA-binding and, in fact, suppressed p300-mediated enhancement of DNA-binding,
perhaps by competition with endogenous wildtype GCN5 (Fig. 17A, Lane 9 vs. 12 and
13), To determine the functional consequences of GCN5 activity on C/EBPB
transactivation, we performed transient transfections in P388 cells with expression
vectors for intact C/EBPB with and without vectors for p300 and GCN5 expression in the
presence of a C/EBP-dependent promoter-reporter (Fig. 18). Consistent with the
previous data showing that GCN5 cooperates with p300 to enhance C/EBPB273-297 DNA-
binding, we found that p300 and GCN5 cooperate to augment C/EBPB transactivation of
a 2X-C/EBP promoter-reporter (Fig. 18).

We previously found that the KXKK motif in C/EBPB (Fig. 8B) and the co-activators
P/CAF and CBP (Fig. 16 B) inﬂuence C/EBPB protein levels. Consistent with a role for

acetylation in protein stability, a western blot analysis of nuclear extracts from the above

described transfections revealed that co-expression of p300 with the highest level of

144

    
 
 

'.
”will

CIEBP8213297

  
   

9 10 11 12 13

Lane 5 6 1
norm. DNA. 1.0 1.4 1.0 1. 7 2.0 3.2 (MW!)
Binding Acﬂvly 1.0 2.6 1.1 3 .1 5.2 16.1 (i-bmodlmer)
B
c’EBPBasew + + + + + +
p300 — + +
GCN5 — — £12.01 HO. 5} +[1.0) +(2.0)

 

1‘9 #9

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R°|aﬂvo 1" uill‘ ‘ullli' ill I" i! U“ hum" u‘tﬂu‘unhlm,‘.‘“0‘l‘4"“ “111‘ ‘nllll‘l‘lvl l
Expression 0.57 0.60 0.78 1.08 1.14 2.3

Lane 1 2 3 4 5 6

Figure 3-17 GCN5 synergizes with p300 to enhance C/EBPB213-297 DNA-
binding. (A). Left Panel: EMSA was performed using nuclear extracts of P388 cells
transiently transfected for C/EBPB213-297 expression with and without p300 and GCN5
expression vectors. Two binding reactions for C/EBPB alone included either anti-
C/EBPB (B) or anti-C/EBPy (y). Arrows indicate the positions of C/EBP:DNA
complexes. GCN5 expression vector was transfected in increasing quantities of 0.5 pg
(Lane 6), 1.0 pg (Lane 7), and 2.0 pg (Lane 8). 2.0 pg of GCN5 expression vector was
used in the cells analyzed in Lane 5. Relative DNA-binding activity was determined by
normalizing the phosphorimage value obtained for each DNA:C/EBPB213-297 complex to
C/EBPB213_297 protein expression levels in each nuclear extract. Right Panel: EMSA
was performed using nuclear extracts of P388 cells transiently transfected for
C/EBPB213-297 expression with and without p300, GCN5, and GCNSAHAT expression
vectors. (B). C/EBPB was detected in a western blot of nuclear extracts (50 pg) from
P388 cells transiently transfected for C/EBPB213-297 expression with and without p300 and
GCN5 expression vectors using C-terminus-speciﬁc anti-C/EBPB.

145

60

40

20"

Relative Luciferase Expression

   

 

Vector CIEBPB CIEBPB CIEBPB CIEBPB
+ p300 + Gcn5 + p300
+ Gcn5

Figure 3-18 GCN5 Cooperates with p300 to Enhance C/EBPB Transactivation
of the 2X-C/EBP promoter-reporter. Transient transfection of P388 cells with
expression vectors for C/EBPB, p300, and GCN5 were carried out in duplicate with a
C/EBP-dependent reporter as indicated. Ltuninometer values were normalized for
expression from a co-transfected SV40 early promoter-B—galactosidase reporter. These
values were then normalized to a relative value of l for cells receiving “empty”
expression vector (Vector). The data presented are the means of two experiments with
standard error.

146

GCN5 elevated C/EBPB213-297 protein expression approximately 4 times the amount
observed in their absence (Fig. 178, Lane 1 vs. 6).

Differential co-activator regulation of C/EBPﬁ DNA-binding through alternative
lysine residues—We have shown that GCN5 cooperates with p300 to enhance C/EBPB
activity, while its homolog P/CAF inhibits C/EBPB DNA-binding activity. Moreover,
both GCN5 and P/CAF modulation of C/EBPB DNA-binding was dependent upon their
acetyltransferase activities. Since these co-activators differentially modulate C/EBPB
DNA-binding activity through acetylation, we wanted to investigate whether these
acetyltransferases mediate their activity through the use of alternative lysines. To
examine the use of alternative lysines, we generated several lysine to arginine
substitutions in the KXKK motif, which was shown to be critical for p300-enhanced
DNA-binding and transactivation of C/EBPB (Fig. 8A) and has been shown to be an
acetylation substrate in other transcription factors, such as p53 (Gu and Roeder 1997),
GATA-l (Boyes, Byﬁeld et al. 1998), and E2F (Martinez-Balbas, Bauer et al. 2000). We
performed transient co-transfections of P388 cells with expression vectors for wildtype
C/EBPB (KXKK), C/EBPBKZMR (RXKK), C/EBPBKKZ16'217RR (KXRR), and
C/EBPBKxxz14,216,217RRR (RXRR) with and without expression vectors for p300 and
P/CAF. In control transfections, overexpression of p300 enhanced wildtype C/EBPB
DNA-binding activity (Fig. 19A, Lane 1 vs. 4), while overexpression of P/CAF reduced
C/EBPB DNA-binding activity (Fig.19A, Lane 1 vs. 5). C/EBPBKzMR (RXKK) was still
capable of increased DNA-binding activity in the presence of p300 (Fig. 19A, Lane 6 vs.
7), but P/CAFs ability to reduce its binding was marginal (Fig. 19A, Lane 6 vs. 8). While

C/EBPBm16,217RR(KXRR) was not subject to p300 enhancement of DNA-binding

147

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“iii; til“ 11““:
n ““1
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“1 l .

 

 

 

 

    

 

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am
ClEBPﬂ+++—---———-———-
RXKK + + +
KXRR
RXRR
P3!!!
PICAF
['"WW
1 ‘ “ " . WB:CIEBPB
in! nml'l'W" w'llllllll“ ..1 Nu
1‘!" I’ mm" ..milllllo.tilllllllW- lllu'n" >|u|:|““‘.““" 1i.|t«rlll.lll‘l-l ..va |.l|l“|\“ 1. lllllllm mlllll‘lll‘L‘ lllIll““H-“"I.k‘ll‘
‘1 ‘ B-tubulln

 
   

ull'u‘l‘li‘ . 1n‘1.hlll“Jill!“ti‘uiilulllllllll’llll- ‘ I" .Llllllllllll' ' -I.l1lllll“l|\‘l."mlttlllllllllll'l’ and

Figure 3- 19 Differential Regulation of C/EBPﬂ Binding Through Alternative
Lysines. (A). EMSA was performed using nuclear extracts of P388 cells transiently
transfected for expression of C/EBPB and C/EBPB lysine to arginine substitution(s) in the
KXKK motif with and without p300 and P/CAF expression vectors. The substituted
residues are indicated by red letters. Two binding reactions for cells transfected for
wildtype C/EBPB expression included either C-terminus-speciﬁc anti-C/EBPB (B) or
anti-C/EBPy (y). Arrows indicate the positions of C/EBP:DNA complexes. (B). Western
blot of nuclear extracts (50 pg) from P388 cells transiently transfected for expression of
C/EBPB and C/EBPB lysine to arginine substitution(s) with and without p300 and P/CAF
expression vectors. C/EBPB and B-tubulin were detected using C-terminus-speciﬁc anti-
C/EBPB and anti-B-tubulin.

ll‘ will" mm It 'smliluim ,1.,.uw-x_ .1

148

activity (Fig. 19A, Lane 9 vs. 10), its DNA-binding activity could still be attenuated by
P/CAF (Fig. 19A, Lane 9 vs. 11). Finally, ﬁmher demonstrating the importance of the
KXKK motif for C/EBPB DNA-binding activity, C/EBPBKKKZM 316,217RRR (RXRR) was
defective in DNA-binding and weakly responsive, if at all, to either co-activator (Fig.
19A, Lanes 12-14). Western analyses of nuclear extracts from these transient
transfections showed similar levels of protein expression for C/EBPB and the various
lysine to arginine mutants (Fig. 19B).

The KKK motif of C/EBPﬂ is critical for endogenous IL-6 expression in response
to LPS and IL-Iﬂ—We previously demonstrated that stable expression of C/EBPB in
P388 cells supports LPS-induced IL-6 expression (Bretz, Williams et al. 1994; Hu, Baer
et al. 1998). To evaluate the function of the KXKK motif on an intact endogenous IL-6
promoter, we generated stable transductants of P388 cells with murine retroviral vectors
expressing the RXKK and RXRR substitutions in the KKK motif of C/EBPB. We
compared the expression of IL-6 in these transductants to P388 cells ectopically
expressing wildtype C/EBPﬂ (P388-CB), as well as a control population transfected with
an “empty” vector (P388-Neo). EMSA of nuclear extracts from populations of these
stable transductants was performed to verify the appropriate expression of wildtype
C/EBPB and the lysine to arginine mutants. When compared to nuclear extracts ﬁom
P388-Neo, nuclear extracts from stable transductants ectopically expressing wildtype
C/EBPB (KXKK), C/EBPBKZMR (RXKK), and C/EBPBKKKzi4,216,217RRR (RXRR) yielded
proteinzDNA complexes corresponding to C/EBPB homodimers and C/EBPB/C/EBPy

heterodimers, as evidenced by the supershiﬁ of EMSA species upon incubation with an

149

antibody speciﬁc for C/EBPB (Fig. 20A, Lane 2 vs. 4, 6, and 8) in comparison to samples
incubated with normal IgG (Fig 20A, Lanes 3, 5, and 7).

Western analysis of nuclear extracts from these stable transductants was conducted to
examine the protein expression levels of wildtype C/EBPB, C/EBPBKZMR, and
C/EBPBKKK214,216,217RRR (Fig. 208). The protein levels of C/EBPB and its two mutant
forms were quite similar, even though the DNA-binding activity of
C/EBPBKKKZM 316 317mm homodimers and heterodimers was reduced in comparison to
wildtype C/EBPB (Fig. 20A, Lane 3 vs. 7). This is consistent with the transient
expression data presented in Figure 19A, which also shows that C/EBPBW14116217RRR
has reduced DNA-binding activity. Populations of stable transfectants expressing
wildtype C/EBPB, C/EBPBKZMR, and C/EBPBKKKZM 316mm“, as well as control P388-
Neo cells not expressing C/EBPB were treated with LPS and IL—lB over a 24 hour time
course during which RNA was isolated. Northern analyses were performed to detect
transcripts of IL-6 and GAPDH. Maximal endogenous IL-6 expression in wildtype
C/EBPB transfectants was observed after 8 hours of LPS and IL-lB stimulation and was
induced 33-fold and 12.5-fold, respectively, in comparison to the maximal IL-6
expression seen in P388—Neo transductants (Fig. 22A and B). Transductants for
C/EBPBKZMR expression supported IL-6 expression in response to LPS and IL-lB (Fig.
21, Lanes 2-9), with maximal IL-6 expression at 2 hours of LPS stimulation and 4 hours
of IL-lB stimulation that was 0.6-fold and 0.67-fold, respectively, of that observed at
maximal IL-6 expression in transductants for wildtype C/EBPB expression (Fig. 22A and
B). Interestingly, these transductants also displayed less sustained induction of IL-6 than

cells expressing wildtype C/EBPB. Transductants for C/EBPBKKK214216317RRR (RXRR)

150

 

 

 

 

. i. we: OIEBPB
a... in... m we: B-Tubulin
min-.1 ml mutu- “rams-nu
Figure 3-20 Stable expression of C/EBPB lysine to arginine substitution

mutants. (A). EMSA of nuclear extracts of P388-Neo, P388-C/EBPB (KXKK), P388-

C/EBPBKZMR (RXKK), and P388-C/EBPBKKK214316317RRR (RXRR) stable transductants.
The substituted residues are indicated by red letters. Binding reactions included either

normal rabbit IgG (N) or anti-C/EBPB (B). Arrows indicate the positions of C/EBPzDNA
complexes. (B). Western blot of nuclear extracts (50 pg) from P388-Neo, P388-C/EBPB,
P388-CEBPBK214R, and P388-CEBPBKKK214316317RRR. C/EBPB and the lysine t0

arginine substitution mutants were detected using anti-C/EBPB. Detection with anti—[3-
tubulin served as a control for loading.

151

“=6 GAPDH

LPS IL-‘IB LPS IL-1ﬂ
”°""'°' o 2 48 242 4 8_24 o2 4&32 4 s 24

 

 

 

Treatment
P388-Neo = . II D

   

 

 

mas—creepsmaqc) F A

 

 

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mcmsmnar) K- ....... .

 

 

 

momemxnn) is III-In " ..q

 

Lan0123456789

Figure 3—21 C/EBPBKmR, and P388-C/EBPBKm14,216,217m are reduced in
their ability to support induction of IL-6 expression in P388 stable transductants.
RNA was isolated over time courses of LPS and IL-lB treatment as indicated. Thirty

micrograms of RNA was analyzed on Northern blots that were hybridized sequentially to
probes for IL-6 and GAPDH.

152

A lL-6 rrRNA Expresslon

 

 

 

 

 

 

 

 

 

 

 

 

 

+pSV00
—I—KXKK
+RXKK
+RXRR
B
lL-6 "RNA Expresslon
0.8 1
‘3 g 0.6 . +pSV(X)
2 g 0 4 +KXKK
an o ‘ ‘
E a +RXKK
& I3 0'2 ‘ +RXRR
0 2
Hours of IL-1 Treatment
Figure 3-22 Quantitation of induced Il-6 expression in P388 stable

transductants. (A). LPS-stimulated and (B). IL-lB-stimulated IL-6 expression was
quantitated using a Storm Phosphimager (Molecular Dynamics). IL-6 values were
normalized to the values obtained for GAPDH expression in the same samples. The
following transductants were analyzed: P388-Neo [pSV(X)], P388-C/EBPB (KXKK),
P388-CEBPBK214R (RXKK), and P388-CEBPBKKK214316317RRR (RXRR).

153

expression exhibited an even more dramatic reduction of LPS- and IL-lB-induced
endogenous IL-6 expression, as maximal IL-6 expression at 8 hours of LPS stimulation
and 4 or 8 hours of IL-IB stimulation was 0.17-fold and 0.2-fold, respectively, of that
observed at maximal IL-6 expression in transductants for wildtype C/EBPB expression
(Fig. 22 A and B). In agreement with biochemical data demonstrating a role for the
C/EBPB KXKK motif in DNA-binding, these data suggest that this motif plays an

important functional role in C/EBPB transcriptional regulation of IL-6 in P388 cells.

154

Discussion

In this chapter, we examined the importance to C/EBPB activity of the highly
conserved acetylation motif, KXKK, that is adjacent to the DNA-binding domain of
several transcription factors, including p53 (Gu and Roeder 1997), GATA-l (Boyes,
Byﬁeld et al. 1998), and E2F (Martinez-Balbas, Bauer et al. 2000). Similar to
acetylation—mediated effects on DNA-binding observed in the transcription factors in the
above studies, we provide several lines of direct and indirect evidence to suggest that
p300 enhances C/EBPB DNA-binding, in a manner dependent upon lysine residues 216
and 217 of C/EBPB. Moreover, mutation of the KXKK motif significantly reduced
C/EBPB—dependent endogenous IL-6 expression in P388 cells, as well as transactivation
of IL-6 and MCP-l promoter-reporters. The p300 homolog CBP was similar in activity
to p300, as it was also found to augment C/EBPB DNA-binding. However, the
acetyltransferase activity of p300 was dispensable for its ability to augment
C/EBPﬂactivity. Apparently, the critical acetyltransferase activity for co-activator
enhancement of C/EBPB activity is provided by GCN5, which acts in cooperation with
p300.

Surprisingly, the GCN5 homolog P/CAF had an opposing role on C/EBPB, as it
reduced C/EBPB DNA-binding. This activity was dependent on the acetyltransferase
function of P/CAF. A speculative model for the differential regulation of C/EBPB
activity by GCN5 and P/CAF is shown in Figure 23. Under optimal conditions for IL-6

and MCP-l transcription, GCN5 would cooperate with p300 to acetylate C/EBPB at

155

lysines 216 and 217 (Fig. 23A). Overexpression of GCN5 alone had a marginal impact
on C/EBPB DNA-binding (Fig 17A, Lane 1 vs. 5) in comparison to p300 expression (Fig.
17A, Lane 1 vs. 4). This suggests that p300 is the limiting factor in GCN5-mediated
enhancement of C/EBPB activity in P388 cells. Presumably, GCN5 is capable of
acetylating C/EBPB by associating with p300 (Fig. 23A), which has previously been
shown to directly interact with C/EBPB (Mink, Haenig et a1. 1997; Schwartz, Beck et al.
2003). Therefore, while ectopically expressed p300 may interact with endogenous GCN5
in order to further augment C/EBPB DNA-binding, ectopic expression of GCN5 may
have a minimal effect on C/EBPB DNA-binding due to the insufﬁcient expression of
endogenous p300. Perhaps, p300 is necessary to position GCN5 acetyltransferase
activity in close proximity to the KXKK motif of C/EBPB. P/CAF, which is also known
to interact with p300 (Yang, Ogryzko et a1. 1996), could potentially reduce C/EBPB
DNA-binding and IL-6 and MCP-l expression by other mechanisms in addition to
acetylating C/EBPB itself (Fig. 23B). It could compete with the GCN5 binding domain
of p300 to displace GCN5 (Fig. 23D). P/CAF could also block GCN5-mediated
acetylation of C/EBPB by displacing GCN5zp3OO complexes from C/EBPB by directly
binding to the p300 interaction domain within C/EBPB (Fig. 23C). While P/CAF
interactions with C/EBPB have not been reported, P/CAF has been shown to directly
interact with other transcription factors, such as E2F (Martinez-Balbas, Bauer et al.
2000), TALl (Huang, Qiu et a1. 2000), and the HIV Tat protein (Dorr, Kierrner et al.
2002). The fact that a P/CAF acetyltransferase-deﬁcient mutant was unable to suppress

C/EBPB/C/EBPB213-297 DNA-binding (Figs. 15A, Lane 2 vs. 4 and 5, and 16A, Lane I vs.

4 and 5) or to block CBP-mediated augmentation of C/EBPBZIHW DNA-binding (Fig.

156

16A, Lane 3 vs. 6 and 7) suggests that P/CAF facilitates its activity through the
acetylation of C/EBPB at an alternative lysine residue(s) (Fig. 23B), rather than
displacing factors associated with positive effects on DNA-binding (Fig. 23C and D).
This is also supported by data that show that lysine to arginine substitutions at amino
acids 216 and 217 of C/EBPB block p300 augmentation of C/EBPB DNA-binding, but
permit P/CAF-mediated inhibition of C/EBPB DNA-binding (Fig. 19A, Lane 9 vs. 10 and
11). In addition, unlike GCN5, which requires p300 overexpression to potentiate its
effects (Fig. 17, Lane 5 vs. 6-8), P/CAF suppression of C/EBPB DNA binding does not
require exogenous p300 or CBP expression (Figs. 14, Left panel, 15A, Lane 2 vs. 4, and
16A, Lane I vs. 4). Thus, a model where P/CAF directly interacts with C/EBPB is most
likely (Fig. 23B).

The ability of p300 and CBP to augment C/EBP13213-297 activity was surprising, as
p300 was previously found to interact with the N-terminus of C/EBPB (Mink, Haenig et
al. 1997). A more recent study has shown that p300 can interact with the DNA-binding
domain of C/EBPB in addition to N-terminal elements (Schwartz, Beck et al. 2003),
which may explain p300- and CBP-mediated augmentation of C/EBPB213_297 DNA-
binding, which lacks the N-terminal interaction domain of p300. Moreover, these
ﬁndings suggest that the lysine residues primarily responsible for changes in co-activator-
mediated DNA-binding are localized to the highly conserved bZIP domain of C/EBPB.
Consistent with this premise, we found that p300 expression had a greater impact on the
DNA-binding activity of C/EBPB213-297 homodimers than C/EBPBmgm/C/EBPY

heterodimers. C/EBPy retains both terminal lysines of the KXKK motif found in

157

IL—6/MCP—l

 

l_' H.-6/MCP-1

 

 

IL-6/MCP-l

 

IL-6/MCP-1

 

Figure 3-23 Hypothetical models of differential C/EBPB acetylation by GCN5
and P/CAF. Black “stars” denote acetylation of a single lysine. (A). GCN5-mediated
enhancement of C/EBPB DNA—binding and gene activation. (B). DNA-binding
inhibition through a dominant inhibitory site in C/EBPB. (C). p300/GCN5 displacement.
(D). P/CAF-mediated inhibition of C/EBPB DNA-binding by GCN5 displacement. ’

158

C/EBPB, but lacks lysine residues 221 and 227, also within the bZIP region of C/EBPB.
While the positive charge of residues 221 and 227 of C/EBPB is highly conserved
throughout the C/EBP family, these residues are arginines at their homologous positions
in the bZIP region of C/EBPy. Interestingly, we found that lysine to arginine
substitutions at amino acids 221 and 227 in a C/EBPBKKK214216,217RRR (RXRR)
background dramatically reduced its DNA-binding activity (data not shown). Such sites
may also be substrates for acetylation by coactivator acetyltransferases and could explain
the differential impact of p300 on the DNA-binding activity of C/EBPB homo- and
heterodimers.

Recently, lysine residues 216 and 217 of C/EBPB have been implicated as acetylation
substrates in transcriptional regulation of Id-l in the pro-B cell line Ba/F 3 (Xu, Nie et al.
2003). STATS-induced recruitment of HDACl and consequent deacetylation of C/EBPB
increased Id-l transcription. This regulatory mechanism, as well as the in vitro
acetylation C/EBPB by CBP and P/CAF required lysines 216 and 217. While our studies
also implicate P/CAF-mediated acetylation in the reduction of C/EBPB DNA-binding
activity, we have found that lysines 216 and 217 are not required for that effect (Fig.
19A, Lane 9 vs. 10 and 11). Also in contrast to the conclusions of Xu et al., we suggest a
positive role for CBP in augmenting DNA-binding (Figs. 5, 13, 14, and 16). While Xu et
al. showed that mutation of lysines 216 and 217 of C/EBPB reduced its in vitro
acetylation by P/CAF and CBP, their results do not preclude other sites of acetylation.
Furthermore, although overexpression of the double lysine mutant of C/EBPB in Ba/F3
cells enhanced IL-3-independent endogenous Id-l expression, a direct linkage of this to

the activity of P/CAF or CBP was not presented. It may be the case that the discrepancy

159

between the conclusions reached by the two studies reﬂects differences in the cell types
used and/or differences in the ﬁmction of C/EBPB on the Id-l promoter in comparison to
promoters that we have utilized in our studies.

Interestingly, we observed an increase in the protein levels of ectopically expressed
C/EBPB213-297 when co-transfected with GCN5 (Fig. 17B), and that C/EBPB protein
levels decreased with mutation of lysines 216 and 217 (Fig. 8A). Furthermore, P/CAF
acetyltransferase activity appears to reduce C/EBPB213-297 protein levels, as C/EBP13213-297
protein levels were elevated in co-transfections with an expression vector for a
P/CAFAHAT in comparison to co-transfections with a vector for wildtype P/CAF (Fig.
16B). These observations suggest that acetylation of lysine residues in the bZIP of
C/EBPB play an important role in the regulation of C/EBPIS protein stability, in addition
to directly modulating the transcriptional activity of C/EBPB. Previous studies have
shown that the acetylation of the transcription factors E2Fl and SREBPl enhances the
stability of these proteins (Martinez-Balbas, Bauer et al. 2000; Giandomenico, Simonsson
et al. 2003). Moreover, p300 was recently found to exhibit intrinsic ubiquitin ligase
activity, which catalyzed the polyubiquitination and subsequent degradation of the tumor
suppressor protein p53 in the presence of the ubiquitin ligase MDM2 (Grossman, Deato
et a1. 2003). Interestingly, the residues in p53 that are acetylated are identical to the
residues that are ubiquitinated (Ito, Kawaguchi et al. 2002). Moreover, MDM2 supports
p53 degradation by promoting its deacetylation. Acetylation of particular lysines in the
C/EBPB KXKK motif by GCN5 may block the ubiquitination of either those same
residues or lysines in their proximity, thereby inhibiting proteasome-mediated

degradation of C/EBPB.

160

It is curious that acetylation similarly modulates the activities of both intact C/EBPB
and the C/EBPB bZIP protein (C/EBP13213-297), while a potential deﬁcit in acetylation
under conditions of IL-lB stimulation selectively limits the activity of C/EBPleHm in
supporting the induction of IL-6 and MCP-l expression (Figs. 1 and 2). The KXKK motif
is, indeed, retained in both forms of C/EBPB. Our studies presented in Chapter 2
highlight the importance of features within the N-terminal sequences of C/EBPB, such as
serine 64, for optimal activity on the MCP-l and IL-6 promoters. Perhaps, the absence of
critical stimulatory motifs normally supplied by N-terminal sequences makes the
acetylation state of the bZIP region a more critical determinant of activity. One
prediction of this notion is that the acetylation status of the bZIP region may strongly
inﬂuence the ability of LIP, a truncated form of C/EBPB that initiates at Met 153 and
lacks N-terminal activation domains (Descombes and Schibler 1991), to function as a
repressor of C/EBP transactivation and to function as a transactivator of the lL-6
promoter (Hu, Tian et al. 2000).

It is striking that the two highly homologous co-activators, GCN5 and P/CAF,
differentially regulate C/EBPB DNA-binding through acetylation. Our current data
suggests that this difference in activity is mediated through the use of alternative lysines
in C/EBPB. It will therefore be important to more clearly ascertain the speciﬁc residues
in C/EBPB that are acetylated and the signaling pathways that are responsible for

regulating these two mechanisms.

161

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Summary and Future Directions

We have found multiple functional motifs in C/EBPB that are important for the
transcriptional regulation of IL—6 and MCP-l. In Chapter 2, we demonstrated the
importance of an N-terminal functional determinant in Activation Domain Module 11
(ADM2) of C/EBPB that is highly conserved within the C/EBP family. This determinant,
serine 64, was critical for LPS-induced expression of IL—6 and MCP-l in P388 B
lymphoblasts. Mutation of C/EBPB serine 64 dramatically inhibited MCP-l expression,
and to a lesser extent, IL-6 expression in stably transduced P388 cells. This site has been
identiﬁed as a substrate for MAPK-independent, Ras-inducible phosphorylation (J. D.
Shuman and P. F. Johnson, unpublished data), implicating a new kinase signaling
pathway in C/EBPB-mediated IL-6 and MCP-l transcription in response to LPS. In
addition, an internal regulatory domain of C/EBPB was found to inhibit C/EBPB activity
in a promoter-speciﬁc manner, as the presence of RBI and RD2 suppressed C/EBPB
activity in LPS-induced MCP-l expression, while permitting LPS-induced IL-6
expression in a B lymphoblast cell line. In fact, the differential activity of this region is
highlighted by the fact that removal of RDl and RD2 reduced C/EBPB activity on an IL-
6 promoter-reporter. This is consistent with a previous report, which demonstrated that
threonine 189 within RD2, a substrate for MAPK-induced phosphorylation of C/EBPB,
was important for enhancing IL-6 promoter activity. Collectively, these studies suggest
opposing roles for the C/EBPB negative regulatory region in MCP-l and IL-6

transcription.

167

Future investigations should address the mechanism(s) by which the N-tenninal
activation motif and internal negative regulatory domain of C/EBPB differentially impact
MCP-l and IL-6 expression. The amino acid sequence surrounding serine 64 is nearly
identical to a proline-directed phosphoacceptor site in the N-terminal transactivation
domain of c-Jun. Subsequent to Jun Kinase-mediated phosphorylation of c-Jun, this site
ﬁmctions as a substrate for the prolyl isomerase Pinl, which alters the function of the

protein through conformational change. Presumably, phosphorylation at serine 64 could
modulate C/EBPB activity by creating a substrate for Pin 1, thus triggering a
conformational change that would alleviate the inhibitory effects mediated by RD] and
RD2 on the MCP-l promoter.

To test this hypothesis, one could generate a serine to alanine substitution at residue
64 in a C/EBPB mutant lacking RBI and RD2, such as C/EBPBA105-212. If the two lesions
proved compensatory, that is mutation of serine 64 in a RD1/RD2-deleted background no
longer reduces C/EBPB activity on the MCP-l promoter, a linkage between these two
ﬁrnctional motifs would be suggested. An additional approach would be to examine the
effects of Pinl expression on C/EBPB and C/EBPBSMA activity on the MCP-l promoter,
as C/EBPBSMA should be unresponsive to Pinl activity. In addition, we would co-
transfect P3 88 cells with expression vectors for C/EBPB and C/EBPBWA in the presence
and absence of a F LAG-tagged Pinl expression vector to determine whether C/EBPB and
Pin 1 physically interact, and whether serine 64 is essential for Pinl recognition. Extracts
from transfected cells would be analyzed by co-immunoprecipitations of FLAG-tagged
Pinl with western detection of C/EBPB and C/EBPBSMA, as well as oo-

immunoprecipitations of C/EBPB and C/EBPBSMA with western detection of the FLAG-

168

tag (currently, there are no commercial antibodies available for Pinl detection). C/EBPB
serine 64 has been identiﬁed as a Ras-induced phosphoacceptor site (J. D. Shuman and
RF. Johnson, unpublished data). Since binding of Pinl depends on phosphorylation of
target proteins on speciﬁc Ser/Thr-Pro motifs, we would transfect P388 cells for
oncogenic Harvey-Ras (Ha-Ras) or dominant-negative Ha-Ras (DN-Ras) expression,
which would be expected to increase or decrease C/EBPB phosphorylation, respectively.
In the absence of an extracellular signal for kinase activation and C/EBPB
phosphorylation, Ha-ras and DN-Ras activity may be necessary to promote or prohibit
Pinl-mediated regulation of C/EBPB activity. However, the fact that ectopically
expressed C/EBPB can activate a proximal MCP-l promoter-reporter in P388 cells
without a stimulus suggests that such a signaling pathway may be constitutively activated
in P388 cells. Therefore, a more suitable cell line, whereby the default state of C/EBPB
activity is inactive on the MCP-l promoter, may be necessary to investigate the ability of
Pinl to regulate C/EBPB function.

Furthermore, C/EBPBSMA was found to be less effective in IL-6 activation in
comparison to wildtype C/EBPB, albeit to a lesser extent than MCP-l activation. Since
region encompassing RD] and RD2 appears to be important for enhancing, rather than
reducing, IL-6 expression, we propose that C/EBPB serine 64 may perform at least two
functions, one speciﬁc to MCP-l and the other operative on both the MCP-l and IL-6
promoters: 1) it could counteract the inhibition of C/EBPB activity mediated by RD] and
RD2 in MCP-l regulation, and/or 2) it may contain some intrinsic positive activity that

plays a more general role in IL-6 and MCP-l expression. For example, a region

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encompassing ADM2 of C/EBPB was found to mediate interactions between C/EBPB
and various factors, including the coactivator p300.

In Chapter 3, we demonstrated that a motif, KXKK, located adjacent to the DNA-
binding domain of C/EBPB was critical for IL-6 and MCP-l expression in P388 B
lymphoblasts. Our ﬁndings strongly suggest that C/EBPB is acetylated, as the
overexpression of the acetyltransferases p300, CBP, and GCN5 augmented C/EBPB
and/or C/EBPBZIHW DNA-binding activity. This identical motif is acetylated in
numerous transcription factors, including p53, E2Fl, and GATA-l, and plays a similar
role in coactivator-mediated augmentation of DNA-binding. While p300 HAT activity
was dispensable for the enhancement of C/EBP13213-297 DNA-binding and C/EBPB
transactivation of a C/EBP-dependent promoter, p300 cooperated with GCN5 to augment
C/EBPBQO DNA-binding and C/EBPB transactivation of a C/EBP-dependent
promoter. The activity of GCN5 was dependent upon its acetyltransferase activity.
Furthermore, lysine to arginine substitutions at amino acids 216 and 217
(C/EBPBKKM, 217m) blocked p300-mediated enhancement of C/EBPB DNA-binding and
transactivation in transient transfections of P388 cells, while the mutation of residues that
comprise the KXKK motif signiﬁcantly reduced the stimulation of the endogenous IL-6
promoter in P3 88 cells in response to LPS. Unexpectedly, we found that P/CAF, a GCN5
homolog, actually reduced C/EBPB and C/EBP13213-297 DNA-binding in a HAT-dependent
manner. The fact that C/EBPBm16,217RR DNA-binding activity was reduced in the
presence of P/CAF overexpression, while being unresponsive to p300 suggests that these
coactivators differentially impact C/EBPB DNA-binding through the acetylation of

alternative lysines, or that P/CAF-mediated effects are indirect.

170

While our data implicate GCN5 and P/CAF acetyltransferase activity in C/EBPB
acetylation, we have yet to clearly demonstrate the direct acetylation of C/EBPB by these
factors. We have demonstrated that co-transfection of P3 88 cells with expression vectors
for FLAG-tagged C/EBPB and p300 increased C/EBPB acetylation over that seen on
transfection with the vector for FLAG-tagged C/EBPB by itself. To more directly
establish p300-mediated acetylation of C/EBPB, the experiment previously discussed
could be repeated with expression vectors for both C/EBPB and C/EBPBm16 .217er-
Mutation of these lysine residues in the KXKK motif of GATA-l was found to reduce its
acetylation by p300. Moreover, mutation of these identical residues in C/EBPB blocked
p300 augmentation of C/EBPB DNA-binding and transactivation, thereby implicating
these residues as potential candidates for acetylation. Therefore, increased acetylation of
wildtype C/EBPB relative to C/EBPBKK216,217RR would implicate lysines 216 and 217 as
major sites for acetylation. In addition, we could demonstrate acetylation of C/EBPB by
in vitro labeling of recombinant C/EBPB or C/EBPBm16,217RR using recombinant puriﬁed
p300 as an acetyltransferase and l4C-acetyl Coenzyme A as the donor of labeled acetate.
If the level of acetylation for C/EBPB and C/EBPBKKM 217m is similar, then identiﬁcation
of the authentic acetylation substrate would be necessary. Rather than performing
mutagenesis of every lysine residue within the bZIP of C/EBPB, we would use mass
spectrometry to identify potential acetylation sites. In all of the above described
experiments, use of expression vectors and/or p300AHAT and GCN5 recombinant

proteins could directly test the role of GCN5 in acetylation at lysines 216 and 217. The

171

use of other C/EBPB mutants in the KXKK motif with P/CAF reagents could test the
potential role of P/CAF at this site.

The fact that p300 HAT activity was dispensable for augmentation of C/EBP0213-297
DNA-binding activity suggests that C/EBPB may be acetylated by additional acetylases,
such as GCN5 and P/CAF, which are recruited by p300. While C/EBPBmw mm; was
not subject to p300 enhancement of DNA-binding activity, its DNA-binding activity
could still be attenuated by P/CAF, which suggests that P/CAF regulates C/EBPB DNA-
binding through the acetylation of alternative lysines. Mass spectrometry of in vitro-
acetylated C/EBPB could identify candidate lysines for mutagenesis studies, where we
would generate various lysine to arginine substitutions in the bZIP of C/EBPB that would
speciﬁcally block the activity of either GCN5 or P/CAF. In addition, it would be
interesting to determine whether the differential impact on C/EBPB DNA-binding
facilitated by GCN5 and P/CAF in P388 cells correlates with differences in C/EBPB
activity on the IL-6 and MCP-l promoter. GCN5 cooperation with p300 was previously
shown to enhance the transactivation potential of C/EBPB on the 2X-C/EBP promoter-
reporter. To establish functional differences between GCN5 and P/CAF, P388 cells
would be transiently co-transfected with the 2X-C/EBP promoter-reporter and a vector
for C/EBPB expression in the presence and absence of an expression vector for P/CAF.
If the activity of P/CAF were in accordance with its impact on C/EBPB DNA binding,
one would expect a reduction in C/EBPB transactivation of the promoter-reporter. This
type of experiment would also be performed with the IL-6 and MCP-l promoter-reporters
comparing the effects of P/CAF to p300 plus GCN5 expression. In addition, the

importance of GCN5 and P/CAF activity in C/EBPB-mediated activation of the

172

endogenous IL-6 and MCP-l promoters could be tested by attempting to abolish the
expression of either GCN5 or P/CAF using small interfering RNA duplexes (siRNA) in
P3 88 cells stably transduced for C/EBPB expression and assessing the effect on LPS— and
IL-1 B-induced IL-6 and MCP-l expression. If GCN5 were important for the stimulation
of IL-6 and MCP—l gene expression, one would expect a reduction in IL-6 and MCP-l
transcription with the inhibition of GCN5 expression, while the inhibition of P/CAF
expression would potentially enhance IL-6 and MCP-l expression. It should be noted,
however, that GCN5 and P/CAF may play important roles in the regulation of factors
other than C/EBPB that are critical for IL-6 and MCP-l transcription. Perhaps more
importantly, the expression of transcriptional co-activators is critical for the normal
function of a variety of vital cellular processes. Therefore, abolishing either GCN5 or
P/CAF expression may impact a number of cellular processes aside from IL-6 and MCP-
1 regulation, which would clearly complicate the interpretation of any data generated.
Finally, acetylation seems to play an important role in regulating C/EBPB protein
levels, perhaps through protein turnover, as p300 cooperation with GCN5 elevated
C/EBP0213-297 protein levels in P388 cells. Furthermore, mutation of lysines 216 and 217
of C/EBPB reduced its protein expression relative to wildtype C/EBPB. The acetylation
of the transcription factors E2F1 and SREBP was shown to enhance the stability of these
proteins. Interestingly, a recent report demonstrated that p300 contains intrinsic ubiquitin
ligase activity, in addition to acetylase activity. Acetylation of C/EBPB may block
protein degradation by preventing ubiquitination. The fact that C/EBP0213-297 protein
levels are modulated by the expression of acetylases suggests that potential targets for

ubiquitination are located between amino acids 213 and 297 of C/EBPB. Therefore, one

173

would predict that p300 and GCN5 expression should reduce the ubiquitination of
C/EBPB. To test this hypothesis, P388 cells would be transiently co-transfected for
expression of C/EBPB with and without expression vectors for p300 and/or GCN5. To
detect ubiquitination of C/EBPB in the presence or absence of p300 and/or GCN5, an
expression vector for F LAG-tagged-ubiquitin (F LAG-Ub) would also be co-transfected.
If p300 expression reduces ubiquitination, one would expect to see less C/EBPB
immunoprecipitated from nuclear extracts of transfectants co-expressing C/EBPB and
p300 using anti-FLAG than from nuclear extracts ﬁom cells expressing C/EBPB alone.
The acetylation and ubiquitination substrates of p53 are believed to be identical. If this
were also the case for C/EBPB, the substitution of lysines 216 and 217 for arginines
would presumably block ubiquitination, thereby increasing protein expression levels.
The fact that this mutation results in the opposite outcome suggests that acetylation of
lysines 216 and 217 may be important for blocking ubiquitination of other lysines rather
than directly competing for ubiquitination. If the above described immunoprecipitation
experiment were carried out comparing C/EBPBKKZIQZURR to wildtype C/EBPB, one
would expect to observe more C/EBPBm16,217RR immunoprecipitated from nuclear
extracts of transient transfections co-expressing C/EBPBKszgnRR and FLAG-Uh than
wildtype C/EBPB from nuclear extracts of transfections co-expressing wildtype C/EBPB
and FLAG-Ub. Not surprisingly, the C-terminus of C/EBPB contains numerous lysines
that are highly conserved within the C/EBP family that may serve as ubiquitination
substrates. Therefore, lysine mutations in this region that augment C/EBPB expression
levels would be good candidates as sites for ubiquitination of C/EBPB, and co-expression

with p300 and GCN5 should block the ubiquitination of such residues.

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