STABILITY, ACTIVITY AND GENOME-WIDE ASSOCIATION OF THE DROSOPHILA
RETINOBLASTOMA TUMOR SUPPRESSOR RBF1
By
Pankaj Acharya

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Microbiology and Molecular Genetics
2011

ABSTRACT
STABILITY, ACTIVITY AND GENOME-WIDE ASSOCIATION OF THE DROSOPHILA
RETINOBLASTOMA TUMOR SUPPRESSOR RBF1
By
Pankaj Acharya
The Retinoblastoma protein (RB) is a well known tumor suppressor that controls cell
cycle and developmentally regulated gene expression. Germ-line mutation of RB is closely
linked with retinoblastoma in early childhood and osteosarcoma in adolescence, and is mutated
in about half of all human cancers. During normal growth, interactions between E2Fs and RB
family proteins, including the Drosophila RB homolog, Rbf1 are regulated by phosphorylation
by cyclins and cyclin-dependent kinases (CDKs) and proteolytic destruction by the proteasome.
To better understand the mechanism for RB family protein instability, we characterized Rbf1
turnover in Drosophila, and the protein motifs required for its destabilization. We show that
specific point mutations in a C-terminal instability element (IE) stabilized Rbf1, but sacrifice
repression activity. Rbf1 is destabilized especially in actively proliferating tissues of the larva,
indicating that controlled degradation of Rbf1 is linked to developmental signals. The positive
linkage between Rbf1 activity and its destruction indicates that the instability and activity
relation is similar to that observed in the case of transcriptional activators such as VP16 and
Myc.
Physical and functional targets of RB and its paralogs p107/p130 have been studied
largely in cultured cells, but the full biological context of this family of proteins’ activities will
likely be revealed only in whole organismal studies. To identify direct targets of the major
Drosophila RB counterpart in a developmental context, we carried out ChIP-Seq analysis of
Rbf1 in the embryo. The association of the protein with promoters is developmentally controlled;

early promoter access is globally inhibited, while later in development Rbf1 was found to
associate with promoter-proximal regions of approximately 2,000 genes. In addition to the
conserved cell cycle-related genes, a wholly unexpected finding was that Rbf1 targets many
components of the insulin, Hippo, JAK/STAT, Notch and other conserved signaling pathways.
Rbf1 may thus directly affect output of these essential growth-control and differentiation
pathways by regulation of receptor, kinase and downstream effector expression. Rbf1 was also
found to target multiple levels of its own regulatory hierarchy. Bioinformatic analysis indicates
that different classes of bound genes exhibit distinct promoter motifs, suggesting that the context
of Rbf1 recruitment involves diverse transcription factors, which may allow for independent
regulation of Rbf1 bound genes. Many of these targeted genes are bound by Rbf1 homologs in
human cells, indicating that a conserved role of retinoblastoma proteins may be to adjust the set
point of interlinked signaling networks essential for growth and development.

To my Dad, Mr. Dhruba Prasad Acharya, my Mom, Mrs. Kopila Acharya,
my wife, Kunjana Acharya
my son, Anshuman Acharya

AND

To my mentor, Dr. David N. Arnosti

iv 
 

ACKNOWLEDGEMENTS

This thesis is by far the most significant scientific achievement of my entire life and it
would not have been possible without the support of people who believed in my success. I would
like to express my sincere gratitude to my mentor Dr. David Arnosti, for his enthusiasm,
patience, support, and exceptional guidance during my graduate study. I appreciate his unique
style of inspiration and challenge to become a better and positive thinker, a better orator, a better
researcher and a cheerful person. I would have been lost without him. I am also grateful to my
co-advisor and committee member Dr. Bill Henry for his continuous support and regular
discussions. I would like to thank my committee members Dr. Robert Britton, Dr. Michele
Fluck, Dr. Susan Conrad and Dr. Kyle Miller for their insightful ideas and constructive
comments that helped me shape my research and this thesis.
I thank Dr. Bob Hausinger for his advice. I am thankful to all the past (Walid Fakhouri,
Yang Zhang, Li Li, and Ahmet Ay) and present (Sandhya Payankaulam, Rupinder Sayal, Jackie
Dresch, Liang Zhang, Kurtulus Kok, Yiliang Wei, Rewatee Gokhale, and Anne Sonnenschein)
for making Arnosti lab an excellent place to work with their insights, helpful discussions and
mental support. I am also thankful to Nitin Raj and Satyaki Sengupta for their inputs in my
project. I am greatly indebted to Dr. Krishna Mohan Shrestha (and family) for supporting me as
an elder brother during my initial years in the USA. I am also thankful to all my friends here in
USA and back in Nepal for being there in good and bad times.
Finally I would like to express my deepest gratitude to my dad Dhruba Prasad Acharya
and my mom Kopila Acharya for their love, constant support and belief in education. I also thank

v 
 

my sisters Shailu and Kanchan for their love and support. Finally, I don’t have enough words to
thank my wife Kunjana for her support, patience, understanding, encouragement and
unconditional love thoughout my career as a graduate student. Last but not least, I would like to
thank my little one, Anshu, for bringing greatest joy in my life by always being there with a
smile when I returned home tired.

vi 
 

TABLE OF CONTENTS
LIST OF TABLES………………………………………………………………………………ix
LIST OF FIGURES……………………………………………………………………………...x
KEY TO ABBREVIATIONS………………………………………………………………….xii
CHAPTER I
Introduction
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
X.

Introduction…………………………………………………………………….....1
The Retinoblastoma family of proteins or the ‘pocket’ proteins………………....2
Tissue expression patterns of the Retinoblastoma family of proteins……………5
Regulation of the pocket proteins………………………………………………...6
Role of retinoblastoma proteins in differentiation and development……………10
Role of retinoblastoma proteins in tumorigenesis……………………………….13
Mechanisms of retinoblastoma protein-mediated transcriptional repression……13
Link between transcriptional regulation and turnover of transcription factors….18
Promoter occupancy of pRB family proteins……………………………………20
Involvement of RB family proteins in regulation of conserved signaling
pathways…………………………………………………………………………24
References……………………………………………………………………………………….32
CHAPTER II
Paradoxical instability-activity relationship defines a novel regulatory pathway for
Retinoblastoma proteins
Abstract…………………………………………………………………………………..51
Introduction………………………………………………………………………………52
Materials and Methods…………………………………………………………………...55
Results……………………………………………………………………………………60
Discussion………………………………………………………………………………..90
References………………………………………………………………………………..94
CHAPTER III
Evidence for autoregulation and cell signaling pathway regulation from genome-wide
binding of the Drosophila retinoblastoma protein.
Abstract…………………………………………………………………………………..99
Introduction……………………………………………………………………………..101
Results…………………………………………………………………………………..104
Discussion………………………………………………………………………………150
Materials and Methods………………………………………………………………….156
References………………………………………………………………………………165

vii

CHAPTER IV
Conclusions and Future Perspectives
Evidence of linkage between turnover and repression activity of Rbf1………………..172
Targeting of various conserved signaling pathways by Rbf1…………………………..174
E2F-independent regulation of target gene promoters by Rbf1………………………..176
References………………………………………………………………………………………178
APPENDIX A
Nuclear localization of Rbf1 mutants………………………………………………………...182
References………………………………………………………………………………………188
APPENDIX B
Generation of epitope tagged CSN genomic rescue constructs…………………………….190
Materials and Methods…………………………………………………………………………190
References……………………………………………………………………………………...192

viii

LIST OF TABLES
Table 2-1:

Rbf1 repression, stability, and localization………………………………………66

Table 2-2:

rbf14 rescued by transgenic Rbf1…………………………………………………76

ix

LIST OF FIGURES
Figure 1.1:

Schematic drawings of Drosophila and human retinoblastoma family proteins. ...3

Figure 1.2:

Schematic drawing of insulin signaling pathway………………………………..25

Figure 1.3:

Schematic drawing of Notch signaling pathway……….………………………..28

Figure 1.4:

Schematic drawing of Hippo signaling pathway………………………………...29

Figure 2.1:

Identification of an instability element (IE) in Rbf1……………………………..61

Figure 2.2:

Conserved lysine residues in IE play critical roles in accumulation and stability of
Rbf1………………………………………………………………………………69

Figure 2.3:

Expression of wild-type and IE mutant forms of Rbf1 in the Drosophila larva…71

Figure 2.4:

Rbf1 requires the IE for transcriptional repression………………………………74

Figure 2.5:

Rbf1 IE is not essential for E2F interactions and promoter binding…………….79

Figure 2.6:

Rbf1 IE harbors positive and negative regulatory elements……………………..84

Figure 2.7:

Severe developmental consequences of expression of hyperactive Rbf1……….86

Figure 2.8:

Mutations in the conserved IE of p107 enhance expression……………………..89

Figure 3.1:

Rbf1 exhibits dynamic promoter occupancy…………………………………...105

Figure 3.2:

Rbf1 promoter-proximal occupancy of diverse classes of genes suggests
autoregulatory effects…………………………………………………………...112

Figure 3.3:

Rbf1 occupies multiple nodes in conserved signaling pathways……………….115

Figure 3.4:

Rbf1 exhibits a strong promoter-proximal targeting bias………………………122

Figure 3.5:

Rbf1 target genes represent diverse Gene Ontology (GO) categories………….125

Figure 3.6:

Transcription factor motifs enriched in Rbf1-bound peaks…………………….127

Figure 3.7:

Enrichment of Rbf1-associated motifs indicates distinct promoter subclasses in
the Rbf1 regulon………………………………………………………………..135

Figure 3.8:

Promoter occupancy by Rbf1 correlates with downregulation of target genes...141

Figure 3.9:

Divergence and conservation of RB regulon…………………………………...146
x

Figure A.1:

Nuclear localization of Rbf1 mutants…………………………………………..183

LIST OF SUPPLEMENTARY FIGURES

Supplementary Figure 2.1:

Effect of proteasome inhibitor MG132 on endogenous Rbf1
protein levels……………………………………………………..64

Supplementary Figure 3.1:

Enrichment of Rbf1-bound promoters peaks at 12-18 hr………107

Supplementary Figure 3.2:

Validation of selected promoters for Rbf1 occupancy…………108

Supplementary Figure 3.3:

Validation of specificity of Rbf1 antibodies……………………110

Supplementary Figure 3.4:

Repression of InR and Rab23 promoters by Rbf1……………...120

Supplementary Figure 3.5:

Determination of threshold for motif analysis………………….130

Supplementary Figure 3.6:

Prevalence of E2F-, DREF- and RAM-like motifs on Rbf1- bound
and not bound promoter regions………………………………..131

Supplementary Figure 3.7:

E2F responsiveness of InR promoter…………………………...138

xi

KEY TO ABBREVIATION
Akt

Protein Kinase B

AR

Androgen Receptor

ATP

Adenosine Triphosphate

BMI1

B lymphoma Mo-MLV insertion region 1 homolog

BRG1

BRM-related Gene product 1

BRM

Brahma

CBP

CREB Binding Protein

CDK

Cyclin Dependent Kinase

CDKN2A

Cyclin Dependent Kinase Inhibitor 2A

CHD

Chromodomain Helicase DNA-binding

ChIP

Chromatin Immunoprecipitation

CHK2

Checkpoint Homolog 2

CMV

Cytomegalovirus

COP9

Constitutive Photomorphogenic 9

CSN

COP9 Signalosome

DCB

Dihydrocytochalasin B

dILP

Drosophila Insulin-like Peptides

DNA

Deoxyribonucleic Acid

DNMT1

DNA Methyltransferase 1

DNMT3a

DNA Methyltransferase 3a

DNMT3b

DNA Methyltransferase 3b

DP

E2F Dimerization Partner

xii

dREAM

DP, RB-like, E2F and MuvB complex

E(bx)

Enhancer of bithorax

E1A

Early region 1A

E1

Ubiquitin activating enzyme 1

E2

Ubiquitin activating enzyme 2

E3

Ubiquitin activating enzyme 3

E2F

Adenovirus E2 promoter binding factor

EBNA3C

Epstein-Barr virus nuclear antigen 3C

EZH2

Enhancer of Zeste 2

FOXO

Forkhead box-containing protein, O subfamily

GR

Glucocorticoid receptor

HCC

Hepatocellular carcinoma

HDAC

Histone Deacetylase

HPV E7

Human Papilloma Virus Early 7

IE

Instability Element

InR

Insulin Receptor

IRS

Insulin Receptor Substrate

ISWI

Imitation Switch

Ken

Ken and Barbie

L

Lobe

l(3)mbt

lethal (3) malignant brain tumor

L3MBTL1

lethal (3) malignant brain tumor like 1

LOH

Loss of Heterozygosity

xiii

MBD

Methyl-CpG-binding domain

MDM2

Murine double minute 2

MEFs

Mouse Embryonic Fibroblasts

Nedd4

Neural precursor cell-expressed developmentally down regulated gene 4

Nedd8

Neural precursor cell-expressed developmentally down regulated gene 8

NICD

Notch Intracellular Domain

NURF

Nucleosome Remodeling Factor

P/CAF

p300/CBP Associated Factor

PCNA

Proliferating Cell Nuclear Antigen

PDK1

Phosphatidylinositol-Dependent Kinase 1

PI3K

Phosphatidylinositol 3-Kinase

PIC

Pre-Initiation Complex

PIP2

Phosphatidylinositol-4,5-biphosphate

PIP3

Phosphatidylinositol-3,4,5-triphosphate

PP1

Protein Phosphatase type 1

PP2

Protein Phosphatase type IIA

PPAR

Peroxisome Proliferator-Activated Receptor

PRC1L

Polycomb-repressive complex-1-like

PRC2

Polycomb-repressive complex-2

PTEN

Phosphatase and Tensin Homolog

Pzg

Putzig

RB, pRB

Retinoblastoma Protein

Rbf1

Retinoblastoma Factor 1

xiv

Rbf2

Retinoblastoma Factor 2

RBP1

Retinoblastoma Binding Protein 1

Rheb

Ras-Homolog Expressed in Brain

RNA

Ribonucleic Acid

S2 cells

Schneider 2 cells

S6K

p70 ribosomal S6 Kinase

SCF

Skp/Cullin/F-box complex

SIK2

Salt Induced Kinase

SIRT1

Sirtuin 1

SMYD2

SET and MYND domain containing 2

Su(dx)

Suppressor of deltex

SUV39H1

Suppressor of variegation 3-9 homolog 1

Suv4-20h1

Suppressor of variegation 4-20 homolog 1

SV40

Simian Virus 40

SWI/SNF

Switch/Sucrose nonfermenting complex

Tip60

TAT interacting protein 60

TKO

Triple Knock Out

TOR

Target of Rapamycin

TSC

Tuberous Sclerosis Complex

TSS

Transcriptional Start Site

UTX

Ubiquitously transcribed tetraticopeptide repeat, X chromosome

VP16

Virion Protein 16

xv

CHAPTER I
INTRODUCTION
I. Introduction
Named after its role in pediatric retinoblastoma, the Retinoblastoma protein (pRB) is a
tumor suppressor that controls transcription of cell cycle specific and developmental genes, and
thereby regulates cell cycle, differentiation, apoptosis and growth [1-6]. RB1, the gene encoding
this protein, was the first tumor suppressor gene to be cloned [7, 8]. Germline mutation of RB1 is
associated with retinoblastoma in childhood [9] and osteosarcoma in adolescence [10] and is one
of the two most mutated genes in human cancers [11]. The E2F family of transcription factors
recruit pRB to the target gene promoters, thereby repressing their transcription [12, 13], and
controlling the cell cycle. pRB’s cell cycle control is frequently disrupted in wide variety of
cancers [11, 14-16]. Though less complex than its human counterpart, the Drosophila
retinoblastoma network is conserved [17]. We utilized this less complex network to study the
structure and function of Rbf1, the Drosophila ortholog of pRB, and its genome wide association
with target gene promoters. In this study I describe research that has contributed to our
understanding of pRB regulation in two important areas. First, we show that a degron, which we
named IE (instability element), is present in the C-terminal region of Rbf1, and that the IE is also
responsible for its activity on certain promoters. Second, I show that Rbf1 targets conserved
signaling pathway genes that were previously not known to be regulated by this tumor
suppressor protein, suggesting widespread involvement of cell-cycle links to central
developmental signaling events.

1

II. The Retinoblastoma family of proteins or the ‘pocket’ proteins
The Retinoblastoma protein (pRB) is a transcriptional corepressor that regulates a wide
variety of cellular processes such as cell cycle, growth, differentiation, and apoptosis. The
negative regulation of cell cycle is most well studied [1-4, 6, 18]. Apart from pRB, there are two
other closely related proteins in mammals, p107 (or RBL1) and p130 (or RBL2). These three
proteins are collectively also known as “pocket proteins” because of a conserved pocket region
that consists of two conserved A and B subdomains, with a less conserved spacer between them
[19]. The A and B domains are characterized by tandem cyclin folds [20-22], which are known
to mediate protein-protein interactions. The pocket also contains a conserved “LxCxE binding
motif” that interacts with LxCxE sequences present on several cellular proteins such as
chromatin remodelers, replication factors, transcription factors, kinases and others. Several
viruses produce proteins targeting this “LxCxE binding motif” of pRB to inactivate the tumor
suppressor for their proliferation. These proteins include the Adenovirus E1A, SV40 TAg and
HPV E7 gene products that function as viral oncoproteins to compete with cellular proteins for
binding to pRB [21, 23].
Amino acid sequence alignment reveals that p107 and p130 are more similar to each
other than either are to pRB [24]. The spacer sequence that separates the A and B boxes of the
pocket region is longer in p107 and p130 than in pRB. Furthermore, the spacer contains a
cyclin/cdk binding site in both p107 and p130 but not in pRB [25-27], which help these pocket
proteins form stable complexes with cdks.
All three pocket proteins bind to heterodimers of E2F and DP transcription factors and
control cell cycle progression. In mammals, there are nine E2Fs, E2F1-3a, E2F3b, E2F4-8 and
three DPs, DP1, DP2 and DP4 [28]. Studies have shown that p107 or p130 deficient cells show

2

Figure 1.1

Figure 1.1 Schematic drawings of Drosophila and human retinoblastoma family proteins.
Comparison of the structure of fly and human pRB family proteins is shown here. The A and B
boxes of the pocket region are shown in green and purple respectively. A putative degron that is
present in Rbf1, p107 and p130 is indicated in orange. A cyclin/CDK binding site found only in
p107 and p130 is shown in red. The amino acid residue numbers are shown above the structure
of each protein. For interpretation of the references to color in this and all other figures, the
reader is referred to the electronic version of this dissertation.

3

similar cell cycle defects as pRB deficient cells, thus it seems that to a certain extent there is
functional redundancy among these proteins [29]. Further in vivo evidence for functional
redundancy is provided by mutant phenotypes of knockout mice; the developmental defects
worsen when p107 [30] or p130 [31] deficiencies are combined with loss of pRB. Although
functional redundancies exist among these three pocket proteins, pRB is by far the most
important one with unique functions. Homozygous mutation in RB1 gene is embryonic lethal
[32], however, p107 or p130 mutant mice have normal development [33]. Although pRB, p107
and p130 bind to E2F transcription factors, they differ in preferences for E2Fs. pRB prefers to
bind E2F1, E2F2, E2F3a, E2F3b and E2F4, while p107 and p130 prefer to bind E2F3b, E2F4
and E2F5 [28]. In a pRB and E2F4 null background, however, p107 and p130 regulate E2F1-3
[34].
Comparison of pRB in human, mouse, frog and chicken, shows high (66%) amino acid
conservation in the A and B boxes of pocket region. However, the spacer region has only 33%
identity. Interestingly, a 20 amino acid sequence (SKFQQKLAEMTSTRTRMQKQ) at the Cterminus is 100% conserved in pRB in all four organisms [35]. Drosophila has two
retinoblastoma orthologs, Rbf1 and Rbf2 [36]. The overall sequence similarity puts Rbf1 closer
to p107 and p130 than pRB, however Rbf1 is more similar to pRB because of the absence of a
cyclin / cdk binding site in the spacer and sequence of the B box [37]. Rbf2 is more similar to
Rbf1 than other mammalian pocket proteins, and shows closer similarity to mammalian p107 and
p130 than pRB [36].
The RB-E2F network is much simpler in Drosophila than in mammals. There are only
two E2Fs, E2F1 and E2F2 and only one DP. E2F1 is an activator and E2F2 is a repressor. Rbf1
binds to both E2F1 and 2 whereas Rbf2 binds to only E2F2 [36]. In Drosophila, a single copy of

4

Rbf1 is sufficient for normal growth and development. Null mutants in Rbf1 are larval subviable, never reaching the late pupal stages of development [38]. However, Rbf2 null mutant
flies are viable and fertile. Mutation of Rbf2 in Rbf1 mutant background revealed that these two
fly pocket proteins have some functional redundancy [39]. Some animals have even more
reduced RB systems: C. elegans has only one pRB ortholog, lin-35. This gene is dispensable for
viability, although the animals have a multivulval phenotype. By sequence homology, lin-35 is
closer to Rbf1, p107 and p130 than pRB [40].
III. Tissue expression patterns of the Retinoblastoma family of proteins
Mammalian pRB, p107 and p130, despite their functional redundancies, are differentially
expressed in tissues and during different stages of the cell cycle. The total pRB levels are
abundant throughout the cell cycle; however the phosphorylation status changes. p130, on the
other hand, is highly expressed in quiescent and differentiated cells, but at low levels in
proliferating cells. p107 is expressed at low levels in non-proliferating cells, but the levels
increase in the proliferating cells [41-47]. These three mammalian pocket proteins are expressed
in dynamic patterns during mouse development [48, 49]. pRB transcripts are expressed during
neurogenesis, hematopoiesis, myogenesis, lens development and in the ganglion cell layer of the
embryonic retina before and during differentiation. In contrast, p107, but not pRB is expressed in
the heart, lung, kidney and intestine. However, both pRB and p107 are co-expressed in liver and
the central nervous system [49]. In the central nervous system, p107 is restricted to proliferating
cells as mentioned in the previous studies [41, 43-46]; while, pRB

is expressed in both

proliferating and differentiating cells. Interestingly, p130 transcripts are found at low levels
throughout embryogenesis [49]. The retinoblastoma family proteins are also differentially

5

regulated in the mammary luminal epithelium of mice [48]. These studies underscore the distinct
roles of pRB, p107 and p130 during development.
Similarly, the Drosophila counterparts, Rbf1 and Rbf2 are also differentially and coexpressed during development [36, 50]. Rbf1 protein levels are relatively unchanged throughout
embryogenesis and also in larvae and adult flies, however, Rbf2 levels peak between four and
ten hours of embryonic development before declining, and the protein is undetectable in adult
flies [36, 50]. Proteins and transcripts of both Rbf1 and Rbf2 are ubiquitously expressed during
early embryogenesis; however they are differentially expressed and confined to certain tissues
during later stages. Both pocket proteins are confined to epidermis, gut and central nervous
system during embryogenesis [50]. Interestingly, rbf1 and rbf2 are differentially expressed in
central nervous system [50] consistent with the distinct expression patterns of pRB and p107 in
central nervous system [49]. These studies in mice and flies showing distinct expression patterns
in developing embryo suggest that the pocket proteins play unique roles during development.
IV. Regulation of the pocket proteins
The retinoblastoma family proteins are regulated by various modifications, of which
phosphorylation is the most important one [51-53]. Hypophosphorylated forms of pRB, p107 and
p130 bind to E2F/DP and repress target genes during G0 and early G1 phase. Their
phosphorylation states are progressively increased during the transition from G1 to S phase of
cell cycle. This hyperphosphorylation causes dissociation of pocket proteins from E2F/DP,
relieving transcriptional repression [51-53]. Phosphorylation of pRB, p107 and p130 is executed
by Cyclin D/CDK4, Cyclin D/CDK6, Cyclin A/CDK2, Cyclin E/CDK2, CHK2 (checkpoint
homolog 2) and RAF-1 (c-Raf) [51]. During late mitosis, pRB is dephosphorylated by
phosphoprotein phosphatase type 1 (PP1), recycling it for regulation of subsequent cell cycles

6

[54, 55]. Another phosphatase, PP2A has been shown to dephosphorylate pRB, p107 and p130
[55-57]. Based on sequence analysis, there are 16 putative cyclin/CDK phosphorylation sites in
human pRB [58, 59]. Upon mutation of 11 out of 16 cyclin/CDK phosphorylation sites, pRB
becomes resistant to cyclin/CDK regulation, and is able to block cell proliferation in fibroblasts
and tumor derived cell lines [58, 60, 61]. Out of those 11 sites, 7 are in the C terminal region
[62]. It is hypothesized that a conformational change is created by the phosphorylation at the C
terminus. This conformational change in turn displaces histone deacetylase (HDAC) bound to
pRB and finally disrupts the E2F-pRB interaction [63]. Similarly, the Drosophila counterpart,
Rbf1 has 5 putative cyclin/CDK phosphorylation sites, four of which are in the C terminal
region. Serine/threonine to alanine point mutations of those sites rendered Rbf1 insensitive to
cyclin/CDK regulation [64]. Taken together, phosphorylation is a very important mode of
regulating the control of retinoblastoma family proteins during the cell cycle.
Another type of modification of human pRB is acetylation at lysines 873 and 874 [6567]. DNA-damage-dependent acetylation at K873/874 interferes with pRB-E2F1 interaction,
retains pRB in a hypophosphorylated state, keeps E2F bound to pRB pocket, however releases
E2F1 bound to C-terminal region and allows E2F1 activity [65]. Acetylation of the C-terminus
of pRB by p300/CBP, which is a histone acetyl transferase, blocks pRB phosphorylation by
Cyclin E/CDK2 [66]. Not only p300, but also p300-Associated Factor (P/CAF) can acetylate
pRB, resulting in pRB-mediated terminal cell cycle exit and induction of late myogenic gene
expression [67]. A recent study showed that acetylation of pRB by P/CAF is required for its
nuclear localization and keratinocyte differentiation [68]. SIRT1 (sirtuin 1) binds to pRB and
mediates NAD dependent deacetylation. This deacetylase activity of SIRT1 ensures resumption
of cell cycle when required by making pRB susceptible to cyclin/CDK mediated phosphorylation

7

[69]. Acetylation of pRB might be beneficial in differentiation and cellular stress, such as DNA
damage, to make sure cell cycle is arrested. However, an alternative pathway for pRB acetylation
is also possible: Tat interacting protein 60 (Tip60) targets pRB for proteasomal mediated
degradation by acetylating its C-terminus. p14ARF, an alternative reading frame product of the
cyclin-dependent kinase inhibitor 2A (CDKN2A) locus, retards Tip60 induced acetylation of
pRB [70]. p14ARF is a positive regulator or p53 tumor suppressor whereas p16INK4a, another
product of CDKN2A locus positively regulates pRB [71].
In addition to the above modifications, sumoylation is another type of modification in
pRB. Sumoylation is a small ubiquitin-like modification of proteins and has been shown to play
important roles in several biological processes and human diseases [72]. pRB can be sumoylated
at lysine 720, close to the LxCxE binding motif [73]. The function of sumoylation in the context
of pRB is not known, but in the case of p53 and RBP1 (retinoblastoma binding protein 1), this
modification has been implicated in induction of senescence [74, 75].
Two recent studies showed that pRB can be methylated by Set7/9[76] and SMYD2 [77].
Set7/9, a histone mono-methyltranserase, is responsible for methylation of pRB at lysine 873,
which in turn promotes transcriptional repression. K873 methylation also induces cellular
senescence and differentiation [76]. However, SMYD2 methyltransferase methylates pRB at
K860, which is in proximity with pRB acetylation sites (K873 and K874). This methylation at
K860 is elevated in G0 phase, during cell cycle exit and during first stages of differentiation.
Furthermore, SMYD2 mediated methylation promotes interaction between pRB and the
corepressor L3MBTL1, a human homolog of Drosophila tumor suppressor l(3)mbt [77]. Taken
together, methylation of pRB at different residues seems to enhance its tumor suppressor
activity.

8

A final level of regulation involves protein turnover. Some studies have shown the
importance of this event in the mammalian setting. Caspase cleaves pRB at consensus caspasecleavage site

884DEADG888

present towards the C-terminus. Absence of this site makes mice

resistant to TNFα induced apoptosis and this in turn renders the mice prone to cancer [78]. The
Ubiquitin-proteasome pathway is another major proteolytic pathway [79]. Ubiquitin is a
conserved 76-amino acid peptide, which is covalently tagged to lysine residues. The
polyubiquitylated proteins are destined for degradation [79]. pRB is ubiquitylated in vitro and in
vivo by Mdm2, a ubiquitin ligase for p53, and targeted for degradation [80]. However, besides
proteolysis, ubiquitin-tagged proteins are also involved in certain nonproteolytic cellular events,
such as nuclear localization, endocytosis and cofactor recruitment [81].
In addition to the above mentioned modifications of retinoblastoma family of proteins, in
chapter two, I describe our findings about a novel turnover-mediated regulation of Drosophila
retinoblastoma tumor suppressor Rbf1. We found a degron-containing 59-amino acid region in
the C-terminus of Rbf1 just C-terminal to the pocket’s B box. Furthermore, this region is also
critical for Rbf1’s activity. Point mutations in certain amino acid residues made the protein lose
its activity and accumulate to higher levels. We named the region as IE for instability element.
We also found single point mutations in K774 that make the protein hyperactive, suggesting that
this region has additional potential regulatory activities that do not affect protein levels. We
extended our study to the human ortholog, p107 and found that its IE is responsible for its
turnover. Our findings suggest that in addition to phosphorylation, acetylation, methylation,
sumoylation, ubiquitylation and caspase cleavage, retinoblastoma proteins might be generally
regulated by IE dependent turnover.

9

V. Role of retinoblastoma proteins in differentiation and development
A variety of developmental phenotypes underscore the roles of pRB and related proteins
in tissue differentiation processes. Ectopic expression of a genomic construct of pRB caused the
transgenic mice to be dwarf by embryonic day 15 compared to their nontransgenic littermates
[82]. Heterozygous pRB+/- mice develop normally and do not develop retinoblastoma. However,
when pRB is completely knocked out from mice, they die between embryonic day 13 and 15
with several defects [4, 32, 83, 84]. pRB-/- embryos show defects in neural, hematopoietic and
eye development that are caused by enhanced apoptosis [32, 83, 84]. In addition, pRB-/- embryos
have abnormal placenta with excessive proliferation of trophoblast cells and severe disruption of
the normal labyrinth architecture. Interestingly, wild-type placenta is sufficient to carry the pRB
deficient embryos to term, however, they don’t survive long after birth [85].
In contrast to the severe RB mutant effects, p107-/- or p130-/- mice show no apparent
phenotypes [33, 86] suggesting that either these pocket proteins are not important for
development or retinoblastoma family of proteins have overlapping functions. However, mice
with the combination of p107-/- and p130-/- mutations are born with short limbs, short snout,
reduced rib cage size and die soon after birth because of defective tracheal cartilage mediated
respiratory failure [33]. This result confirms that the members of pocket family of proteins have
overlapping functions. This notion is further supported by the fact that quiescent pRB-negative
or p130-negative cells have elevated levels of p107 [29, 33]. Furthermore, triple knockout (pRB-/,p107-/-,p130-/-) cells are resistant to senescence-inducing signals such as DNA damage, contact
inhibition, serum starvation, and they had shorter cell cycle compared to the wild type, single or
double knock out cells [87, 88]. Interestingly pRB-/- MEFs that have elevated levels of p107 are

10

sensitive to senescence-inducing signals, however, acute loss of pRB causes increase in the
number of cells entering S phase probably because there is a lag time between inactivation of
pRB and induction of p107 [89]. pRB/p107 or pRB/p130 null embryos have similar phenotypes
as pRB-/- littermates, but die a couple of days earlier. This aggravating phenotype suggests that
p107 and p130 can partially substitute for pRB during development [86, 90, 91]. Interestingly,
some phenotypes exhibited by pRB-/- mice can be partially rescued in E2F1, E2F2 and E2F3
mutant backgrounds [92-94]. The current model is that E2F1, E2F2 and E2F3 have some
redundancy and knocking out pRB causes these E2Fs to go unchecked, causing various
biological consequences. Furthermore, these three E2Fs are significant regulators of pRB
function and must be kept under control for normal development [92]. The differentiated roles of
pocket proteins can also be inferred from gene expression changes. The majority of genes
misexpressed in pRB-deficient cells include genes encoding DNA replication and cell cycle
regulatory proteins, but the genes misexpressed in p107/p130-deficient cells are mostly devoid of
DNA replication and cell cycle regulatory genes [95]. Taken together, the molecular and genetic
evidence indicates that the three mammalian pocket proteins play critical and distinct roles
during development.
Similar to the phenotype observed with the mammalian pRB, null mutants of Drosophila
rbf1 are larval lethal. The embryos do hatch into larvae, but die at early larvae stage [96].
Although Rbf1 is dispensable for early cell cycles, it plays an important role in controlling G1 to
S phase transition during development. The lethality in rbf1 null mutants is caused by the
deregulation of the cell cycle and elevation of apoptosis in embryos [38, 96]. E2F1 and E2F2 are
the transcription factors that recruit Rbf1 to the target gene promoters. E2F1 is an activator, so
removing Rbf1 from the system is thought to activate target genes, many of which are involved

11

in the cell cycle. Conversely, overexpression of E2F1 should have the same effect. Nicholas
Dyson’s group showed that overexpression of E2F1 in the fly eye causes a rough eye phenotype,
a result of disruption of eye development due to post-mitotic cells re-entering cell cycle [97].
This phenotype is further worsened by rbf1 mutation [38] which supports the notion that Rbf1
suppresses E2F1 activity. Consistent with this notion, knocking down E2F1 in an rbf1
background rescues the early larval lethality and late differentiation phenotypes [96].
Polyteny in Drosophila adds more complexity to the cell cycle and this process involves
Rbf activity. Polyteny is caused by endoreduplication cycles in many larval and adult tissues
[98]. Endoreduplication in a process involving more than one round of DNA replication without
cytokinesis that leads to polyploidy. Giant chromosomes in Drosophila salivary glands are well
known examples of result of endoreduplication [99]. Drosophila follicle cells undergo
endoreduplication cycles to reach 16n ploidy. This is important for localized amplification of the
chorion gene cluster, which is necessary for eggshell formation. Endoreduplication in follicle
cells is regulated by Rbf1/E2F; in absence of Rbf1 or E2F or in presence of mutant E2F that
cannot recruit Rbf1, the cells reach 32 ploidy [100].
Eye-specific overexpression of Rbf1 causes rough and small eye phenotypes. Rbf1
mutant proteins that are resistant to cyclin/CDK phosphorylation worsen the eye phenotypes
[64]. In chapter II, I show that similar severe phenotypes are induced by a single point mutation
towards the C-terminus makes Rbf1 hyperactive. Interestingly, rbf2 mutant flies are viable and
fertile and they only exhibit a phenotype in a background with reduced expression of rbf1 [39].
This result suggests that Rbf1 and Rbf2 may have functionally redundant roles in development,
similar to the mammalian counterparts. Molecular studies support this overlapping function;
Rbf1 and Rbf2 co-occupy many of the same genes, and a knockdown study in Drosophila S2

12

cells, showed that some target genes show significant perturbations only when both proteins are
knocked down [101].
VI. Role of retinoblastoma proteins in tumorigenesis
Germline mutation of pRB is closely linked with retinoblastoma in early childhood [9]
and osteosarcoma in adolescence [10] and is one of the two most mutated genes in a variety of
sporadic and familiar human cancers [11]. Mutations in p107 or p130 were not observed in a
recent genome wide study of somatic copy-number alteration in a wide variety of human cancers
[102]. Although deletions of chromosomal regions containing p107 or p130 have been reported
in cancers, these large deletions contain many other genes that may also play a role in
tumorigenesis [102-105]. Further experimental evidence suggests that pRB is a potent tumor
suppressor but p107 and p130 are not because mutations in pRB cause tumors to develop in
chimeric mice but p107, p130 or p107-/-;p130-/- mutant mice do not show any tumors [33, 86,
106]. However, p107 or p130 mutations in pRB null background worsen the tumor phenotype
compared with pRB null mutation alone [86, 90, 91, 107-110], which indicates that they may act
as tumor suppressor in certain contexts. In Drosophila, overexpression of the Notch ligand Delta
in the eye causes enlarged eye phenotypes, which when coupled with activation of two
Polycomb group proteins, Pipsqueak and Lola, induces metastatic tumors. This induction of
metastatic tumors is associated with silencing of the rbf1 gene by these Polycomb group
epigenetic silencers [111]. This study corroborates Rbf1’s role as a tumor suppressor because its
silencing causes metastatic tumors in flies.
VII. Mechanisms of retinoblastoma protein-mediated transcriptional repression
The retinoblastoma tumor suppressor protein is a transcriptional co-repressor that
regulates cell cycle, differentiation, apoptosis and growth [1-6]. pRB and related pocket proteins

13

interact with DNA indirectly by binding sequence specific transcription factors, in particular the
E2F family of transcription factors [28]. Bound to target genes, these corepressors use a variety
of mechanisms to repress gene expression. One pathway involves direct antagonism of E2F
protein activation. These corepressor proteins can physically bind to and block the C-terminal
transactivation domain of E2Fs [112-115]. However, a R661W mutant pRB protein that is
defective in binding E2F [2, 116] still retains some growth repressive activity in cell culture
[117], which suggests that retinoblastoma tumor suppressor’s interaction with E2F is not the only
mechanism utilized to regulate transcription and cell growth. pRB does not act solely by
blocking E2F activity; when it is tethered to a promoter, the corepressor can repress reporter
gene activity independent of E2F [118].
This activity reflects the ability of retinoblastoma family proteins to mediate active
repression of transcription by recruiting histone modifiers such as histone deacetylases and
methylases [119-124]. Covalent modification of lysine residues on histones N-terminal tails with
acetyl group is critical for gene activation [125]. This modification neutralizes the positive
charge of histones causing the DNA to unwrap, hence opening the promoter for transcriptional
initiation. This action is reversed by removal of the acetyl moiety by histone deacetylase
(HDAC) complexes [126]. Because histone deacetylation causes the chromatin to compact,
restricting access to transcription factors, it is considered to be a hallmark of repressed chromatin
[127, 128]. Several groups have shown association of HDACs with mammalian retinoblastoma
family proteins, and shown that these enzymes are recruited to promoters by pocket proteins to
repress the target genes [119, 120, 127, 129-131]. In Drosophila, Rpd3, an HDAC, was shown to
be associated with dREAM complex, a 700 kDa multi-subunit repressor complex containing nine
proteins including Rbf, E2F2 and DP [132, 133]. Consistent with the interaction of

14

retinoblastoma proteins and HDACs, E2F target gene promoters are hypoacetylated during early
G1 phase [134] when pRB family members are actively silencing these target genes.
Modification of certain lysine (and sometimes arginine) residues of histones with methyl
group causes the genes to be either activated or repressed. H3K4, H3K36, H3K79, H3R2,
H3R17, H3R26 and H4R3 methylation are the mark of activation and H3K9, H3K27, and
H4K20 are the hallmarks of heterochromatin [125, 135]. SUV39H1 is a histone methylase that
specifically methylates H3K9 [136], which helps recruit HP1 [137], a mark of silenced
chromatin. pRB associates with SUV39H1 and HP1 on target gene promoters [122]. Loss of all
three pRB, p107 and p130 showed increased acetylation of histone H3 and decreased
trimethylation of H4K20 globally [138]. In the same study, the authors showed that the
mammalian pocket proteins associate with the H4K20 tri-methylating enzymes Suv4-20h1 and
Suv4-20h2, suggesting a role of pRB family proteins in H4K20 tri-methylation and promotion of
heterochromatin formation [138]. Methylation reactions are also linked to the activity of the
Polycomb complex proteins [139]. Polycomb proteins are critical for development and mutations
in these proteins cause developmental defects leading to homeotic phenotypes [140, 141]. There
are two Polycomb-repressive complexes (PRCs), PRC1 and PRC2. Studies show that the PRC2
recruits the PRC1 to promoters of target genes [142, 143]. pRB family proteins recruit Polycomb
genes BMI1 (a subunit of PRC1L) and EZH2 (a catalytic subunit of PRC2) to the promoter of
p16 gene causing repression of p16 gene expression through H3K27 tri-methylation by PRC2
and ubiquitination of H2A by PRC1L ubiquitin ligase complex [144]. In summary, these results
suggest that the retinoblastoma tumor suppressor proteins use diverse chromatin modifying
enzymes to effect gene silencing.

15

In mammals, pRB is involved not only in histone methylation but also in DNA
methylation. DNA methylation occurs at CpG dinucleotides and is essential for normal
development, and frequently perturbed in disease[145]. Methylation of cytosine is carried out by
DNMT1, DNMT3a and DNMT3b [146-148], and this modification of the DNA provides a
binding site for proteins possessing the methyl-CpG-binding domain (MBD). These proteins
interact with HDACs, histone methyl transferases, and corepressors such as Sin3A to silence
genes [149]. E2F target genes are known to be silenced via promoter hypermethylation in tumors
and during terminal differentiation [145, 150-155]. Interestingly, DNMT1 co-purifies with pRB,
E2F and HDAC1, and these proteins can synergistically repress transcription of a reporter gene
[156].
RB corepressors are also found to interact with SWI/SNF remodeling complexes. These
complexes play extensive roles in the regulation of gene expression. Unlike the above-mentioned
chromatin modifying proteins, chromatin remodeling complexes hydrolyze ATP to move
nucleosomes along DNA and exchange or remove nucleosomes [157]. Originally identified in
yeast, SWI/SNF complexes are evolutionarily conserved and orthologs have been reported in
flies, plants and mammals [157-159]. There are four chromatin remodeling complex families:
SWI/SNF, INO80, ISWI and CHD [157], which are suggested to act by either a “twist-diffusion”
model, in which the changes in DNA twist are propagated from linker DNA into the nucleosomal
DNA, or “reptation bulge” mechanism whereby a portion of the nucleosomal DNA is displaced
away from the histone core, to form a “bulge” which can then be propagated [160, 161]. In either
case, the alterations in chromatin structure generated by chromatin remodeling complexes
activate or repress gene transcription [162]. pRB, p107 and p130 physically associate with

16

SWI/SNF complexes containing either Brahma (BRM) or BRM-related gene product (BRG1)
ATPases, which repress E2F-responsive genes and induce cell cycle arrest [52, 163-166].
These interactions are conserved in Drosophila; and E2F1 genetically interacts with the
components of BRM complex brahma, moira and osa [167]. Furthermore, BRM complex
physically interacts with Rbf1 [168]. In addition, Rbf containing transcriptional repressor
complex, dREAM, is shown to be associated with nucleosome remodelers such as ISWI and
NURF301 [132] , another ATP dependent chromatin remodeler [169].
An additional level of RB activity is represented by interactions with general
transcription factors. Activation of gene expression involves formation of a stable pre-initiation
complex (PIC), in which co-activators recruit histone and nucleosome remodelers to promoter
regions, facilitating the recruitment of RNA polymerase II and the general transcription factors to
form the PIC [170]. The PICs are believed to be assembled in a step-by-step manner. First
TFIID binds to TATA element followed by binding of TFIIA, TFIIB, TFIIF and RNA
polymerase at transcription initiation site [171, 172]. pRB family proteins can directly disrupt the
assembly of transcriptional initiation complexes by blocking the formation of TFIID-TFIIA
complex [173]. pRB is also known to interfere with RNA polymerase III mediated transcription.
TFIIIC complex binds to promoters of classes 1 and 2 genes, and then recruits TFIIIB. TFIIIB is
an initiation factor that recruits RNA polymerase III. pRB disrupts the TFIIIC-TFIIIC interaction
and prevents the formation of PIC [174-176]. However, pRB and RNA polymerase II co-occupy
promoters of class 3 genes such as U6 snRNA, suggesting that it might not interfere with the
formation of PIC [177].
In summary, pRB and Rbf1 regulate target genes by diverse means, including direct
inhibition of E2Fs, recruitment of histone modifying proteins, recruitment of nucleosome

17

remodelers and preventing formation of PIC through interference with the basal transcription
machinery.
VIII. Link between transcriptional regulation and turnover of transcription factors
Transcription factors that regulate cell growth, such as Myc, p53, Jun, Fos and E2F are
unstable proteins [178-183] that are turned over through the ubiquitin proteasome pathway [184].
This pathway is one of two major intracellular proteolytic pathways in the cells [79]. Ubiquitin
mediated protein turnover involves a three reaction cascade [185]. In the first reaction, the
ubiquitin activating enzyme, E1, hydrolyses ATP and binds ubiquitin to form a complex of E1ubiquitin. Then, E1 transfers the activated ubiquitin to one of several E2 conjugating enzymes.
The E2-ubiquitin then forms a complex with an E3 ubiquitin ligase and a substrate protein,
which is ubiquitylated at one or more lysine residues to generate a polyubiquitylated substrate.
The polyubiquitylated substrate is targeted to the 26S proteasome, where it is deubiquitylated.
The ubiquitins are recycled and the protein is degraded [185].
For transcriptional activity of the activators such as p53, β-Catenin, Rpn4, Glucocorticoid
receptor (GR), c-Jun, Hif1α, VP16, Myc, Gcn4, Ste12, Androgen receptor (AR), Estrogen
receptor, Gal4 and the Notch intracellular domain, proteolytic destruction is required for full
transcriptional activity [184, 186, 187]. Interestingly, the transcriptional activation domains and
degrons overlap in most of these unstable transcriptional activators [188]. Although the
mechanism of how degradation and transcriptional activation are linked, a model proposed by
Muratani and Tansey suggests that, when activators interact with general transcription
machinery, they recruit ubiquitin ligases to the site of transcription. These ubiquitin ligases then
ubiquitylate several factors such as the activators, RNA polymerase II and histones, which in

18

turn recruit the 26S proteasome. This results in turnover of activators and promotion of
transcriptional elongation by RNA pol II [188].
Although established for activators, this instability-activity link has not been established
for any repressors. In chapter II I discuss our findings that suggest there is an instability element
(IE) in the C-terminus of Rbf1 that contains a degron and is critical for activity of the repressor.
We also note that the instability mechanism is conserved in the p107 human homolog. This
finding suggests that the turnover of Rbf1 is required for its activity and that the instabilityactivity relationship holds for repressors as well as activators, possibly representing a general
property of these proteins in multicellular organisms.
RB instability is not only linked to activity, but is also regulated by diverse mechanisms.
pRB is targeted to ubiquitin-dependent or ubiquitin-independent proteasome mediated turnover
by several cellular and viral proteins [189]. MDM2 is a cellular oncoprotein and E3 ubiquitin
ligase for p53 that is overexpressed in many human cancers [190], leading to p53 degradation in
ubiquitin-proteasome pathway. Interestingly, David Livingston’s group has shown that MDM2
also interacts with pRB [191]. Further study showed that MDM2, like viral oncoproteins, binds
to hypophosphorylated pRB and the interaction blocks pRB-E2F binding, hence suppressing
pRB’s repression activity [192]. MDM2 interacts with C8 subunit of 20S proteasome and
promotes pRB-C8 interaction [193]. Two different groups have demonstrated that MDM2 targets
pRB to ubiquitin-dependent [80] and ubiquitin-independent

[193] proteasome mediated

turnover. Another cellular oncoprotein, gankyrin, is shown to interact with pRB and promote
proteasome mediated turnover [194].
In addition to these cellular pathways, viral proteins have dramatic effects on RB
stability. E7 viral oncoprotein of human papilloma virus (HPV) binds to the LxCxE binding

19

motif of pRB [195] targeting the protein to the ubiquitin-proteasome pathway [196, 197].
Another viral oncoprotein, human cytomegalovirus (CMV) pp71, is shown to promote
degradation of pRB, p107 and p130 via ubiquitin-independent but proteasome-dependent
pathway [198]. Epstein-Barr virus nuclear antigen 3C (EBNA3C) destabilizes pRB by forming a
complex with pRB and SCF ubiquitin ligase [199]. Hepatitis C virus NS5B is also shown to
interact with and down-regulate pRB [200]. Similar to pRB, p107 and p130 are also inactivated
through proteasomal degradation by simian virus large T antigen (SV40 LTAg) [201]. All these
above viral oncoproteins utilize LxCxE motif to bind to pRB family of proteins to target them for
turnover. These studies demonstrate that regulation of pRB family protein levels is important for
normal cellular growth, but their elevated turnover promotes tumorigenesis.
IX. Promoter occupancy of pRB family proteins
There are two forms of cell cycle, canonical and noncanonical. The noncanonical cell
cycle involves DNA-synthesis (S-phase) followed by mitosis (M-phase) and in canonical cell
cycle, S- and M-phases are separated by two gap phases, G1 and G2. The noncanonical forms of
embryonic cell cycle are common during development of organisms requiring rapid
embryogenesis, such as insects, amphibians and marine invertebrates [202]. In Drosophila, the
first thirteen cell cycles are synchronous and consist of S-M cycles driven by maternal
components that are independent of zygotic transcription. G2 phase appears in cell cycle fourteen
and its length is regulated by developmental control. After completion of cell cycle sixteen, cells
in different tissues undergo different forms of cell cycle, for example, epidermal cells exit cell
cycle, whereas the cells in nervous system continue S-G2-M cycles. G1 phase is introduced in
the seventeenth cell cycle [202]. Another variant of the cell cycle, endoreduplication (in which
mitosis is not followed by cytokinesis), is common in many larval and adult tissues. Many

20

organisms contain some tissues with polyploid or polytene chromosomes, which is required for
high metabolic activity and are a result of repeated endo cycles [98, 202]. Endo cycles and origin
of replication in follicle cells are regulated by Rbf1/E2F in Drosophila suggesting a role of Rbf1
in regulating origin of replication activity, an S-phase event, and separate from its role as
transcriptional corepressor [100].
pRB family of proteins are also required in tissue- and stage-specific manners, as seen in
studies of different metazoan pRB family members. Although the protein is expressed in the
early embryo, the Drosophila Rbf1 protein is not functionally required for early cell cycle
regulation [36, 38, 50]. In the mouse, early embryonic requirements for pRB are restricted to the
trophectoderm, although the protein is expressed in other tissues [85]. The C. elegans lin-35
(pRB homolog) mutant shows a largely nonoverlapping set of genes that are misregulated in
embryo, L1 and L4 larvae suggesting that the protein targets distinct genes in different
developmental stages [203]. These and other studies emphasize that the multifarious functions of
this protein family will require global studies in a developmental setting. A major objective
along these lines is the identification of functional and physical target genes of pRB family
corepressors.
pRB family of proteins are best known to regulate genes involved in cell cycle regulation,
such as PCNA and DNA polymerase α. However, several studies have shown that the pocket
proteins are also involved in regulation of other pathways, such as apoptosis, DNA repair,
differentiation and development [91, 203, 204].

The diversity of pRB family-mediated

regulation in a developmental context indicates that the regulation of pRB proteins may also be
complex. We know, for instance, that Rbf1 is not involved in early cell cycle regulation in the
embryo, despite the physical presence of the protein [36, 50]. One way to develop a better

21

understanding of how Rbf1 may be differentially regulated is to study target occupancy on a
genome-wide level. It is well known that Rbf1 (or other pRB proteins) binds to target gene
promoters via E2F to execute repression. Nicholas Dyson’s group looked at the presence of Rbf1
at various Rbf1 target genes in S2 cell culture by transcriptomic analysis. Based on up or down
regulation of E2F1 or DP or E2F2 knockdown background, the genes were classified into A
through E genes [101]. Physical and functional targets of the MMB/dREAM complex, with
which Rbf1 is also associated, were identified in Drosophila Kc cells by chromatin
immunoprecipitation (ChIP) followed by tiling array analysis [205]. These studies suggest that
Rbf1, and to a lesser extent Rbf2, interact with distinct classes of genes that show varying
sensitivity to loss of Rbf and E2F proteins. Ferrari et al [206], studied genomewide temporal
occupancy of human pRB, p107 and p130 on E2F target gene promoters in adenoviral E1A
protein overexpressed human lung fibroblast cell line. The E1A protein does two important
things; first it recruits p300, pCAF and H3K18ac to the promoters of cell cycle and growth genes
causing transcriptional activation. Second, it recruits pRB family proteins to the promoters of
antiviral genes causing transcriptional repression [206]. This observation of redeployment of
pocket proteins in response to the expression of E1A protein suggests that these proteins switch
targets depending upon different growth and developmental signals. A recent ChIP-seq study
with human E2F4 protein identified about 16,000 E2F4 binding sites. Among those sites, about
20% were more than 20 kb away from any annotated transcriptional start sites (TSS) suggesting
that E2F4 might function as a long range regulator. This study also suggests possible role of
E2F4 in regulation of micro RNAs [207]. Because all three human pocket proteins are known to
bind E2F4, the potential E2F4 functions suggested in this study might also hold for pRB, p107
and p130. Furthermore, another ChIP-on-chip and expression profiling study shows that p130

22

and E2F4 bind to a common set of widespread transcriptionally downregulated targets [208]. The
genome-wide occupancy of pRB and p130 proteins was also recently reported in growing,
quiescent and senescent human fibroblasts, indicating that these proteins bind to thousands of
putative target genes [209]. However, until now no study has presented a picture of the genomewide occupancy of pRB proteins in a whole organism. As discussed above, the study with E2F4
suggests it may be a long range regulator. Although pRB, p107 and p130 are known to bind
E2F4, no study until now has suggested the pocket proteins behaving as long range repressors. A
model has been proposed that E2Fs bind within a gene and activate the expression of non-coding
antisense transcript, yielding gene silencing [210]. The question arises that is there a possibility
of finding pRB family proteins binding to a non-promoter region and repressing gene silencing?
Furthermore, do pocket proteins bind to promoters in the context of E2F sites only or other
transcription factors also recruit them?
Using specific antibodies developed against the endogenous Rbf1 protein, I carried out
ChIP with embryos and collaborated with Kevin White’s lab for the utilization of massive
parallel sequencing (ChIP-seq) to identify genome-wide targets of Rbf1 in developing
Drosophila embryo. In chapter III, I describe my findings in which I show that there are many
more non-cell cycle targets than genes involved in cell cycle. I also show that almost all of the
Rbf1 binding occurs within 2 kb from the annotated TSS, suggesting that it is a short-range
repressor. Two thirds of Rbf1 binding regions are devoid of E2F sites, suggesting that either the
regions have poor E2F sites or there are other transcription factors recruiting Rbf1 to the
promoters. Furthermore, I found that there is a distinct promoter composition in terms of
transcription factor binding motifs.

23

X. Involvement of RB family proteins in regulation of conserved signaling pathways
Functional assays in model systems have provided evidence for complex regulatory relationships
between RB proteins and conserved signaling pathways. One such pathway involves insulin
signaling; metabolic signals propagated through this pathway control the activity of basic protein
synthetic capacity of cells and hence tissue growth in response to nutritional status [211-213].
The protein components of this signaling pathway are shown in Figure 1.2. PDK4, a component
of this pathway, is a major isoenzyme responsible for changes in pyruvate dehydrogenase
complex activity. The PDK family (PDK1 through 4) is responsible for controlling insulin
responsiveness and the efficacy of glucose utilization. PDKs are considered as nutrient sensors
that transduce signals in response to nutrient status (shortage or abundance) [212, 214, 215]. A
decrease in glucose levels induces muscle PDK4 expression to avoid being hypoglycemic [216218]. Loss of E2F1 in vivo results in increased sensitivity to insulin stimulation and
improvement in glucose oxidation and inactivation of pRB does the opposite [212]. This study
suggests that E2F1-pRB complex is involved in maintaining glucose homeostasis in the body
and insulin sensitivity thus linking pRB proteins in insulin signaling pathway. Another study also
links pRB to insulin pathway, however, it suggests that pRB haploinsufficiency ameliorates
insulin resistance [213]. This study in the mouse shows that partial deficiency in the pRB gene
antagonizes development of obesity and associated metabolic disturbances by increasing fatty
acid oxidation in these tissues through increase in the expression of peroxisome proliferatoractivated receptor (PPAR)α in liver and PPARδ in skeletal muscle [213]. Deregulation of
CDK/pRB pathway is shown to help mammary epithelial cells to cause desmoplasia via
phosphorylation of c-Met tyrosine phosphorylation and stimulation of the phosphorylation of
downstream signaling intermediates S6K and Akt [219]. In plants, the Arabidopsis S6K ortholog

24

Figure 1.2

Figure 1.2 Schematic drawing of insulin signaling pathway. This is insulin signaling pathway
in Drosophila, but the pathway is largely conserved. Insulin like peptide binds to membrane
receptor and the signal is transduced via kinase cascade that involves phosphoinositol 3 kinase
and downstream effectors such as FOXO, TSC and translational regulators including S6K and
4E-BP. Arrows indicate activation and bar-ended lines indicate inhibition.

25

S6K1 interacts with the Retinoblastoma-related 1(RBR1), Arabidopsis ortholog of pRB, through
its LVxCxE motif. This S6K1-RBR1 complex helps nuclear transport of RBR1 and is involved
in repression of cell cycle regulatory proteins such as E2FB, DPA, CDKB1;1 and CDKA [211].
These studies indicate a possibility of crosstalk between retinoblastoma and insulin pathways in
animals and plants.
Links between insulin and TOR signaling and RB function have also been noted in
Drosophila. A recent study from Wei Du’s group showed that inactivation of gigas, a
Drosophila ortholog of TSC2 (Tuberous sclerosis complex 2), in rbf1 mutant background
induces cell death. The authors claim that inactivation of gigas specifically kills rbf1 mutant
cancer cells under stress conditions without affecting normal cells [220]. These studies suggest
close functional interrelation between the insulin signaling pathway and pRB proteins. However,
no study has shown whether pocket proteins are involved in direct repression of the genes
involved in this pathway. In Chapter III I show that Rbf1 binds at the promoter of several genes
involved in this pathway and that Rbf1 directly represses insulin receptor, InR, in a cell culture
study with InR-luciferase reporter.
Additional signaling pathways have been functionally related to RB proteins in knock out
experiments. For instance, Wnt, MAPK, Ras and Notch pathways were found to be upregulated
in pRB, p107 and p130 triple knock out hepatocellular carcinomas (TKO HCCs) [221]. A variety
of genetic experiments link Notch and RB proteins. The protein components of this signaling
pathway are shown in Figure 1.3. Expression of several components of the Notch signaling
pathway was significantly increased in TKO HCCs. It was also shown that Notch signaling in
HCC cells are regulated by E2F-directed transcription [221], suggesting that pRB proteins may
be directly involved in regulation of this pathway. This activation of Notch signaling actually

26

seems to compensate for the loss of pRB proteins in promoting normal cell cycling, because
blocking Notch signaling with DAPT (an inhibitor of γ–secretase that normally cleaves the
internal domain of the Notch receptor during activation), accelerates the tumor progression
[221]. Notch signaling is not confined to tumor suppression, because both tumor suppressive and
oncogenic properties of Notch have been implicated in tumors [222]. A further link between
Notch signaling and RB action comes from study of the fly eye. In Drosophila, overexpression
of the Notch ligand Delta in the eye causes enlarged eye phenotypes. Further activation of two
Polycomb group proteins, Pipsqueak and Lola, induces metastatic tumors, which is caused by
epigenetic silencing of rbf1 gene [111]. A further study also showed that Notch can induce
proliferation of cells in Drosophila eye by inhibiting Rbf1 [223]. Notch-induced inactivation of
RB protein provides a separate path to cell proliferation. A recent study showed that Notch
activates the Hes-1 transcription factor, which represses expression of CKDN1B (p27) an
inhibitor of the CDK2-Cyclin E complex. This kinase complex normally phosphorylates and
inactivates pRB. Thus by inhibiting CKDN1B, Hes-1 promotes pRB hyperphosphorylation,
leading to cell proliferation [224]. Another genetic connection between Notch and Rbf1 is noted
in the context of UTX [225], an H3K27me3 demethylase that antagonizes Polycomb-mediated
silencing. utx mutant tissue has a growth advantage over wild type tissue because of increased
Notch activity, indicating that UTX is a Notch antagonist. Furthermore, the utx phenotype is
overrepresented by the loss of rbf1 [225]. Taken together, these studies suggest that there is
negative cross-talk between pRB and Notch pathways. In Chapter III I show that Rbf1 is
physically present on promoters of several components of Notch signaling pathway, providing a
molecular explanation for negative feedback between pRB and Notch signaling pathways.

27

Figure 1.3

Figure 1.3 Schematic drawing of Notch signaling pathway. This is notch signaling pathway is
in Drosophila, but the pathway is largely conserved. After the binding of Notch ligands (Serrate
and Delta), the membrane-bound Notch receptor is cleaved extracellularly and intracellularly by
several proteases. It is then translocated to the nucleus and forms an activator complex with
proteins such as mastermind, and Su(H) and activates target genes. Arrows indicate activation
and bar-ended lines indicate inhibition. Dashed arrows indicate translocation.
28

Figure 1.4

Figure 1.4 Schematic drawing of Hippo signaling pathway. This is hippo signaling pathway is
in Drosophila, but the pathway is largely conserved. Hippo and Warts are the major kinases of
this tumor suppressor pathway. These kinases regulate the target gene expression by controlling
Yorkie/Scalloped transcription factors. Arrows indicate activation and bar-ended lines indicate
inhibition.
29

Several studies have linked retinoblastoma tumor suppressor with the hippo tumor suppressor
pathway, a recently discovered conserved signaling pathway that regulates organ growth [226229]. The protein components of this signaling pathway are shown in Figure 1.4. Merlin, a
component of hippo pathway, blocks pRB phosphorylation, and inhibiting the increase of cyclin
D1 levels in NIH3T3 cells. Furthermore, Merlin represses an E2F activated luciferase reporter,
possibly by the same pRB phosphorylation mechanism [230]. In another study cells were
arrested at G1 in response to dihydrocytochalasin B (DCB) in a pRB proteins-dependent but p53independent manner. Merlin levels were elevated in these treated cells, indicating a possibility of
requirement of both Merlin and pocket proteins for DCB-induced arrest. Interestingly, pRB triple
knock out (TKO), but not p53 null, MEFs die rapidly when exposed to DCB [231]. Merlin is
positively regulated by pRB by repressing Rac1/Pak1, which suppress Merlin’s activity. This
way pRB regulates cell adhesion by helping Merlin promote cell adhesion [232]. This study
suggests that loss of pRB weakens Merlin’s activity which causes loss of cell-to-cell contacts
eventually contributing to the later stages of metastasis [232]. Another study suggests that pRB
and Hippo pathways may act synergistically in gene regulation [233]. A microarray analysis
using RNA extracted from imaginal discs harboring rbf1 and wts mutants suggests that Rbf and
hippo pathways cooperate to regulate DNA replication and cell cycle genes. In the same study
using reporter assay, authors show that overexpression of E2F1 and Yki, a component of hippo
pathway, showed synergistic upregulation of some target genes compared to E2F1 or Yki alone.
Interestingly this was squelched by cotransfection of Rbf1 overexpressing plasmid. The authors
argue that in the absence of both Rbf1 and wts, hyperactivated E2F1 and Yki synergistically
upregulate a novel set of genes and establish the distinct gene expression signature needed to
overcome terminal cell cycle exit upon differentiation [234]. LATS2, a mammalian ortholog of
30

warts kinase and a component of mammalian hippo tumor suppressor pathway, is required for a
senescent-like state and silencing of E2F target genes [235]. Interestingly, LATS2 is located at
13q11-q12 region of chromosome 13 which is close to 13q14.2 (where pRB is located) and loss
of heterozygosity (LOH) of LATS2 is frequently observed in primary cancers along with pRB
[235, 236]. LATS2 phosphorylates DYRK, a kinase that phosphorylates lin52 that is necessary
for the assembly of DREAM complex, thus helping RB in its repressive activity [235].
All the above studies imply a connection between pRB pathway and other conserved
signaling pathways. In Chapter III I show that Drosophila Rbf1 binds to promoters of many
components of conserved signaling pathways providing a molecular mechanism by which this
protein may direct alternative levels of signaling components, possibly setting different
thresholds of signaling activity in a tissue specific manner.

31

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50

CHAPTER II
Paradoxical instability-activity relationship defines a novel regulatory pathway for
Retinoblastoma proteins

1

Abstract
The Retinoblastoma (RB) transcriptional corepressor and related family of pocket
proteins play central roles in cell cycle control and development, and the regulatory networks
governed by these factors are frequently inactivated during tumorigenesis. During normal
growth, these proteins are subject to tight control through at least two mechanisms. First, during
cell cycle progression, repressor potential is downregulated by Cdk-dependent phosphorylation,
resulting in repressor dissociation from E2F family transcription factors. Second, RB proteins are
subject to proteasome-mediated destruction during development. To better understand the
mechanism for RB family protein instability, we characterized Rbf1 turnover in Drosophila, and
the protein motifs required for its destabilization. We show that specific point mutations in a
conserved C-terminal instability element strongly stabilize Rbf1, but strikingly, these mutations
also cripple repression activity. Rbf1 is destabilized especially in actively proliferating tissues of
the larva, indicating that controlled degradation of Rbf1 is linked to developmental signals. The
positive linkage between Rbf1 activity and its destruction indicates that repressor function is
governed in a fashion similar to that described by the degron theory of transcriptional activation.
Analogous mutations in the mammalian RB family member p107 similarly induce abnormal
accumulation, indicating substantial conservation of this regulatory pathway.
1

The work described in this chapter was published as the following manuscript: Pankaj
Acharya*, Nitin Raj*, Martin S. Buckley*, Liang Zhang, Stephanie Duperon, Geoffrey
Williams, R. William Henry and David N. Arnosti (2010). Paradoxical instability-activity
relationship defines a novel regulatory pathway for Retinoblastoma proteins. Mol Biol Cell,
21(22):3890-3901 (* co-first authors)
51

Introduction
Originally identified as an important player in juvenile retinal cancer, and the first
example of a tumor suppressor protein, the retinoblastoma (RB) gene product has been
recognized as a key regulator of the eukaryotic cell cycle. RB is also inactivated in a significant
proportion of adult onset of human cancers (Knudson, 1978; Classon and Harlow, 2002)
attesting to the centrally important role for RB in proliferation control. Further analyses in
mammals have revealed that other RB related proteins, p130 and p107, contribute to cell cycle
governance, but the partitioning of cell cycle duties among family members is not well defined.
Nonetheless, the RB family and their cognate regulatory networks are well conserved among
metazoans, substantiating the physiological significance of RB family function (van den Heuvel
and Dyson, 2008).
As potent regulators of cellular proliferation, the activities of RB family proteins are
tightly regulated. The canonical pathway for RB family regulation is mediated by cyclin/Cdk
complexes that phosphorylate pocket proteins at key points during the cell cycle. In response,
phosphor-RB dissociates from E2F binding partners, and transcription of cell cycle related genes
such as PCNA

can initiate at the G1/S phase transition (Dyson, 1998). In addition to

phosphorylation control, RB protein activities are also regulated by proteolysis. During in vitro
differentiation of 3T3-L1 adipocytes, p130 levels are transiently decreased relative to p107 by a
proteasome-mediated pathway, and this switch is associated with successful differentiation
(Prince et al., 2002). RB levels can be regulated by the Mdm2 ubiquitin ligase, better known for
its control of levels of the p53 tumor suppressor, and in cancers overexpressing Mdm2, RB
levels are diminished (Sdek et al., 2004; Uchida et al., 2005). The idea that altered RB protein
levels contribute to disease etiology is further highlighted during infection by certain oncogenic

52

viruses that hijack the proteolytic process and induce RB family member turnover to relieve host
control of cellular proliferation (Boyer et al., 1996; Stubdal et al., 1997). Together, these
examples demonstrate that regulation of RB family protein levels are important for normal
cellular growth, but that these processes are often deregulated in disease.
In Drosophila, the RB family (Rbf) is comprised of two members, Rbf1 and Rbf2, and
like their mammalian counterparts, these proteins function as transcriptional corepressors that
interact with the E2F family of transcription factors (Sutcliffe et al., 2003). The Drosophila Rbf
proteins provide canonical cell cycle control functions, and they are similarly regulated by
phosphorylation involving cyclin/cdk complexes (Xin et al., 2002; Frolov et al., 2005; Swanhart
et al., 2007). Rbf proteins are further subjected to influence of their turnover rates. Our recent
studies indicated that proteasome-mediated turnover of both Rbf1 and Rbf2 is prevented through
an association with the COP9 signalosome (Ullah et al., 2007). This linkage may contribute to
COP9 control of cell cycle and development in plants and animals (Wei et al., 2008). The COP9
signalosome consists of 8 subunits (CSN1-8), many of which exhibit limited similarity to
subunits of the 19S regulatory lid of the proteasome, suggesting that the COP9 signalosome may
play a direct role in modulating protein stability, possibly via interactions with the catalytic 20S
core proteasome (Su et al., 2003; Chang and Schwechheimer, 2004). The COP9 signalosome
may also control protein degradation through interactions with and subsequent deneddylation of
the cullin subunits of SCF ubiquitin E3 ligase complexes (Wei et al., 2008). Multiple subunits of
the COP9 signalosome were found to physically associate with Rbf proteins, and the depletion of
any of these subunits lead to destabilization of both Rbf1 and Rbf2 in cultured cells and embryos
(Ullah et al., 2007), suggesting that the entire complex is involved in stabilizing Rbf proteins.
However, it is not known whether the COP9 regulation of Rbf proteins is a constitutive process,

53

or whether this control is regulated during development. The CSN4 subunit of the COP9
signalosome co-occupies cell cycle regulated genes simultaneously with Rbf proteins, suggesting
that processes affecting repressor stability are spatially and temporally linked to repressor
function during gene regulation (Ullah et al., 2007).
While proteasome-mediated destruction of cellular proteins is clearly linked to
downregulation of factor activity, the converse relationship has also been described, notably, that
the potency of transcriptional regulatory proteins is directly linked to processes that mediate their
destruction. This somewhat paradoxical relationship has been described for a variety of
eukaryotic transcriptional activator proteins, including c-Jun, c-Fos, Myc, E2F1, and Gal4, all of
which harbor degradation signals in regions closely overlapping with their activation domains
(Salghetti et al., 1999; Salghetti et al., 2000; Salghetti et al., 2001). Synthetic constructs with
multiple degradation domains exhibit higher levels of transcriptional activation, suggesting that
the correspondence is not just coincidental (Salghetti et al., 1999; Salghetti et al., 2000). One
proposed explanation for the tight correlation between protein lability and increased
transcriptional potency posits that the proteasome, which is essential for turnover of
ubiquitylated substrates, also mediates transcriptional activation functions directly (Gonzalez et
al., 2002; Ferdous et al., 2007). A second mechanism suggests that activator ubiquitylation
serves to recruit co-activator proteins, such as P-TEFb, to increase RNA polymerase elongation
while simultaneously increasing the susceptibility of the activator to proteasome-mediated
destruction (Muratani and Tansey, 2003; Lee et al., 2005; Collins and Tansey, 2006; Daulny et
al., 2008). Although this effect has been observed for transcriptional activator proteins, no
transcriptional repressor has been reported as potentiated by proteolytic susceptibility. In this
study, we provide evidence that the lability of the Drosophila RB-related factor Rbf1 is tightly

54

linked to its function as a transcriptional repressor, and that this evolutionarily conserved feature
may provide an additional level of developmental control of the cell cycle.

Materials and Methods
Expression Constructs and Transgenic Lines
To express Rbf1 proteins under control of the endogenous regulatory sequences, an 8.8kbp genomic locus of Rbf1 was cloned, extending from 2.4 kb upstream of first exon to 2.4 kb
downstream stop (2.1 kb downstream end of the last exon) into pCaSpeR (Schejter and Shilo,
1989) between KpnI and XhoI sites in three steps using PCR amplification of genomic DNA.
Two Flag epitope tags were inserted immediately 5’ of the rbf1 stop codon into an XbaI site. The
genomic construct of Rbf1 ∆728-786 was made by site-directed mutagenesis. For genes used in
S2 cell culture transfection, rbf1 cDNA was PCR amplified and various mutants produced by
site-directed mutagenesis were cloned from pLD02906 (Keller et al., 2005) into KpnI and XbaI
sites of pAX vector (Ryu and Arnosti, 2003). Two Flag epitope tags were inserted 5’ of the stop
codon. For misexpression in the fly, the constructs were cloned into KpnI and XbaI sites of
pUAST (Brand and Perrimon, 1993). For bacterial expression of GST fusion proteins, the pRSF
Duet-1 vector (Novagen, Narmstadt, Germany) was modified to introduce a GST ORF followed
by a ligation independent cloning (LIC) site into its multiple cloning site (MCS I) to generate the
pRSF GST-Tb/LIC vector. rbf1 cDNA was PCR amplified and cloned into this LIC site to
generate the pRSF GST-Rbf1 1-845 construct. The pRSF GST-Rbf1 ∆728-786 construct was
generated by site-directed mutagenesis. For expression of human p107 in S2 cells, the cDNA and
various mutants produced by site-directed mutagenesis were cloned into the pAX vector and
modified with a C-terminal double Flag epitope. The pCaSpeR and pUAST plasmids were used

55

to generate transgenic flies by P-element mediated germline transformation of yw flies. The
transgenic flies were then balanced with SM2 CyO or TM3 Sb balancers.
Luciferase Reproter Assay
Drosophila S2 cells were transfected using Effectene transfection reagent (Qiagen,
Valencia, CA) according to the manufacturer’s protocol. Typically, 1.5 million cells were
transfected with 1 µg of PCNA-Luciferase reporter, 0.25 µg of pRL-CMV Renilla luciferase
reporter (Promega, Madison, WI) and 0.2 µg of one of pAX-rbf1 constructs. Cells were
harvested 72 h after transfection, and luciferase activity was measured using the Dual-Glo
Luciferase assay system (Promega) and quantified using the Veritas microplate luminometer
(Turner Biosystems, Synnyvale, CA). Firefly luciferase activity was normalized to renilla
luciferase activity.
Immunocytochemistry
Drosophila S2 cells were transfected with 400 ng of each rbf mutant using the Effectene
transfection reagent (Qiagen) according to the manufacturer’s protocol.

Cells were grown

directly on cover slips pretreated with 0.01% poly-L-Lysine (Sigma, St. Louis, MO). Three days
after transfection, cells were washed once in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4, 1.47 mM KH2PO4, pH 7.4) and fixed in 4% paraformaldehyde (in PBS) for 30 min at
room temperature. Cells were then washed four times in PBS, permealized in PBS + Triton-X100 (0.4% vol/vol) for 10 min at room temperature, and blocked with 1% bovine serum albumin
(in PBS). Cells were then incubated with M2 anti-Flag antibody (Sigma; final concentration 20
g/ml) in 1% wt/vol BSA in PBS buffer, washed three times in TBST (10 mM Tris-HCl, pH 8.0,
0.15 M NaCl, 0.05% Tween-20) for 5 min at room temperature, and incubated for 1 h at room
temperature with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin G

56

(1:500 dilution) (Boehringer Mannheim, Mannheim, Germany, and Invitrogen, Carlsbad, CA).
Cells were then washed three times in TBST and mounted in Vectashield mounting medium
(Vector Laboratories, Burlingame, CA) containing 1.5 µg/ml 4’,6’-diamidino-2-phenylindole
(DAPI) and incubated overnight at room temperature. Cells were visualized using an Olympus
BX51 fluorescent microscope.
Western Blot Analysis
To measure protein expression in larval tissue, third-instar larvae were collected from
transgenic lines expressing Flag-tagged Rbf1 and Rbf1 ∆728-786, mashed with a plastic pestle,
and sonicated (3 cycles of 12 pulses each) in lysis buffer (50 mM HEPES, pH 7.9, 150 mM
NaCl, 10% glycerol, 0.1 mM EDTA, 12.5 mM MgCl2, Complete mini-EDTA free protease
inhibitor cocktail, Roche, Indianapolis, IN). Imaginal discs were dissected out from ten thirdinstar larvae and extracts were prepared in lysis buffer. Extracts were run on 10% SDS-PAGE
gels and analyzed by Western blotting using M2 anti-Flag (mouse monoclonal, 1:10,000, 5
mg/ml, Sigma; F3165). Antibody incubation was performed in TBST (20 mM Tris-Cl, pH 7.5,
120 mM NaCl, 0.1% Tween-20) with 5% nonfat dry milk. Blots were developed using HRPcongjugated secondary antibodies (Pierce, Rockford, IL) and SuperSignal West Pico
chemiluminescent substrate (Pierce, Rockford, IL) and SuperSignal West Pico chemiluminescent
substrate (Pierce). To measure protein expression in cell culture, 50 µg S2 cell lysates were
resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with M2 anti-Flag mouse
monoclonal at 1:10,000 dilution, mouse monoclonal anti-tubulin (Iowa Hybridoma Bank) at
1:20,000 dilution, anti-Groucho (mouse mAb obtained from Developmental Studies Hybirdoma
Bank and used at 1:50 dilution) and anti-Rbf1 antibody as described previously (Keller et al.,
2005).

57

Treatments with MG132 Proteasome Inhibitor and Cycloheximide
For proteasome inhibitor treatments, S2 cells were transfected with 0.5 µg of pAXrbf1
constructs using the calcium phosphate transfection method. The cells were grown for 5 d then
treated with 50 µg/ml MG132 or the vehicle DMSO for the indicated times. For determination of
Rbf1 protein half-life, 1.5 million S2 cells were transfected using Effectene transfection reagent
(Qiagen) with 10 ng of pAXrbf1 1-845 or 4K-A.1 genes. Seventy-two hours post-transfection
the cells were treated with 100 µM cycloheximide for the indicated times.
Protein-Protein Interaction Studies
For the expression of GST fusion proteins, the appropriate expression constructs were
transformed into Rosetta2 (DE3) E. coli cells (Novagen). Protein expression was induced by 0.5
mM IPTG for 3 h at 37˚C. The proteins were purified on Glutathione sepharose beads (GE
Healthcare, Piscataway, NJ). The [35S]-Met labeled E2F proteins were generated using the TNT
T7 Quick for PCR DNA Kit (Promega). In vitro translated proteins were bound to ~1 µg of
preincubated immobilized GST fusion proteins for 3 h at room temperature. The beads were
washed three times with HEMGT-150 buffer (25 mM HEPES, 0.1 mM EDTA, 12.5 mM MgCl2,
10% Glycerol, 0.1% Tween-20, 150 mM KCl). Bound proteins were eluted by boiling in 1X
Laemmli sample buffer and analyzed by SDS-PAGE and autoradiography. For the
coimmunoprecipitation assays, 200 ng Myc-tagged E2F1 and 200 ng of various Flag-tagged
Rbf1 constructs were cotransfected into S2 cells using Effectene transfection reagent (Qiagen).
Cells were grown for 3 d after which whole cell extracts were prepared and Flag
immunoprecipitation reactions were performed (Anti-Flag M2 affinity gel, Sigma) followed by
anti-Myc Western blotting (mouse monoclonal, 1:3000 dilution, 5 mg/ml, Roche).

58

Chromatin Immunoprecipitation
Chromatin was prepared and analyzed from 0- to 20-h-old embryos as described
previously (Martinez and Arnosti, 2008), except that the chromatin (1 ml) was incubated with 5
µl (5 µg) of Flag antibody (Sigma; F7425) or 2 µl H3 antibody (Abcam, Cambridge, MA; 0.4
µg/µl) overnight at 4˚C. The recovered DNA was dissolved in 40 µl water. 2 µl of each ChIP
sample was used for 28 cycles of PCR. The oligos used for PCR were 5’CCGCAAGCATCGATAATGAGCAGA-3’ and 5’-AGTTGTGCGGGTACTTGGTTTCC-3’ for
the

DNA

primase

promoter;

5’-TGTGGGCTCTCTTCGTGTAGACTT-3’

and

5’-

TGGTTTCTGATTCTCACACACGAC-3’ for the sloppy paired 1 promoter and 5’GTTGAGAATGTGAGAAAGCGG-3’ and 5’-CGAAAAAGGAGAAGGCACAAAG-3’ for an
intergenic region.
Fly Assays
Flies harboring the wild-type or mutant rbf1 forms in the pUAST vector were crossed
with flies containing an eyeless-Gal4/CyO driver (Gilbert et al., 2006), and the offspring were
screened for eye phenotypes. The rbf14 mutant (stock number 7435) was obtained from the
Bloomington Stock Center.
Immunohistochemical Staining of Imaginal Discs
Imaginal discs were dissected in chilled PBS from third-instar larvae of rbf1 and rbf1
∆728-786 flies and fixed in 3.7% formaldehyde in 10 mM potassium phosphate, pH 6.8; 15 mM
NaCl; 45 mM KCl; 2 mM MgCl2 for 30 min at room temperature. Antibody detection was
performed by diaminobenzadine staining using the Vectastain kit (Vector Labs). Primary M2 αFlag dilution was 1:1500. Following the horseradish peroxidase reaction, discs were mounted in
70% glycerol.

59

Results
The Rbf1 C-Terminal Region Encodes an Instability Element
Our previous studies demonstrated that endogenous Rbf1 and Rbf2 proteins are
dependent on the presence of the COP9 signalosome for stability; depletion of COP9 subunits
resulted in a loss of Rbf protein, which was prevented by the addition of proteasome inhibitors,
indicating the involvement of the 26S proteasome pathway (Ullah et al., 2007). To identify
regions involved in Rbf turnover as first step toward understanding the process of Rbf
stabilization, we examined the stability of epitope-tagged transfected Rbf1 proteins in S2 cells.
We focused on Rbf1 because this protein represents the predominant functional RB family
member in Drosophila; rbf1 null mutations are lethal, while rbf2 null mutants have only very
modest phenotypes (Stevaux et al., 2005). Furthermore, previous data suggested that endogenous
Rbf1 levels fluctuate during embryogenesis (Keller et al., 2005; Stevaux et al., 2005). We
initially examined the importance of the conserved central pocket domain, as well as the lessconserved N- and C-terminal regions (Figure 1A; Table 1). In this process, we identified a region
in the C terminus of the protein as an instability element (IE); proteins lacking residues 728-786
accumulated to high levels, and these levels were not further increased by treatment with the
proteasome inhibitor MG132 (Figure 1B). In contrast, Rbf1 proteins containing the IE were
expressed at lower levels, and these levels were enhanced by proteasome inhibition. Rbf1
stability was sensitive to growth conditions; Rbf1 ∆IE proteins were expressed at higher levels
than proteins containing this domain under conditions of higher cell density, longer periods of
cell culture, or with low amounts of transfected DNA (Figure 1C).

This last observation

suggested that the system for Rbf1 turnover can be saturated, and indeed we observed greater
differences between the wild-type and mutant Rbf1 ∆IE proteins in cells expressing lower levels

60

Figure 2.1

61

Figure 2.1 (cont’d.)

62

Figure 2.1 cont’d.
Figure 2.1. Identification of an instability element (IE) in Rbfl. (A) Schematic diagram of Rbf1
proteins expressed in Drosophila S2 cells. The N and C termini are indicated in dark and light
gray respectively; the black box represents the instability element; the E2F-binding pocket
domain is in white. (B) Effect of proteasome inhibitor MG132 on Rbf1 protein levels. Cells were
transfected to express the indicated proteins and treated for 1-8 h with MG132, and protein levels
assayed by Western blot using antibodies to C-terminal Flag epitope tag. The wild-type 1-845
and mutants lacking the extreme C terminus (∆787-845) or the pocket domain deletion mutant
(∆376-727) were expressed at lower levels and were strongly stabilized by this drug, while the
mutants lacking the IE (∆728-786 and 1-727) were expressed at higher levels and were not much
further stabilized by MG132 treatment. (C) Effects of cell density and culture time on differential
expression of wild-type Rbf1 and IE mutant. 400 ng of Rbf1 expression plasmid was tranfected
into S2 cells. At lower initial cell densities (0.75 X 106 / ml) and shorter growth times (3 d),
expression of wild-type Rbf1 (1-845) and a deletion mutant lacking the IE (∆728-786)
accumulate to similar levels. Normalized protein levels are shown below the lanes containing
Rbf1. Cells at higher initial densities (1.5-3 X 106 / ml) grown for longer times (5 d) show higher
levels of the mutant protein relative to the wild-type form. Levels of transfected CtBP protein,
and endogenous tubulin protein, are shown as controls.

63

Supplementary Figure 2.1

64

Supplementary Figure 2.1 cont’d.
Supplementary Figure 2.1. (A) Effect of proteasome inhibitor MG132 on endogenous Rbf1 and
Groucho corepressor protein levels. S2 cells were plated at a density of 1.5 million/ml, grown for
5 days, then treated with proteasome inhibitor MG132 or vehicle (DMSO) as indicated. The
protein levels were assayed by Western blot and quantitated by photon-capture analysis with a
Fuji LAS-3000 Imager. Under these cell culture conditions, the Rbf1 protein but not Groucho
was found to be specifically stabilized upon MG132 treatment. (B) Quantitative assessment of
Rbf1 levels shown in (A). Bars indicate ratios of Rbf1 from MG132 treated / DMSO treated
cells. All values were first normalized to tubulin levels.

65

Table 1. Rbf1 repression, stability, and localization

Rbf1
construct
1-845
1-375
376-845
1-727
Δ728-786
Δ787-845
K754A
K754R
K774A
K774R
3K-A.1
3K-R.1
4K-A.1
4K-R.1
6K-A.1
6K-R.1

Repression
activity ± stdev
100 ± 9
12 ± 1
42 ± 3
16 ± 2
16 ± 4
107 ± 14
65 ± 6
81 ± 9
151 ± 15
125 ± 22
35 ± 11
105 ± 26
22 ± 5
86 ± 7
36 ± 9
110 ± 9

Protein
stability

+
+

+
+
+

Nuclear
localization
+
+
+
+
+
+
+
+
+
+
+
+
+

Constructs marked (-) for nuclear localization were not exclusively nuclear.

66

of each protein (not shown). Under these cell culture conditions we also observed that the
endogenous Rbf1 protein was stabilized by addition of MG132 (Supplementary Figure 1). We
conclude that the C-terminal region encompassing amino acids 728-786 harbors element(s) that
contribute to Rbf1 instability and proteasome responsiveness.
Critical Roles of Lysine Residues within Instability Element
The striking accumulation of wild-type Rbf1 protein in cells treated with the proteasome
inhibitor MG132 indicated that this protein, but not the mutant forms lacking the IE, is subject to
active degradation. We hypothesized that the Rbf1 IE may serve as a target for protein
ubiquitylation as one mechanism explaining the contribution of this region to proteasomemediated turnover. Protein ubiquitylation of lysine residues often directs processing by the 26S
proteasome, therefore we tested whether the lysine residues in the IE are involved in the stability
of Rbf1 (Figure 2; Table 1). Mutant Rbf1 in which three, four, or all of the six lysines were
converted to alanine (K to A) were assessed for expression. All three of these mutant forms
accumulated to significantly higher levels than the wild-type protein. In contrast, mutant Rbf1
proteins harboring charge-conserving lysine-to-arginine substitutions in the same residues did
not over accumulate, suggesting that the positive charge of the side chain, rather than its ability
to be ubiquitylated, is important for low steady state levels (Figure 2A). To determine whether
the change in steady state levels is due to altered stability, we next tested whether the half-life of
wild-type and mutant (4KA) Rbf1 proteins differed by treating S2 cells with the translational
inhibitor cycloheximide. Three days after transfection at a point when our previous data
indicated that Rbf1 (4KA) mutant protein was expressed at higher levels than wild-type Rbf1, S2
cells were treated with cycloheximide and Rbf1 protein levels subsequently measured at 0, 6, and
12 h (Figure 2B, 2C). By 6 h, levels of the wild-type Rbf1 protein, but not the mutant Rbf1

67

(4KA), were significantly decreased, confirming that the heightened accumulation of Rbf1
proteins lacking the IE is caused by reduced rate of Rbf1 degradation (Figure 2D).
To assess whether the Rbf1 IE functions as an instability element in the context of normal
Drosophila development, we devised a rescue construct that expresses epitope-tagged Rbf1
under the control of the endogenous rbf1 regulatory sequences. Developmental expression of the
wild-type Rbf1 and Rbf1 ∆IE (∆728-786) proteins was then assessed by Western blotting. As
shown in Figure 3A (left panel), the overall levels of both proteins were similar in third-instar
larval extracts, suggesting that the deletion mutant accumulated to wild-type levels. However, a
very different picture emerged when we measured protein expression in imaginal disc tissue
from third-instar larvae as shown through Western blots in Figure 3A (right panel) and imaginal
disc staining in Figure 3, B-J. The relationship between this effect and previously characterized
Rbf1 function is especially evident in the eye imaginal disc. The terminally differentiating cells
of the posterior eye disc normally have no transcription of rbf1 and low or nonexistent levels of
Rbf1 (Keller et al., 2005), but the Rbf1 ∆IE mutant also shows staining in these posterior cells,
suggesting an abnormal perdurance of the protein (Figure 3C, D). The marked difference
between the steady-state levels of the two proteins in these contexts indicates that the wild-type
Rbf1 protein is specifically destabilized in the proliferating and differentiating tissue of the
imaginal discs. The tissue-specific stability of the Rbf1 wild-type and mutant proteins suggests
that turnover of Rbf1 is a regulated event and is likely triggered by developmental signals. The
cell density-dependent difference in protein accumulation for wild-type and IE-deleted Rbf1
proteins as described in Figure 1C also supports this hypothesis.

68

Figure 2.2

69

Figure 2.2 cont’d.
Figure 2.2. Conserved lysine residues in IE play critical roles in accumulation and stability of
Rbf1. (A) Mutation of multiple lysine residues within the IE leads to increased protein
accumulation. Lysine residues were changed to alanine (K732A, K739A, K740A for 3K-A; also
K754A for 4K-A; also K774A and K782A for 6K-A) or to arginine. Rbf1 overaccumulation is
not observed with the lysine to arginine substitution. 1.5 X 106 S2 cells were transfected with
100 ng of Rbf1 expression plasmid and grown for five days. The data shown are representative
of three biological experiments. (B and C) Half-life measurements of unstable wild-type and
stable IE mutant proteins. Three days after transfection, cells were treated with cycloheximide
and harvested at the indicated times. Rbf1 protein levels were quantified by photon-capture
analysis with a Fuji LAS-3000 Imager and normalized to tubulin levels. (D) Bar graphs showing
averaged normalized flag:tubulin ratios for the Rbf1 wild-type and 4K-A mutant proteins at the
6-h time point from three biological replicates. At this time point, the difference between the
wild-type and the 4K-A mutant protein levels was statistically significant (p = 0.05).

70

Figure 2.3

71

Figure 2.3 cont’d
Figure 2.3. Expression of wild-type and IE mutant forms of Rbf1 in the Drosophila larva.
Indicated proteins were expressed from the endogenous rbf1 promoter, and expression levels
were assayed in total larval extracts as well as in imaginal discs. (A) Western blot showing
expression of Flag-tagged Rbf1 from third-instar larvae (left panel) and pooled imaginal discs
(right panel) carrying homozygous copies of rbf1 genomic constructs. Equivalent levels of
proteins were noted in whole larval extracts whereas the mutant protein was found to accumulate
to ~fourfold of the wild-type protein in the imaginal discs. The Western blot of whole larval
extracts is representative of four biological replicates for the two lines shown in C, F, I, and D,
G, J; the average difference in protein levels in total larval extracts was 13% ± 2%. (B-J) Rbf1
expression in third-instar larval imaginal discs. (B-D) Eye discs, (E-G) wing discs, and (H-J) leg
discs. Weak background staining was observed in nontransgenic yw flies (B, E, and H), and
specific but weak staining was evident in discs expressing wild-type Rbf1 protein (C, F, and I).
Strong expression was noted in flies expressing the inactive Rbf1 ∆728-786 IE mutant (D, G, and
J). The imaginal disc staining is representative of stainings of three different lines for each
construct; in all cases, the IE mutant protein was expressed at higher levels. For interpretation of
the references to color in this and all other figures, the reader is referred to the electronic version
of this dissertation.

72

The Rbf1 Instability Element Contributes to Repression Potency
In the previous experiment, the rbf1-Flag transgene rescued an rbf14 null mutant,
substituting for both zygotic and maternal Rbf1 protein as demonstrated by its ability to support
viable flies for generations (Table 2 and data not shown). In contrast, the similar construct
expressing Rbf1 (∆IE)-Flag protein was not capable of rescuing the mutation, despite robust
expression in imaginal discs and wild-type expression at the third-instar larval stage. We
therefore hypothesized that the IE is required for Rbf1’s role in regulating activity. To test this
hypothesis, S2 cells were cotransfected with expression plasmids encoding wild-type or mutant
Rbf1 proteins and the effect on repression potency was determined using PCNA-luciferase
reporter construct, which is sensitive to repression by Rbf1 (Stevaux et al., 2002). As expected,
proteins lacking the central pocket domain were inactive; this region of the protein is required for
interaction with the E2F transcription factors that recruit Rbf1 to the promoter (Figure 4A).
Removal of the N-terminal portion of the protein had only a mildly deleterious effect on
repression, consistent with previous studies that suggested it is not required for transcriptional
activity in vivo and in vitro (Hiebert et al., 1992). In contrast, removal of portions of the entire C
terminus revealed multiple effects. First, deletion of the IE region alone had a strong inhibitory
effect on transcriptional repression, and this effect was just as severe as removal of the critical
pocket domain. The Rbf1 ∆IE and pocket deletion mutant proteins did not exhibit aberrant
localization, but remained in the nucleus (Figure 4B). Second, loss of the adjacent C-terminal 59
amino acids (∆787-845) did not abolish repression but did change its subcellular localization so
that the protein was no longer strictly nuclear. These data indicate that this region harbors a
nuclear targeting element governing Rbf1 cytoplasmic/nuclear distribution. As observed for
deletion of the entire IE (∆728-786), removal of portions of this 59-aa region in blocks of 20 was

73

Figure 2.4

74

Figure 2.4 cont’d
Figure 2.4. Rbf1 requires the IE for transcriptional repression. (A) Deletion of the IE (∆728-786)
or E2F binding pocket (∆376-727) compromises transcriptional repressin activity of Rbf1
proteins measured on the PCNA-luciferase reporter gene (bar graph). Under these transfection
conditions, proteins were expressed at similar levels (Western blot). (B) Subcellular localization
of wild-type (1-845) and deletion mutants. DAPI staining indicates DNA in nucleus, and FITC
staining the Rbf1 proteins. Proteins lacking residues 787-845, which include the presumptive
nuclear localization signal, are found predominantly in the cytoplasm. (C) Transcriptional
activity of Rbf1 IE deletion and point mutant proteins assayed on PCNA-luciferase reporter.
Mutant proteins lacking the IE, or with multiple lysine to alanine mutations, were compromised
for transcriptional repression activity. Lysine to arginine mutant proteins exhibited wild-type
repression activity. Error bars indicate SD, and asterisks indicate p < 0.05. (D) Rbf1 repression
of Drosophila Polα-luciferase reporter. Deletion of the IE largely inactivates the protein for
transcriptional repression (top panel). Data in 4A represents two biological replicates, each with
three technical replicates, except for 1-845 and ∆728-786, which represent 16 and 9 biological
replicates respectively. Other transfections include data from at least three biological replicates.
Firefly luciferase activity is expressed relative to Renilla luciferase control. For interpretation of
the references to color in this and all other figures, the reader is referred to the electronic version
of this dissertation.

75

Table 2. rbf14 rescued by transgenic Rbf1.
rbf14 mutant male flies rescued by rbf1 transgene
Strain

Genotype (%)

n

rbf14/Y

FM7/Y

rbf14/+

FM7/+

Rbf1 L1

3.7

19.1

41.2

36.0

1116

Rbf1 L2

3.6

22.6

39.8

34.0

1163

0

30.0

37.4

32.6

697

Rbf1Δ728-786

rbf14 mutant female flies rescued by rbf1 transgene
Strain

Genotype (%)

n

rbf14/Y

FM7/Y

rbf14/rbf14

rbf14/FM7

Rbf1 L1

6.1

39.6

9.8

44.5

164

Rbf1 L2

1.1

36.7

8.5

53.7

188

L1 and L2 are two independent transgenic lines expressing wild-type Rbf1 protein. Rbf1Δ728786 expresses a nonfunctional, proteolytically stabilized form of Rbf1. rbf14 is a complete
deletion mutant of Rbf1. FM7 represents an X-chromosome balancer. rbf14/Y represents
rescued males; rbf14/rbf14 represents rescued females. The larger percentage of flies carrying
the wild-type (+) or balancer (FM7) X-Chromosome indicates that some flies are not rescued.

76

sufficient to inhibit repression activity, suggesting that the function of the IE is distributed over
numerous residues throughout this region (data not shown).
Our previous data indicated that multiple lysine residues within the Rbf1 IE contributed
to Rbf1 stability, thus we tested whether these same residues were involved in the transcriptional
repression mediated by Rbf1. Indeed, as shown in Figure 4C, Rbf1 proteins bearing multiple
lysine to alanine substitutions were less effective repressors, even though these proteins were less
effective repressors, even though these proteins were more stable than the wild-type Rbf1. This
effect was most notable for the Rbf1 4KA mutant whose repression capability was similar to that
mediated by Rbf1 lacking the IE. Surprisingly, alanine substitution of two additional lysine
residues (6KA) reproducibly improved the function of Rbf1 in repression. This observation
raised the possibility that this region harbors elements that throttle Rbf1 repressor potency, as
discussed further below. In contrast to alanine substitution, Rbf1 proteins harboring multiple
lysine to arginine substitutions did not overaccumulate, and significantly, were just as potent as
wild-type Rbf1 for transcriptional repression. Based on these data, we conclude that these
residues contribute both Rbf1 instability and to repressor function. These data further indicate
that modification of these residues is not essential to either process. To test whether the effects
on transcriptional repression of these Rbf1 mutations were evident in other contexts, we
compared transcriptional repression of wild-type and mutant Rbf1 proteins on the Polα promoter,
which has somewhat different requirements for E2F and DP activation compared with the PCNA
promoter (Figure 4D) (Dimova et al., 2003). Deletion of the IE or point mutations within this
region similarly reduced the repression activity on this promoter as well, indicating that the
relationship between protein activity and instability is independent of promoter context. Taken
together, these data strongly indicate that the ability of the Rbf1 protein to act as a transcriptional

77

repressor is tightly associated with its instability, and that the IE in the Rbf1 C terminus is
multifunctional, linking these two features.
The Rbf1 IE Is Not Essential for E2F Interactions and Promoter Binding
Previous studies have shown that both the pocket domains as well as the carboxy
terminus of the human RB protein can make molecular contacts with E2F1 (Lee et al., 2002;
Xiao et al., 2003; Rubin et al., 2005). We reasoned that the reduced activity of the Rbf1
instability element mutants might be a direct result of their inability to physically associate with
the

E2F

transcription

factors.

Therefore,

we

performed

GST

pull-down

and

coimmunoprecipitation (CoIP) assays to test for interactions between Rbf1 and E2F proteins. In
the GST pull-down assays, both GST-Rbf1 1-845 and the IE mutant (∆728-786) displayed
similar binding ability to in vitro translated E2F1 and E2F2 proteins (Figure 5A, lanes 5 and 6).
No interaction was observed with beads alone or GST protein (Figure 5A, lanes 3 and 4).
Similarly in CoIP assays from Drosophila S2 cells, Myc-tagged E2F1 coprecipitated with Rbf1
1-845 and two IE mutants (∆728-786 and 4K-A.1) but not with the pocket domain deletion
mutant (∆376-727) (Figure 5B; top panel, lanes 3-6). These results show that the IE mutants
retain a capacity to interact with both E2F1 and E2F2 proteins.
To assess whether the IE plays a role in Rbf1 promoter occupancy we performed
chromatin immunoprecipitation (ChIP) assays using embryos expressing the Flag-tagged Rbf1
wild-type or ∆IE mutant to test for promoter binding of these proteins at the DNA primase
promoter (Figure 5C). Binding at the intergenic locus and a nontarget gene (sloppy paired 1)
promoter was assesses as negative controls. Interestingly, the DNA primase promoter was found
to be enriched in immunoprecipitates from chromatin derived from embryos expressing both the
wild-type Rbf1 as well as the Rbf1 IE mutant proteins indicating that the Rbf1 IE mutant can still
78

Figure 2.5

79

Figure 2.5 cont’d.

Figure 2.5. Rbf1 IE is not essential for E2F interactions and promoter binding. (A and B)
Physical association between Rbf1 IE mutants and E2F proteins. (A) GST-Rbf1 and E2F
interaction assay. Indicated GST fusion proteins were bound to radio-labeled E2F proteins and
bound proteins were analyzed by SDS-PAGE and autoradiography. GST-Rbf1 1-845 and ∆IE
mutant displayed similar binding ability to both in vitro translated E2F1 and E2F2 proteins
(compare lanes 5 and 6). No interaction was observed with beads alone and GST protein (lanes 3
and 4). Coomassie stained gel showing equal amounts of GST fusion proteins used in binding
assays (bottom panel). The data shown are representative of three biological replicates.

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Figure 2.5 cont’d.
(B) Coimmunoprecipitation assay. Rbf1/E2F1 interactions in cotransfected S2 cells. Cells were
cotransfected with Myc-tagged E2F1 and Flag-tagged Rbf1 expression constructs. Whole cell
lysates were used for Flag immunoprecipitations (IP) and the samples were assayed using
Western blots with anti-Myc antibody (top panel). Myc-tagged E2F1 coprecipitated with Rbf1 1845 and two IE mutants (∆728-786 and 4K-A.1) but not with the pocket domain deletion mutant
(∆376-786) (top panel, lanes 3-6). Mock is IP performed using cell lysate from untransfected
cells (lane 7). The asterisk indicates a nonspecific band that is contributed by the Flag M2 beads
since it appeared in the no extract control where IP was performed in the absence of any cell
lysate (lane 8). Equivalent levels of the heavy chain IgG (marked as HC) were seen in all
samples indicating the use of equal amount of antibody for each IP reaction. The IP samples
were also blotted with the anti-Flag antibody (bottom panel) to verify the amount of Flag-tagged
protein that was captured in each assay. The data shown are representative of two biological
replicates. (C) Promoter occupancy by Flag-tagged Rbf1 wild-type and Rbf1 IE mutant proteins
measured by chromatin immunoprecipitation. Formaldehyde cross-linked chromatin was
prepared from 0 to 20 h embryos expressing the wild-type or mutant Rbf1 protein and
immunoprecipitated using the indicated antibodies. Enrichment of the Rbf-regulated promoter
(DNA primase) was observed by anti-Flag antibody immunoprecipitation reactions with both
wild-type and IE mutant fly embryos but not in reactions using pre-immune IgG (top panel) or at
an intergenic locus (middle panel) and a nontarget gene promoter (sloppy paired 1) (bottom
panel).

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occupy promoters (Figure 5C; top panel). Binding of the IE mutant at this locus was slightly
reduced compared with the wild-type Rbf1 although the association was significantly above
background as no enrichment was observed at an intergenic locus (middle panel) or the nontarget
sloppy paired 1 promoter (bottom panel). It appears that, unlike the Rbf1 pocket deletion mutant,
the reduced activity of the Rbf1 IE mutants cannot be attributed simply to their inability to
interact with E2F proteins or target gene promoters.
The Rbf1 IE Is a Dual-Function Regulator of Repressor Potency
Our data indicates that the Rbf1 IE region influences Rbf1 instability and contributes to
Rbf1 repression potency, providing a link between these two activities. However, during these
analyses we additionally observed that Rbf1 (6KA), harboring substitutions of all lysine residues
with the IE was reproducibly a more potent repressor than Rbf1 (4KA), harboring substitutions
of only the four most N-terminal lysine residues within the IE. This observation raised the
possibility that while most of the lysines play a positive role in Rbf1 repression, one or both of
the C-terminal-most lysine residues (K774, K782) play a negative role, restricting Rbf1 activity.
Therefore, to determine whether the lysine residues within the IE contribute to both positive and
negative regulation of Rbf1 function, we tested the repression activities of Rbf1 proteins with
individual alanine substitutions of each lysine residue within the IE. A subset of these results is
shown in Figure 6A, revealing three outcomes. In one case (K732), alanine substitution did not
affect repressor potency and was indistinguishable from wild-type Rbf1. The second class of
mutants were hypomorphic (K739, K740, K754), exhibiting modest but reproducible inhibitory
effects on repression, consistent with these residues contributing a positive influence on
repressor potency (Figure 6, A and B). In contrast, three mutants, K774A, K774R, and K782A
exhibited hypermorphic phenotypes with modest but reproducibly higher repression activity than

82

the wild-type Rbf1 protein, suggesting that these residues are involved in a negative control of
repressor activity (Figure 6, A and B). In cases where lysine to arginine substitution did not
moderate activity to wild-type levels, such as with K754 and K774, it is possible that the lysine
in question is a target of modification, as a positive charge is not the sole important feature.
However, for mutants with only single point mutations, we did not observe, for mutants with
only single point mutations, we did not observe the robust stabilization of mutant proteins
compared with the wild-type protein (not shown). Together, these data also indicate that the IE
exerts both positive and negative influences on transcriptional activity. Those mutant forms of
Rbf1 lacking all lysines exhibited intermediate repression phenotypes because of two distinct and
opposite effects, with decreased activity caused by mutations in K739, 740, and 754 partially
offset by increased activity mediated by the mutation of K774 and K782.
To test the physiological importance of these positively and negatively-acting residues
for repressor regulation in Drosophila, we expressed Rbf1 isoforms in the developing eye
imaginal disc using an eyeless-Gal4 driver system (Figure 7, A-H). As noted in previous studies,
misexpression of the wild-type Rbf1 protein induced rough eyes in a large percentage of
offspring. The mutant form of Rbf1 (∆728-786) lacking the IE was completely inert, despite
robust expression of the protein in the fly (not shown), consistent with a role for the IE in
repression. Individual point mutations that had modest effects on repression in cell culture assays
similarly showed modest effects on eye development, exhibiting milder phenotypes, and lower
penetrance that the wild-type Rbf1. In contrast, the hypermorphic K774A mutant, which
exhibited elevated repression activity in cell culture assays, induced dramatic phenotypes (Figure
7, E-H). A large percentage of offspring expressing this protein exhibited very severe eye
defects, including complete loss of the eye or developmental abnormalities including antennal

83

Figure 2.6

84

Figure 2.6 cont’d.
Figure 2.6. Rbf1 IE harbors positive and negative regulatory elements. (A) Transcriptional
repression activity of Rbf1 lysine point mutant proteins. Examples of mutant proteins that show
either enhanced or reduced repression activity. Mutation of K754 to alanine or arginine
attenuates repression activity while K774 to alanine mutant exhibited enhanced repression
activity with respect to the wild-type protein (top panel). Under these transfection conditions,
proteins were expressed at similar levels (lower panel). Error bars indicate standard deviations,
and asterisks indicate p <0.05 compared with wild-type Rbf1. (B) The lysine point mutants were
classified as neutral, hypo-, or hypermorphic based on the indicated t test results. (C) Schematic
representation of the Rbf1 IE indicating the location of lysine residues that play a positive or
negative role in Rbf1-mediated repression.

85

Figure 2.7

86

Figure 2.7 cont’d.
Figure 2.7. Severe developmental consequences of expression of hyperactive Rbf1. cDNAs of
rbf1 wild-type and IE hypermorphic and hypomorphic mutants were misexpressed in the eye
imaginal disc using the eye-Gal4 driver. (A-H) representative eyes exhibiting wild-type, mild,
moderate, severe, and four very severe phenotypes. (I) Bar graphs representing frequency with
which flies carrying the eye-Gal4 driver and UAS-rbf1 gene were recovered, as well as frequency
with which these latter flies exhibited a phenotype (“WT” normal eye, “RE” rough eye of any
degree of severity, “Cy wings” indicates flies that lacked the Gal4 driver, did not express the
rbf1 transgene, and had wild-type eyes). Note that ∆728-786 and 1-727, which lack the IE and
were inactive in cell culture, never showed a phenotype, and that the hyperactive K774 mutants
exhibited a partially lethal phenotype, as judged by lower recovery of flies containing the eyeGal4 driver. (J) Severity of eye phenotype in flies exhibiting rough eyes. Mutants are shown in
order of increasing severity; point mutants in the IE that decreased function in cell culture assays
also exhibited weaker eye phenotypes, and hypermorphic K774 alleles exhibited much stronger
phenotypes.

87

outgrowths and fewer transgenic individuals were recovered relative to nonexpressing controls,
suggesting lethality (Figure 7, I and J). Thus, the effects of the mutant forms of Rbf1 on eye
development mirror exactly the relative potencies of these proteins as measured in cell-based
repression assays indicating that Rbf1 is subjected to both positive and negative regulation of
repressor potency via the C-terminal IE in vivo. This result additionally demonstrates the
importance of limiting Rbf1 repression activity during development.
Conserved Instability Domain of Mammalian p107
The correlation between Rbf1 activity and instability in Drosophila prompted us to
examine whether similar regulation affects mammalian RB proteins. The overall level of amino
acid conservation is highest between the “pocket” domains of RB family members, but there are
clearly conserved blocks of residues in the C-terminal region. The primary structure of the C
terminus of Rbf1 most closely resembles that of p107, including the amino acids residues located
in the instability element of Rbf1 (Figure 8A). To directly compare Rbf1 and p107, we
transfected S2 cells with wild-type p107 and mutant forms in which conserved lysine and
arginine residues were replaced with alanine, as well as a deletion of the region most similar to
the Rbf1 IE (amino acids 964-1024). Similar to the stabilization effects noted with Rbf1, mutant
p107 exhibited increased accumulation compared with the wild-type protein (Figure 8B),
suggesting that the C-terminal region of p107 harbors an instability element that funnels p107
into similar turnover pathways even in this heterologous system.

88

Figure 2.8

Figure 2.8. Mutations in the conserved IE of p107 enhance expression. (A) Similarities between
Rbf1 IE and homologous region of p107, which is most similar to Rbf1. Asterisks mark basic
residues mutated in each protein to stabilize expression. (B) Genes for Flag-tagged wild-type
p107 or IE mutants were transfected into S2 cells and expression quantitated by Western blot.
The 60-aa region deleted from p107 in ∆964-1024 is similar to the Rbf1 IE. Endogenous tubulin
levels are shown as controls.

89

Discussion
During Drosophila development, cell-cycle regulation deviates considerably from the
classical four-stage G1/S/G2/M pattern, exhibiting rapid direct S-M cycling early in
development, stepwise acquisition of G2 and G1 phases, and endoreplication. These alternative
cycles involve a variety of regulatory features, including constitutive inactivation of Rbf proteins
by phosphorylation, transcriptional regulation of the rbf1 and rbf2 genes, and regulated
degradation of the E2F1 protein. Here we provide evidence that this regulatory richness also
includes a novel developmentally-triggered degradation of Rbf1 that paradoxically appears to be
required for repression activity. Our study indicates that Rbf1 lability is tightly linked to
repression activity, both in a cellular as well as a whole organismal context. The IE identified in
the C terminus of this protein appears to be a complex domain with dual functions, so that even a
few lysine to alanine mutations can dramatically enhance protein stability while inhibiting
transcriptional activity, while other lesions enhance the protein’s activity (Figures 1, 3, and 4).
Not only is the turnover of Rbf1 required for effective gene regulation, but it appears that
this turnover can be developmentally cued, presumably to be coordinated with the engagement of
Rbf1 with regulation of the cell cycle (Figure 3). Highly proliferative imaginal disc tissue
appears to provide one such context, where levels of wild-type, but not an instability element
mutant, Rbf1 protein decrease sharply, presumably in response to the engagement of this protein
during cell cycling. In the eye imaginal disc, the Rbf1 protein levels drop sharply in the
posterior, where cells are becoming terminally differentiated. Presumably, Rbf1 is activated and
consumed in the coordinated cells divisions that occur in the two stripes flanking the
morphogenetic furrow; the absence of any further transcription leads to global depletion of Rbf1.
The Rbf1 protein lacking the IE accumulates inappropriately in differentiating cells.
90

How might the repression activity of Rbf1 be linked to protein turnover? Protein lability
has previously been found to underlie the action of some eukaryotic transcriptional activators
(Salghetti et al., 2001; Kim et al., 2003). The activation domain of the VP16 protein was found
to be subject to modification by ubiquitylation, enhancing the transcriptional potency of this
factor as well as destabilizing it. This process is thought to affect other transcriptional activators
as well (Salghetti et al., 2000). The exact mechanism by which ubiquitylation enhances
transcriptional activation is poorly understood. The ubiquitin tag may serve a dual purpose of
facilitating interactions with the transcriptional machinery as well as attracting the 26S
proteasome. Alternatively, the proteasome itself, or portions of this multi-protein complex, may
directly enhance transcription; chromatin immunoprecipitation experiments have placed the “lid”
of the proteasome on specific genomic locations (Gonzalez et al., 2002; Ferdous et al., 2007).
Until now, there have been no examples of a connection between transcriptional
repression and turnover. If it is the modification of the protein with ubiquitin that potentiates
Rbf1’s repressor activity, this moiety may allow efficient interaction with the transcriptional
machinery, similar to the manner in which SUMOylation of PPAR-γ enhances interaction with
NCoR corepressors to silence inflammatory genes (Pascual et al., 2005). Ubiquitylation would in
this case attract the 26S proteasome in a competing, parallel reaction that enables Rbf1 turnover.
Alternatively, Rbf1 recruitment of the proteasome may allow this complex to directly mediate
repression, in a way opposite to that produced by activation domains.
The C terminus of Rbf1 appears to represent a regulatory nexus for this protein; in
addition to the instability/repression activity described here, key residues appear to provide a
damper to modulate its overall activity (Figure 6), and phosphorylation within this region by
cyclin kinases can inactivate the protein (Xin et al., 2002). The deep conservation of residues

91

within the Rbf1 IE argues strongly for similar activities in mammalian pocket proteins; indeed,
mutations of key residues in p107, the closest homolog of Rbf1, strongly stabilize the levels of
this protein (Figure 8). In addition, the spectrum of mutations associated with the human
retinoblastoma gene indicates that the C-terminal region correlating to the Rbf1 IE may similarly
contain critical functions for the mammalian RB protein. One common class of genetic lesion
associated with retinoblastomas are nonsense mutations that cause a truncation of the C terminus
of the RB protein, and several cancer-associated missense mutations have similarly been mapped
to the region corresponding to the Rbf1 IE (Lohmann, 1999).
Previous studies have shown that the RB C terminus interacts with the E3 ligase Skp2
and the anaphase promoting complex (APC/C) to regulate turnover of the p27 cyclin kinase
inhibitor (Ji et al., 2004; Binne et al., 2007). This pathway has been suggested to represent a
transcription-independent mechanism by which RB controls the cell cycle, and indeed RB was
shown not to be subject to APC/C degradation (Binne et al., 2007). Our results indicate that a
clean separation of transcription and proteolytic control in the context of RB proteins may be
oversimplified; here we see evidence for a separate route of proteolytic regulation that modulates
transcriptional regulatory potential and protein stability of Rbf1, and possibly related mammalian
pocket proteins. Interestingly, the regulation of this pathway may involve the evolutionarily
conserved COP9 signalosome. Our previous biochemical studies indicated that the COP9
signalosome regulatory complex is physically associated with Rbf proteins and limits turnover of
these repressors (Ullah et al., 2007). From the results of the current study, we postulate that
COP9 antagonizes the function of the Rbf1 IE, perhaps by blocking the access of ubiquitinmodifying E3 ligases that would otherwise potentiate Rbf1 activity and turnover. Alternatively,
inhibition of E3 ligases may involve the enzymatic activity of COP9, whereby this complex

92

downregulates E3 ligases by deneddylation of their cullin subunits (Wei et al., 2008). How the
instability of pocket proteins potentiates their activities, and how these processes relate to
developmental control of retinoblastoma family proteins and cancer, will be an area of active
investigation.
Acknowledgements
We thank Nick Dyson for sharing the PCNA and Polα luciferase reporter genes, Maxim
Frolov for providing the Myc-E2F1 construct, Stefan Gaubatz for providing the p107 expression
construct, Min-Hao Kuo and Jianjun Luo for aid with fluorescence microscopy, Jaclyn Peraino
for generating Rbf1 single lysine point mutants, and John Wang for reagents and friendly advice
on protein localization assays. We also appreciate the assistance from Satyaki Sengupta for his
helpful discussions. This work was supported by an undergraduate summer training grant from
the Michigan State University Genetics Program (to S.D.), the Michigan State University Gene
Expression in Development and Disease Group, and a grant from the National Institute of Health
(to D.N.A. and R.W.H.).

93

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CHAPTER III
Evidence for autoregulation and cell signaling pathway regulation from genome-wide
2

binding of the Drosophila retinoblastoma protein
Abstract

The retinoblastoma (RB) tumor suppressor protein is a transcriptional corepressor with
essential roles in cell cycle and development. Physical and functional targets of RB and its
paralogs p107/p130 have been studied largely in cultured cells, but the full biological context of
this family of proteins’ activities will likely be revealed only in whole organismal studies. To
identify direct targets of the major Drosophila RB counterpart in a developmental context, we
carried out ChIP-Seq analysis of Rbf1 in the embryo. The association of the protein with
promoters is developmentally controlled; early promoter access is globally inhibited, while later
in development Rbf1 is found to associate with promoter-proximal regions of approximately
2,000 genes. In addition to conserved cell cycle-related genes, a wholly unexpected finding was
that Rbf1 targets many components of the insulin, Hippo, JAK/STAT, Notch and other
conserved signaling pathways. Rbf1 may thus directly affect output of these essential growthcontrol and differentiation pathways by regulation of expression of receptors, kinases and
downstream effectors. Rbf1 was also found to target multiple levels of its own regulatory
hierarchy. Bioinformatic analysis indicates that different classes of bound genes exhibit distinct
promoter motifs, suggesting that the context of Rbf1 recruitment involves diverse transcription
factors, which may allow for independent regulation of Rbf1 bound genes. Many of these
targeted genes are bound by Rbf1 homologs in human cells, indicating that a conserved role of
retinoblastoma proteins may be to adjust the set point of interlinked signaling networks essential
for growth and development.
99

2

The work described in this chapter is resubmitted (after reviewer’s comments) to Genome
Research as the following manuscript: Pankaj Acharya, Nicolas Negre, John Johnston, Heming
Wang, Yiliang Wei, Kevin P. White, R. William Henry, and David N. Arnosti

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Introduction
The retinoblastoma tumor suppressor protein (RB) is an evolutionarily conserved
transcriptional corepressor that controls cell cycle, differentiation, development, autophagy and
apoptosis (Lipinski and Jacks 1999; Bosco et al. 2001; Nevins 2001; Classon and Harlow 2002;
Jiang et al. 2010). Germ-line mutations of RB are closely linked to retinoblastoma in early
childhood and osteosarcoma in adolescence, and somatic mutations in the RB gene are extremely
frequent in human cancers (Lohmann 1999; Sherr and McCormick 2002; Tang et al. 2008). The
vertebrate RB protein and the related family members, p107 and p130, are recruited to promoters
by interactions with E2F/DP heterodimers (van den Heuvel and Dyson 2008). Interactions
between E2F transcription factors and RB family proteins are regulated by cyclin/CDK-directed
phosphorylation during the cell cycle, and RB-E2F interactions can also be affected by viral
proteins (Nevins 1992a; Nevins 1992b; Dick 2007; Flowers et al. 2010). Though less complex
than its human counterpart, the Drosophila retinoblastoma network is functionally conserved and
consists of two RB proteins, Rbf1 and Rbf2, two E2F proteins, E2F1 and E2F2, and one DP
protein (Du and Pogoriler 2006). Drosophila Rbf proteins are regulated by phosphorylation,
similar to the vertebrate RB proteins (Xin et al. 2002; Frolov et al. 2005). Rbf1 activity is also
regulated during development by proteosome-dependent degradation, which is dependent on a Cterminal instability element that is simultaneously required for corepressor activity (Acharya et
al. 2010). The instability mechanism is conserved in the p107 human homolog, indicating that
this novel linkage between protein lability and repression function may be a general property of
these proteins in multicellular organisms (Acharya et al. 2010).
RB proteins are involved in regulation of both canonical and noncanonical forms of the
cell cycle. The canonical cycling involves the separation of DNA-synthesis (S-phase) and

101

mitosis (M-phase) by two gap phases, G1 and G2. However, during development, noncanonical
mitotic programs are common. In Drosophila, the first thirteen embryonic cell cycles are
synchronous, consisting of only S and M phases. G2 appears in cell cycle fourteen and G1 in cell
cycle seventeen (Foe 1989; Edgar and O'Farrell 1990; Shibutani et al. 2007). Endoreplication (in
which mitosis is not followed by cytokinesis), is another variant common in many larval and
adult tissues (Spradling and Orr-Weaver 1987). Endoreduplication in follicle cells is regulated by
Rbf1/E2F (Bosco et al. 2001). This diversity of cell cycle regulation suggests that Rbf1/2 and its
partners may be differentially utilized or regulated in different settings. Consistent with this idea,
Rbf1 stability is decreased in proliferating larval imaginal discs, and E2F1 is specifically turned
over during early S-phase in embryos and larvae (Shibutani et al. 2007; Shibutani et al. 2008;
Acharya et al. 2010).
RB proteins are also required in tissue- and stage-specific manners, as seen in studies of
different metazoan RB family members. Although expressed, the Drosophila Rbf1 protein is not
functionally required for early cell cycle regulation in the embryo (Du and Dyson 1999; Stevaux
et al. 2002; Keller et al. 2005). In the mouse, early embryonic requirements for RB are restricted
to the trophectoderm, although the protein is expressed in other tissues (Wu et al. 2003). The C.
elegans lin-35 (RB homolog) mutant shows a largely nonoverlapping set of genes that are
misregulated in embryo, L1 and L4 larvae (Kirienko and Fay 2007). These and other studies
emphasize that the multifarious functions of this protein family will require global studies in a
developmental setting. A major objective along these lines is the identification of functional and
physical target genes of RB family corepressors.
Among the best-characterized targets of RB family proteins are genes such as PCNA and
DNA pol alpha, which are involved in cell cycle regulation, however, RB proteins also regulate a

102

variety of genes involved in other pathways, such as apoptosis, DNA repair, and differentiation
(Lipinski and Jacks 1999; Classon and Harlow 2002; Kirienko and Fay 2007). In Drosophila,
functional targets of Rbf and E2F proteins were identified by transcriptomic analysis of
Drosophila S2 cells (Dimova et al. 2003). Physical and functional targets of the MMB/dREAM
complex, with which Rbf1 is also associated, were identified in Drosophila Kc cells (Georlette et
al. 2007). These studies suggest that Rbf1, and to a lesser extent Rbf2, interact with distinct
classes of genes that show varying sensitivity to loss of Rbf and E2F proteins. Targets of
mammalian RB, p107 and p130 proteins were identified by chromatin immunoprecipitation
(ChIP) in human lung fibroblasts; the proteins are observed to be redeployed in response to
expression of the RB-binding adenoviral E1A protein (Ferrari et al. 2008). The genome-wide
occupancy of RB and p130 was also recently reported in growing, quiescent and senescent
human fibroblasts, indicating that these proteins bind to thousands of putative target genes
(Chicas et al. 2010). However, until now no study has presented a picture of the genome-wide
occupancy of RB proteins in a whole organism during development. Using highly specific
antibodies developed against the endogenous Rbf1 protein, we carried out ChIP to study Rbf1
protein occupancy through developmental time, and employed parallel sequencing (ChIP-seq) to
identify genome-wide targets of Rbf1 in the Drosophila embryo. These results identify a
diversity of potential Rbf1 targets and promoter composition, and suggest that in addition to
known links to cell cycle, this protein may play a direct role in the control of numerous
conserved signaling pathways that are linked to metabolic regulation and growth.

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Results
Rbf1 exhibits developmentally-regulated promoter occupancy
In previous studies, we and others showed that Rbf1 protein is expressed throughout
embryogenesis, although the corepressor is not required for early cell cycles (Du and Dyson
1999; Stevaux et al. 2002; Keller et al. 2005). To investigate possible temporal control of Rbf1
binding to target gene promoters, we performed ChIP using 0-6, 6-12, 12-18 and 18-24 hr old
embryos. Enrichment of Rbf1 protein was studied at selected promoters from different classes of
Rbf1-responsive genes, described previously (Dimova et al. 2003). In all cases, Rbf1 occupancy
was low in early embryos with a peak in 12-18 hr embryos (Figure 1 and Supplementary Figure
1). These results indicate that Rbf1 promoter association is developmentally regulated; the lack
of early Rbf1 association coincides with the rapid early cell cycles that lack G1 and G2 phases.
Characterization of genome-wide Rbf1 association
Although many well-characterized targets of RB family proteins are genes involved in
cell cycle regulation and DNA replication, genes involved in other processes are also
functionally regulated by these corepressors (Classon and Harlow 2002; Dimova et al. 2003;
Kirienko and Fay 2007). To develop a global understanding of the genomic targets Rbf1, we
utilized ChIP-seq technology. We prepared chromatin from 12-18 hr embryos where robust
signals had been detected by conventional ChIP (Figure 1 and Supplementary Figure 1), and
generated separate DNA libraries from two Rbf1 immunoprecipitation experiments and one
experiment using preimmune antibodies. The anti-Rbf1 libraries yielded ~14 and 18 million
reads, while the preimmune serum library generated considerably fewer reads (1.2 million), as
expected for non-specific interactions of the preimmune serum with the Drosophila chromatin.

104

Figure 3.1

105

Figure 3.1 cont’d.
Figure 3.1. Rbf1 exhibits dynamic promoter occupancy. Rbf1 occupancy of regulated
promoters measured by chromatin immunoprecipitation was low in 0-6 hr embryos and peaked
at 12-18 hr. Formaldehyde cross-linked chromatin was prepared from embryos of different ages
and immunoprecipitated using the indicated antibodies. No specific enrichment was found at a
non-target gene promoter (sloppy paired 1). “No Ab”, immunoprecipitation carried out without
antibody; “IgG”, nonspecific mouse polyclonal antibodies; “α-H3”, anti-histone H3 antibody;
“α-Rbf1”, rabbit anti Rbf1 antibody.

106

Supplementary Figure 3.1

Supplementary Figure 3.1. Enrichment of Rbf1-bound promoters peaks at 12-18 hr.
Quantitation of enrichment of Rbf1-bound promoters shows that the dynamic promoter
occupancy of Rbf1 is maximum at 12-18 hr. The PCR products shown in Figure 1 were
measured on a Fuji LAS3000 imager and quantitated using Multi Gauge software (Fuji).

107

Supplementary Figure 3.2

Supplementary Figure 3.2. Validation of selected promoters for Rbf1 occupancy. To
independently assess enrichment of Rbf1 on novel target genes, several genes were selected and
their enrichment in ChIPed chromatin was tested by PCR. DNA prim is a positive control; the
intergenic region on chromosome 3 and sloppy paired 1 are negative controls. The enrichment of
the Rbf1 target gene promoters tested is significantly above the background. “Preimmune”,
serum from the rabbit used for later generation of α-Rbf1 antibody; “α-H3”, anti-histone H3
antibody; “α-Rbf1 226.3” and “α-Rbf1 226.4”, different bleeds of rabbit anti-Rbf1 antibodies.
108

About 60% of the reads were uniquely alignable to the Drosophila melanogaster
genome; other reads were found to map to more than one site, or did not align at all using the
strict criteria employed. 1236 peaks were found in both anti-Rbf1 immunoprecipitations, while
an additional ~1000 peaks were also found, mainly in one of the two ChIP experiments that
exhibited more robust peak intensities. The preimmune control ChIP results showed an even
distribution of very low peaks as would be expected from nonspecifically precipitated material
(data not shown). The overall low background found with the preimmune immunoprecipitation
and reproducibility between the two biological replicates provides a high level of confidence for
many of the peaks. Of the 1236 high-confidence peaks recovered in both of the biological
replicate experiments, about 95% could be mapped to within 2 kb of predicted transcriptional
start sites of known genes. These peaks were proximal to transcriptional start sites of 1890 genes.
Additional strong candidate genes were found in the library that yielded 18 million reads,
generating a total of 3188 genes.
Rbf1 target genes indicate regulation of RB pathway at multiple levels
Clear signals were observed on a number of promoters that we expected to find in this
data set, including DNA pol alpha, DNA primase, and PCNA, known physical targets of Rbf1
(Stevaux et al. 2002; Dimova et al. 2003). To validate the process of Illumina sequencing and
peak calling used to generate this data, we selected a set of promoters to independently analyze
by direct PCR; positive and negative signals were confirmed in all cases (Supplementary Figure
2 and data not shown). We also carried out experiments with a different antibody to test the
reproducibility of these ChIP results. Chromatin was prepared from flies harboring transgenic
Flag-epitope tagged Rbf1, (Acharya et al. 2010) and immunoprecipitations were carried out
using either anti-Rbf1 or Flag antibodies (Supplementary Figure 3). In each case, ChIP signals

109

Supplementary Figure 3.3

Supplementary Figure 3.3. Validation of specificity of Rbf1 antibodies. To assess the
specificity of Rbf1 antibodies, ChIP experiment was performed with embryos from transgenic
flies harboring Flag epitope tagged Rbf1. Some genes identified in ChIP-seq analysis along with
previously known target (DNA primase) and intergenic region were selected for PCR. A similar
significant enrichment of the Rbf1 target gene promoters was noted for each antibody.
“Preimmune”, serum from the rabbit used for later generation of α-Rbf1 antibody; “α-H3”, antihistone H3 antibody; “α-Rbf1”, rabbit anti-Rbf1 antibody; “Flag”, anti-Flag antibody.

110

coincided exactly. We found that diverse classes of genes were targeted by Rbf1, including a set
of genes that indicates that Rbf1 regulates its own functional output at multiple levels (Figure 2
and Supplementary Table 1 as in Acharya et al, 2012). The autoregulatory properties of Rbf1
were suggested by particular genes, such as cyclin A, B3, E, and cdk4/6, which encode the kinase
complexes that downregulate Rbf activity, the rbf1 gene itself, and cyclin-dependent kinase
subunit 30A, a component of the Cdk1-cyclin B kinase complex that phosphorylates numerous
proteins involved in DNA replication, translation, and chromatin structure (Holt et al. 2009).
Consistent with this notion, negative feedback loops of regulation of RB and p107 have been
reported in mammalian cells (Burkhart et al. 2010a; Burkhart et al. 2010b). A peak was also
associated with the 5’ promoter of the l(3)mbt gene, whose protein product is a member of a
conserved MMB/dREAM transcriptional regulatory complex that also involves the Rbf1 protein
(Lewis et al. 2004). Other MMB/dREAM components were also targeted by Rbf1, consistent
with autoregulation of the entire complex by Rbf1 and MMB/dREAM (Lewis et al. 2004;
Tabuchi et al. 2011). Rbf1 peaks were also associated with additional chromatin-regulatory
components, including the E(bx) gene, which encodes the NURF301 component of a SWI/SNF
nucleosome remodeling complex that has been shown to antagonize the action of RB in C.
elegans development (Andersen et al. 2006). These data indicate that Rbf1’s direct
transcriptional regulation is likely to control RB pathway output at five levels; Rbf activity via
cyclins and kinases that directly phosphorylate the corepressor, production of rbf1 transcripts, of
cofactors that work together with Rbf proteins, of factors that antagonize Rbf activity, and
kinases that are implicated in cell-cycle regulation of downstream genes. The potential effect of
this regulatory structure is that changes in Rbf protein abundance or activity will reset levels of
other components that would magnify or dampen the control of the entire Rbf regulon.

111

Figure 3.2

112

Figure 3.2 cont’d.
Figure 3.2. Rbf1 promoter-proximal occupancy of diverse classes of genes suggests
autoregulatory effects. Strong peaks were noted on cell-cycle related genes, such as DNApolα60 and cyclin-dependent kinase 30A (A, B). Autoregulation is suggested by occupancy of the
Rbf gene (C). The 5’ region of the Rbf1-related corepressor l(3)mbt is also associated with Rbf1
(D). The promoter of the dNURF 301/E(bx) gene, a chromatin remodeling component for RB
function in development, is also bound (E). Numerous components of cell signaling pathways,
including the insulin receptor, InR, are also targeted by Rbf1 (F). Relative peak intensities are
shown on the Y-axis. Representative individual peaks are visualized on the UCSC genome
browser. Bent arrows indicate the direction of transcription of the genes and absence of arrows
indicates 3’ region of the gene. For interpretation of the references to color in this and all other
figures, the reader is referred to the electronic version of this dissertation.

113

Rbf1 target genes include multiple components of conserved signaling pathways
One of the most striking observations about the roster of genes occupied by Rbf1 was its
extensive and hitherto unappreciated occupancy of genes involved in essential, conserved
signaling pathways. One of the strongest Rbf1 peaks is found proximal to the insulin receptor
promoter (Fig. 2F). Further investigation of the 3188 gene data set revealed that Rbf1 peaks were
extensively associated with other components of insulin signaling, including three of the four
Drosophila PI3 kinase genes, S6 kinase, and Thor/4E-BP (Fig. 3 and Supplementary Table II as
in Acharya et al, 2012). JAK/STAT signaling components identified as Rbf1 targets include the
signal mediator JAK kinase, the STAT92E transcriptional effector, as well as regulators of this
pathway, Ken, E(bx), Pzg and STAM. A large number of the genes in the Hippo growth-control
pathway, including those for the central Hippo and Warts kinases, are bound by Rbf1.
Components of the Notch, in particular regulatory proteases that process the Notch protein, were
also found among Rbf1 target genes. Wingless, Hedgehog, NF-B, TGF-, TOR, EGFR/Ras,
and JNK pathway components were also bound by Rbf1. In total, we identified 137 signaling
pathway genes that were bound by Rbf1; 75 of these were clearly identified in both ChIP-seq
samples, with a further thirteen present as peaks in both datasets, but just below the cutoff in one
of the ChIPs. Association of Rbf1 with signal pathway genes was confirmed in additional
biological ChIP experiments (Supplementary Figures 2 and 3, and data not shown). The
promoter-proximal positions of most Rbf1 peaks, as well as the comparatively small number of
regions bound by Rbf1 overall, suggest that these binding events are not simply the effect of
large numbers of promiscuously bound Rbf1 proteins. Genes for almost half of insulin signaling
and more than half of Hippo signaling components were occupied by the Rbf1 protein, compared
with the genome-wide average of 22%, indicating a strong enrichment. The extensive physical

114

Figure 3.3

115

Figure 3.3 cont’d.

116

Figure 3.3 cont’d.

117

Figure 3.3 cont’d.

118

Figure 3.3 cont’d.
Figure 3.3. Rbf1 occupies multiple nodes in conserved signaling pathways. Genes in the
insulin (A), JAK/STAT (B), Notch (C) and Hippo/Warts/Yorkie (D) signaling pathways were
targeted at multiple levels by Rbf1. Proteins of genes targeted by Rbf1 are indicated in red. For
interpretation of the references to color in this and all other figures, the reader is referred to the
electronic version of this dissertation.

119

Supplementary Figure 3.4

Supplementary Figure 3.4. Repression of InR and Rab23 promoters by Rbf1. Drosophila S2
cells were cotransfected with InR, Rab23, PCNA, or Act5C luciferase reporters, with (+) or
without (-) an Rbf1-expressing plasmid. Cells were harvested 72 h after transfection, and
luciferase assay was performed. Results of four (Act5c luciferase), and six (InR, Rab23, and
PCNA luciferase) biological replicates with three technical replicates each were pooled.
Asterisks indicate p < 0.0001.

120

interaction between Rbf1 and components of diverse signaling pathways suggests a novel means
by which cell cycle information may be integrated with the information related to metabolic
status, organ and tissue size, and differentiation states. The occupancy of genes located at
multiple levels of signaling, such as the insulin receptor and the S6 kinase genes indicate that
Rbf1 may be in a position to regulate these pathways in a complex, multifactorial manner. To
test whether Rbf1 indeed represses the some of these novel targets, we created InR (insulin
signaling), and Rab23 (hedgehog signaling) reporter constructs and measured their sensitivity to
Rbf1 overexpression compared with Act5C, and PCNA controls. As expected, Rbf1 effectively
repressed the PCNA reporter and not the Act5C reporter. Significantly, strong inhibition of InR
and Rab23 promoters was observed, indicating that Rbf1 can functionally regulate in S2 cells at
least some of the novel targets identified (Supplementary Figure 4).
Rbf1 exhibits a strong promoter-proximal targeting bias
As noted above, most Rbf1 peaks were located within 2 kb of transcriptional initiation
sites; mapped on a finer scale, we found that there was a strong preference for binding centered
at -205 bp (Figure 4). Unlike other corepressors such as CtBP and Groucho, the strong
preference of Rbf1 for the 5’ ends of genes suggests that Rbf1 can only exhibit activity near the
initiation site, or that transcription factors that it interacts with, such as E2F proteins, can only
activate

effectively

from

promoter-proximal

locations.

Interestingly,

Rbf1-associated

MMB/dREAM complex proteins are also found to bind in promoter-proximal locations; neither
this complex nor Rbf1 alone appear to frequently interact with distal cis-regulatory sequences
(Georlette et al. 2007). Rbf1 peak intensities (representing the number of sequences recovered
for particular genomic positions) spanned approximately an order of magnitude, with relatively
few outliers showing more than ten fold higher than the median value. Overall peak heights were

121

Figure 3.4

122

Figure 3.4 cont’d.
Figure 3.4. Rbf1 exhibits a strong promoter-proximal targeting bias. (A) The distribution of
peaks relative to the nearest transcription start site (TSS). The majority of peaks are centered 205
bp 5’ of the TSS. Distances were grouped into 100 bp bins and points fitted with a smooth curve.
(B) Distribution of peak intensities. Most peaks had an intensity within a few fold of the average,
although some peaks were >10 fold higher.

123

not correlated to the types of genes targeted, as genes in functionally related classes exhibited
peaks of a range of intensities. As discussed below, the sequences underneath the peaks
presented a very heterogeneous picture, indicating that different transcription factors may
provide alternative pathways for recruiting Rbf1.
Cell cycle and DNA replication related genes represent only a minority of bound sites
The previous functional assessment of genes regulated by Rbf in cell culture had
highlighted genes related to cell cycle and DNA replication, although a much broader spectrum
of gene functionalities was indicated by analysis of genes regulated and bound by the Rbf-related
MMB/dREAM complex (Dimova et al. 2003; Georlette et al. 2007). To characterize the nature
of Rbf1 direct targets, we performed Gene Ontology (GO) analysis on genes associated with the
highest confidence peaks using the DAVID annotation analysis system (Huang da et al. 2009).
Out of 1890 Rbf1 target genes, 42% were enriched for GO terms. Rbf1 peaks were associated
primarily with protein-coding genes, but 12 annotated noncoding RNA genes were also
associated with the cofactor. Approximately one quarter of the genes were enriched for cell cycle
and DNA replication categories, the areas that showed the most significant enrichment of all
groups of genes (Figure 5 and Supplementary Table III as in Acharya et al, 2012). Other
categories that were enriched included processes, such as chromatin modification and
transcription, cellular systems, such as cytoskeleton, and developmental programs, including
oogenesis and neurogenesis. A large number of smaller categories comprised 36% of the target
genes. Previous studies have shown that RB family members have particular roles in distinct
developmental settings, such as the role for RB in mouse trophectoderm development, vulval
development in C. elegans, and osteoblast differentiation (Wu et al. 2003; Bender et al. 2007;
Berman et al. 2008). The smaller number of non-cell cycle related genes previously found to be

124

Figure 3.5

Figure 3.5. Rbf1 target genes represent diverse Gene Ontology (GO) categories. 42% of
1890 Rbf1 target genes were enriched for GO terms. Of these, only about a quarter were
associated with Cell Cycle and DNA replication, while the majority of targets grouped into other
gene regulatory and developmental processes. The GO terms are arranged in the pie chart in
decreasing order of significance of enrichment from Cell Cycle and DNA replication to Other
DNA/RNA Metabolic Process. “Others” indicates numerous smaller groups of enriched genes.
The p-values for the categories are as follows: Cell Cycle, 7E-16, Cytoskeleton, 5E-13;
Chromatin Modification, 2E-12, Recombination/Repair, 7E-09; Phagocytosis, 1E-03; Apoptosis,
5E-03; Transcription, 2E-02; Neurogenesis, 2E-02; and other DNA/RNA Metabolic Process, 4E02.
125

functionally regulated by Rbf1, Rbf2 and E2F proteins indicate that perhaps in cell culture, many
physiological targets of these proteins are relatively quiescent (Dimova et al. 2003). Our data
suggest that taken in a developmental context, Rbf1 function may be distributed over a very wide
set of diverse cellular processes.
Enrichment of transcription factor motifs in Rbf1-bound peaks
Rbf1 does not bind to DNA directly, but is instead recruited by transcription factors,
generally of the E2F family. RB has been reported to interact with other types of transcription
factors, including MyoD, NeuroD1, GATA1, and components of the RNA polymerase III basal
transcriptional machinery (Gu et al. 1993; Felton-Edkins and White 2002; Batsche et al. 2005;
Kadri et al. 2009). In consideration of the wide diversity of genes targeted by Rbf1, we sought to
understand whether particular subsets of these genes would be characterized by distinct
constellations of protein binding sites in the promoter proximal regions. We extracted sequences
representing the 200 bp sequences from the center of each peak and performed de novo motif
discovery analysis using MEME (Bailey and Elkan 1994). We sought the top five
overrepresented motifs for 5 to 15-mers and noted that variations of four motifs occurred most
frequently (Figure 6A). We searched the motifs in the JASPAR and TRANSFAC databases, and
one of the motifs was similar to the E2F site, as expected. Another bears clear similarity to the
DREF site, which is bound by the DNA Replication-related Element-binding Factor involved in
expression of a wide variety of proliferation-related genes, and whose function in the context of
cell cycle regulation has been investigated on the PCNA and DNApol alpha promoters
(Yamaguchi et al. 1995; Takahashi et al. 1996; Seto et al. 2006). We also found a motif similar
to the binding sequence of the mammalian Forkhead transcription factor FOXJ2, and one novel
motif that did not closely match any other sequence in the database, which we designated RAM

126

Figure 3.6

127

Figure 3.6 cont’d.

128

Figure 3.6 cont’d.
Figure 3.6. Transcription factor motifs enriched in Rbf1-bound peaks. (A) The four most
overrepresented motifs identified by the MEME motif discovery tool, including one previously
unknown motif (RAM). (B) Rbf1-associated motifs are highly enriched compared to average
occurrence in DNA of the same A/T composition. The sequences under Rbf1 peaks were
scrambled five times and specific motifs with p < 0.0001 were identified. E2F sites showed the
highest level of enrichment in specifically bound regions compared to randomized DNA
sequences. (C) E2F, DREF and RAM motifs preferentially associate with Rbf1-bound
promoters. Presence of motifs in Rbf1-bound sequences was compared to presence in Rbf1unbound promoters. FOXJ2 sites are not restricted to Rbf1-associated promoters, and may
represent a motif for a broadly acting factor. Note that the canonical DREF sites are 8-mers
(Yamaguchi et al. 1995). In our data the eighth nucleotide was not conserved. (D) Diversity of
motif composition of peaks. 42% of total peaks contained only one of the four different motifs
(E2F, DREF, FOXJ2 or RAM). A quarter of the peaks had a combination of two different
motifs; 6%, a combination of three and 1% contained all four motifs. Only 36% or peaks had an
identifiable E2F motif (small insert). Strikingly, a quarter of the peaks did not have any of the
four motifs. The heterogeneity of sequences in Rbf1-bound peaks suggests that E2F may not be
the only transcription factor that recruits Rbf1 to target gene promoters. Peaks used in this
analysis were drawn from the 1236 bound regions found in both Rbf1 ChIP biological
experiments. A peak with multiple E2F motifs, but no other motif types, was counted as one type
of motif; a similar treatment applies for the other three motifs. “None” means the peaks did not
contain any motifs for E2F, DREF, FOXJ2 or RAM. For interpretation of the references to color
in this and all other figures, the reader is referred to the electronic version of this dissertation.

129

Supplementary Figure 3.5

Supplementary Figure 3.5. Determination of threshold for motif analysis. To determine
optimal p values to minimize false positive calls and provide reasonable sensitivity, MAST
analysis was performed on sequences under peaks with different thresholds for E2F, DREF,
FOXJ2 and RAM motifs (A-D). The analysis was repeated five times on sequences of identical
A/T composition that had been scrambled. The threshold p < 0.0001 showed the highest fold
enrichment, thus further analyses (in Figure 6 B, C and D) were carried out with this value.
130

Supplementary Figure 3.6

131

Supplementary Figure 3.6 cont’d.
Supplementary Figure 3.6. Prevalence of E2F-, DREF- and RAM-like motifs on Rbf-1
bound and not bound promoter regions. Scores for each Rbf1-bound peak (black bars) and
Rbf1-unbound 200 bp promoter region centered at -200 bp (white bars) obtained from MAST
analysis show that Rbf1-bound promoters have better E2F, DREF and RAM motifs (in each
case, Wilcoxon rank sum test p < 2.2e-16). However, there was no significant difference in
scores for FOXJ2 motifs in Rbf1-bound or unbound promoters (Wilcoxon rank sum test p =
0.16). The significant enrichment of weak E2F, DREF, and RAM sites among bound genes
suggests that there may be a higher fraction of Rbf1-bound promoters that utilize these proteins
than indicated by the use of our stringent cutoff criteria. An alternative statistical analysis of the
peaks indicated that the prevalence of these motifs in Rbf1-bound and -unbound promoters is
significant (Supplementary Table IV as in Acharya et al, 2012).

132

(Rbf associated motif). The motifs occur much more frequently than they would in scrambled
sequences of similar composition, indicating that these motifs have a high information value
(Figure 6B). The threshold for calling each of the motifs (p = 0.0001) was selected to provide a
high discrimination between the bound sequences and scrambled DNA of similar composition
(Supplementary Figure 5). Promoters selected from those in the genome that did not exhibit Rbf1
binding were tested for frequency of these motifs; E2F, DREF, and RAM motifs were
considerably less enriched on these promoters than on those bound by Rbf1, indicating that these
sequences may play a role in recruiting of Rbf1, or coregulation of the associated promoters (Fig.
6C). FOXJ2 sequences were not preferentially enriched on Rbf1 bound promoters, although
these sequences occur at a higher frequency than would be expected by chance, thus it is likely
that these motifs are relevant to promoter function in general.
Regarding overall promoter composition, just under one half of the peaks contained at
least one copy of one of the four motifs identified. A quarter of the peaks had a mixture of two of
the motifs, and a small percentage had three or all four of the motifs (Fig. 6D). One-fourth lacked
any of these motifs; these promoters may contain novel motifs that are not found in many genes,
or they may contain low-affinity canonical sites that fell below the threshold used here (see
Materials and Methods). Interestingly, of the peaks that contained recognizable motifs, only
about a third contained the E2F motif, although this has been presumed to be the chief route by
which Rbf proteins are recruited to promoters (Figure 6D insert). There may be other factors
involved in recruiting Rbf1, or low-affinity E2F sites may be important on some genes. Indeed,
Rbf1-bound regions as a group tend to be enriched in E2F-like sites (Supplementary Figure 6).

133

Enrichment of motifs in different promoter subclasses
In light of the diverse cellular processes represented among the targets of Rbf1, we
studied whether different classes of genes exhibited distinct promoter composition (Fig. 7).
Sorting genes by GO category, we noted that genes involved in Phagocytosis, Chromatin
Modification and Cell Cycle were among those most highly enriched in E2F motifs, while
Neurogenesis and Oogenesis GO categories are depleted of these motifs. The novel RAM motif
co-occurred frequently with E2F sites, except for certain GO categories such as Chromatin
Modification while DREF sites, which had been previously shown to help regulate cell cyclerelated genes such as PCNA and DNApol alpha, were not overall enriched on this class of gene,
instead showing strong association with genes involved in Apoptosis. FOXJ2 was also
differentially distributed, showing some correlation with RAM sites, but it was not highly
enriched in any subcategory (Fig. 7A). The overall impression obtained from this analysis is that
Rbf1-bound regions vary strongly in their average composition; it is likely that functional classes
of genes are coordinately regulated by unique combinations of factors that interact with these
motifs. Factors responsible for Rbf1 recruiting may vary as well, possibly placing some Rbf1bound promoters out of reach of the canonical cell-cycle regulatory pathways.
We also analyzed the association of motifs with groups of genes drawn from our set of
Rbf1-associated promoters that are defined by other properties, rather than the GO categories
identified by DAVID. The separate categories analyzed included a set of genes affected by RNAi
knockdown of Rbf1, Rbf2, E2F1, and E2F2 in S2 cells (Dimova et al. 2003), genes misexpressed
by knockdown of the l(3)mbt malignant brain tumor protein that interacts with Rbf-containing
complexes (Janic et al. 2010), functional targets of the MMB/dREAM complex identified by
knockdown in Kc cells, physical targets of the Rbf1-associated MMB/dREAM complex

134

Figure 3.7

135

Figure 3.7 cont’d.
Figure 3.7. Enrichment of Rbf1-associated motifs indicates distinct promoter subclasses in
the Rbf1 regulon. (A) Heat map for association of motifs with different GO categories. E2F
sites were present in a significant fraction of bound regions as a whole, especially in GO
categories Phagocytosis, Chromatin Modification, and Cell Cycle. Genes involved in
Neurogenesis and Oogenesis tend to be depleted of E2F motifs. RAM sites tend to occur on a
subset of E2F-containing sequences, but Chromatin Modification and Nucleotide Metabolic
Process genes are depleted of RAM motifs. Apoptosis and Transcription/Translation genes are
associated with DREF motifs. Chromatin Modification, Phagocytosis and Oogenesis genes are
depleted of FOXJ2 motifs, while a larger fraction of Neurogenesis and Oogenesis genes lack all
of the four motifs. (B) Heat map for association of motifs with selected functional and physical
targets of Rbf1 including functional targets of Rbf in S2 cells (Rbf); functional and physical
targets of the Rbf- and Myb- containing dREAM complex; functional targets of l(3)mbt, a
corepressor and a binding partner of Rbf; and physical targets of Rbf1 identified in this study that
are involved in signaling pathways. The E2F motif alone is preferentially associated with Rbf1
functional targets in S2 cells (Rbf) and dREAM functional targets, however, a combination of
E2F and RAM sites is found preferentially on l(3)mbt functional and dREAM physical targets.
Signaling pathway Rbf1 target genes are depleted of all four motifs, suggesting a distinct
promoter signature. “Percent” indicates fraction of genes in a selected GO category containing at
least one occurrence of the indicated motif within the Rbf1 peak. The category “all” represents
all 1236 peaks present in both ChIP replicates. The category “other” represents small clusters of
genes found to be overrepresented in numerous GO categories (see Fig. 5). “Signaling pathway”
represents 136 genes found in diverse conserved Drosophila signaling networks.

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Figure 3.7 cont’d.

For interpretation of the references to color in this and all other figures, the reader is referred to
the electronic version of this dissertation.

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Supplementary Figure 3.7

Supplementary Figure 3.7. E2F responsiveness of promoters of selected genes in signaling
pathways. Drosophila S2 cells were cotransfected with InR, PCNA, Merlin, Rab23, Hippo, Dad,
p53-proximal, or Stat92E luciferase reporters, with (+) or without (-) a plasmid overexpressing
E2F1. Only PCNA luciferase expression was elevated by E2F1.

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components identified by ChIP (Georlette et al. 2007), and the set of genes that we identified in
this study that are components of conserved signaling pathways (Fig. 7B). Grouping genes in
these five (non-exclusive) categories, we found that the E2F motif is especially enriched for
functional targets of Rbf1 and MMB/dREAM identified in S2 cells. As noted above, the motif is
associated with cell cycle-related genes, which would be expected to be expressed in these
mitotically active cells. The E2F motif is also associated with phagocytosis-related genes, which
may also be preferentially active in the S2 cell line, which has properties of hemocytes (insect
macrophages). E2F sites were less enriched, and RAM sites were more enriched, on physical
targets of MMB/dREAM and genes misexpressed in l(3)mbt brain tumor samples; these genes
may represent developmentally regulated targets of Rbf1, as opposed to genes that are tightly
integrated into regular mitotic control pathways (Lee et al. 2010). In the fifth group, genes
involved in conserved signaling pathways, we noted that Rbf1-bound regions were depleted of
all four motifs, suggesting a distinct promoter signature for these genes. Further bioinformatic
analysis of the group as a whole did not find enrichment of new motifs, although weak E2F sites
were found (data not shown).
E2F proteins have been observed to bind diverse sequences in vivo (Bieda et al. 2006).
To directly test the possibility that some of the promoters with weak or nonexistent E2F sites
may nonetheless interact with this transcription factor, we overexpressed E2F1 and measured its
ability to activate a diverse panel of signaling pathway gene promoters. E2F1 strongly activated
the PCNA promoter as expected, however, none of the signaling pathway gene promoters tested
were activated by E2F1, indicating that these promoters are E2F-independent (Supplementary
Figure 7). Some promoters such as InR were slightly repressed, possibly because E2F1 may
drive expression of rbf1 gene itself. Taken together, we see evidence that Rbf1 binding takes

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place in the context of a rich diversity of motifs among the different categories of genes targeted,
suggesting that regulation of promoters by this corepressor may involve separate regulatory
programs, consistent with recent studies (Dimova et al. 2003; Kirienko and Fay 2007; Lee et al.
2010).
Correlation of Rbf1-target gene expression and Rbf1-binding
The RB protein is generally thought to mediate transcriptional repression, but mutant
studies have also identified genes that are downregulated upon loss of the protein (Dimova et al.
2003; Georlette et al. 2007; Kirienko and Fay 2007; Flowers et al. 2010). We therefore examined
whether targeted promoters tend to show a loss of activity at the timepoint when we see peak
Rbf1 binding, consistent with corepressor activity. Using recently described transcriptome data,
from ModENCODE, we grouped embryonic expression of genes into eight distinct profiles using
fuzzy c-means clustering, a method that identifies genes with the most similar patterns of change
(Figure 8A-H and Supplementary Table VI as in Acharya et al, 2012). 54% of the genes bound
by Rbf1 were enriched in three of these clusters, which all show downregulation of expression
during the 12-18 hour time period for which the Rbf1 data was collected. To test whether this
enrichment in certain clusters is significant, we randomly selected 2000 genes not targeted by
Rbf1 and determined whether there was significant enrichment in any of the eight clusters. No
significant enrichment was found for any of the five repetitions of this procedure (data not
shown). The composition of genes in these three clusters showed a strong bias toward cell cyclerelated genes (Fig. 8I), indicating that on these targets Rbf1 is likely to play a common repressive
role. Thus, at least for a large fraction of targeted genes, Rbf1 is implicated in transcriptional
repression.

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Figure 3.8

141

Figure 3.8 cont’d.

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Figure 3.8 cont’d.
Figure 3.8. Promoter occupancy by Rbf1 correlates with downregulation of target genes. AH show the expression patterns of all embryonic genes clustered into eight groups by similarity
of expression. The Y-axis represents standard Z score (see Materials and methods) for the
expression change. The X-axis represents age of the embryos in hours. The boxes indicate the 12
to 18 hr time window that was used for ChIP-seq. The enrichment of Rbf1 targets is indicated by
numbers above the boxes. The Rbf1 target genes are enriched in clusters B, D and G, which
comprise of 54% of the targets, and 33% of total genes. Genes in clusters B, D and G tend to be
downregulated between 12 to 18 hours, consistent with a repressive role for Rbf1. The RNA-seq
data for 0 to 24 hour embryos (in two hour windows) was obtained from modENCODE,
converted to relative changes in expression, and grouped using fuzzy c-means clustering.
Clusters are arranged according to timing of increased expression from early (A) through late
(H). Distribution of GO categories of Rbf1 targets in clusters A-H are shown in I. Statistics for
each cluster is shown in Supplementary Table VI (as in Acharya et al 2012). For interpretation of
the references to color in this and all other figures, the reader is referred to the electronic version
of this dissertation.

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Interestingly, some genes bound by Rbf1 (in particular cluster F) show upregulation
during the 12 to 18 hr time window. The composition of these genes showed a strong bias away
from cell cycle-related genes and are enriched in the highly heterogenous “other” GO category
(Fig. 8I). This increase in expression may occur despite Rbf1 promoter occupancy because of the
heterogeneity of cells in the embryos, so that only a fraction of nuclei have Rbf1 occupancy.
Alternatively, genes that are highly active may be also be intermittently occupied by Rbf1, or
Rbf1 may have true activator function in certain settings (Flowers et al. 2010).
Divergence and Conservation of Rbf1 regulon
To determine whether the genomic targets of the Drosophila Rbf1 protein represent
deeply conserved regulatory interactions, we compared human orthologs of the Rbf1-occupied
genes with those bound by human RB and p130 proteins in fibroblasts (Chicas et al. 2010). The
1890 Drosophila genes identified in our study correspond to 2310 human orthologs. We
compared these genes to those bound by RB or p130 in growing, quiescent, or senescent
fibroblasts (Figure 9). Close to half of the orthologs were identified as RB targets under at least
one condition, while just over 60% of the orthologous genes were bound by p130 (Fig. 9A-E).
Among the genes bound by RB or p130, the GO categories DNA Replication, Cell Cycle, DNA
Damage/Repair and Chromatin Modification were under all conditions enriched. The GO
category Cytoskeleton, which was found to be enriched in the Rbf1 targets in the Drosophila
embryo, was actually depleted from genes bound by RB in growing and quiescent cells,
however, it was enriched in genes bound by RB in senescent cells, as well as p130. This result
indicates that certain categories of genes can be selectively occupied depending on the state of
the cells, underscoring the differential regulation of subsets of RB/p130 targets. Finally, GO
categories such as Oogenesis, Phagocytosis, and Neurogenesis that were overrepresented among

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Drosophila targets were slightly or not at all enriched in the set of human genes. The common
binding of genes involved in chromatin modification, cell cycle, DNA repair and replication by
RB family members suggests that they represent deeply conserved functions of this family of
proteins. Other categories of genes may represent lineage-specific innovations or tissue-specific
binding interactions that are not present in the cell culture system.
The intriguing targeting of many conserved signaling genes by Rbf1 in the Drosophila
embryo let us examine whether RB/p130 show similar binding preferences. The vast majority
(106/137) of signaling components bound in the fly were also found to have RB and/or p130 at
the promoter in human fibroblasts. These corepressors were found at 111 additional signaling
pathway genes that lacked Rbf1 occupancy in the embryo (Supplementary Table IX as in
Acharya et al, 2012). The high proportion of conserved signaling pathway genes targeted by RB
family proteins suggests that this proposed regulatory connection may represent an essential link
between multiple cellular components of growth control and differentiation.

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Figure 3.9

146

Figure 3.9 cont’d.

147

Figure 3.9 cont’d.

148

Figure 3.9 cont’d.
Figure 3.9. Divergence and conservation of RB regulon. 2310 identifiable human orthologs of
Rbf1 targets were compared with RB targets in growing (A), quiescent (B) and senescent (C)
cells and p130 targets in quiescent (D) and senescent (E) cells. The overlaps in (A), (B) and (C)
were further compared with each other (F) and the overlaps in (D) and (E) were compared with
each other (G). Comparison of the total genes in (F) and (G) with each other (H), shows that
most of the targets of Rbf1 and RB, as well as Rbf1 and p130, are the same. In all overlaps in AE, GO terms DNA Replication, Cell Cycle, DNA Repair, and Chromatin Modification were
enriched, indicating that these genes may form a conserved ancient regulon of RB proteins. The
overlap of human homologs of Rbf1 targets in Drosophila embryos and RB and p130 targets in
human cell culture suggests that many genes have retained regulation by RB proteins since
divergence of these organisms. Other categories of genes may represent divergence of RB family
function, or context-dependent differences in binding. Human homologs for Rbf1 targets were
compared with published RB and p130 targets in growing, quiescent and senescent human lung
fibroblasts (Chicas et al. 2010). For interpretation of the references to color in this and all other
figures, the reader is referred to the electronic version of this dissertation.

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Discussion
Our analysis of genomic occupancy of Rbf1, the major retinoblastoma protein in the
Drosophila embryo, provides intriguing new pictures of activities of this conserved corepressor.
Previous genetic studies of this factor showed that the protein is not required in early
embryogenesis, despite the presence of this protein (Du and Dyson 1999; Stevaux et al. 2002;
Keller et al. 2005). Consistent with this picture, our temporal analysis indicates that there is a
widespread, perhaps universal regulation of Rbf1 binding during this period of development,
limiting access to promoter regions. Although phosphorylation of RB proteins is a well-studied
pathway that regulates contact of the corepressor with E2F proteins, there is no evidence that RB
proteins from early embryos show a preferentially hyperphosphorylated, slower-migrating form,
thus additional forms of regulation may be important for this developmentally controlled binding
(Stevaux et al. 2002; Keller et al. 2005).
Our study identified 1890 promoters that are bound by Rbf1; this is an order of
magnitude higher than the number of genes identified as functional targets of Rbf/E2F factors in
RNAi experiments conducted on cultured S2 cells (Dimova et al. 2003). A large majority of the
genes misregulated in S2 cells after RNAi knockdown of Rbf1 were bound by the corepressor in
the embryo, indicating that many of these genes are indeed direct Rbf1 targets, but the question
remains how to interpret the other identified binding events. Some may represent fortuitous
associations that do not materially contribute to gene regulation, as has been suggested for some
ion change data for each gene. We chose a fuzzy c-(Georlette et al. 2007; Li et al. 2008),
the tight promoter localization puts the corepressor in a position that is very likely to influence
basal promoter activity and indeed we show evidence that at least some of these promoters can
be repressed by Rbf1. We favor the idea that only a fraction of the genes that we identified are

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affected in cell culture because S2 cells do not represent the complex mixture of differentiated
tissues that we sampled in the embryo. Activation of some of these genes may require costimulatory signals that are lacking in the cell culture system. Alternatively, the RNAi
knockdown may have not been extensive enough to uncover the true scope of Rbf1 regulation.
One of the most surprising findings of our study is the extensive occupancy of multiple
nodes of conserved signaling pathways by Rbf1 (Fig. 3 and Supplementary Table II). This aspect
highlights one feature of RB biology that this ChIP-seq analysis in developing embryos has
brought to the fore. Previous studies that examined the regulons or direct physical targets of RB
proteins have not emphasized this striking aspect of the system (Dimova et al. 2003; Georlette et
al. 2007; Kirienko and Fay 2007; Ferrari et al. 2008; Chicas et al. 2010), which may be partially
due to the heavy reliance on cell culture systems for this information; certain promoters may
only be bound in a developmental context. However, this aspect of RB biology may also have
been overlooked in part because GO categories do not specifically identify individual signaling
pathway genes. We reanalyzed the genes identified as RB and p130 targets in human fibroblasts
and found that over 200 signaling pathway genes are bound in these cells (Chicas et al. 2010 and
data not shown). An additional feature of the human cell data set is that RB and p130 in cultured
cells appear to occupy a higher percentage of total promoters than does Rbf1 in the Drosophila
embryo, which tends to obscure the enrichment of any particular set of genes.
There is abundant evidence linking RB and conserved signaling pathways. The Hippo
growth control pathway has been recently found to control Rbf1 activity itself, suggesting that
there are homeostatic feedback loops regulating Rbf and Hippo levels (Nicolay et al. 2011;
Tschop et al. 2011). Previous functional studies linked RB regulation to individual components
of the insulin signaling and S6 kinase pathways in mammals and plants (Hsieh et al. 2008;

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Annicotte et al. 2009; Mercader et al. 2009; Henriques et al. 2010). Recent studies have also
highlighted the functional interaction of RB regulatory pathways with insulin signaling; (Hsieh et
al. 2008; Mercader et al. 2009; Henriques et al. 2010). The direct targeting of signaling
component gene promoters by RB family members may provide one means for a molecular
linkage of these conserved pathways.
For example, the integration of RB and insulin signaling would provide a means by
which the sensitivity of this pathway would be controlled through differential expression of the
insulin receptor, downstream kinases, and targets such as 4E-BP, a regulator of translation.
Occupancy of target genes in the Wnt, Hh, EGFR, JNK, TGF-β, PI3K/Akt, insulin, AMPK,
Notch, Hippo, JAK/STAT, NF-B, and TOR pathways indicates that Rbf1 has the potential to
exert broad and concerted regulation of multiple signaling systems. Of the ~300 genes that we
identified as core constituents of these pathways in Drosophila, about 46% exhibit significant
promoter-proximal signals for Rbf1 occupancy, more than twice the frequency for genes at large.
It is possible that Rbf1 controls all promoters involved in signaling pathways in a unified
manner, or that some promoters are especially sensitive to the levels/activity of Rbf1 protein;
determining how promoters of individual components of these pathways respond to this
corepressor will be a first step to quantitatively modeling the interaction of these systems. One
feature of the signal transduction genes bound by Rbf1 is the relative paucity of genes encoding
extracellular signaling proteins; despite rich representation of receptors and intercellular
components, very few ligands involved in the signaling pathways were among the observed
targets of Rbf1. Perhaps Rbf1 is involved more in setting the cellular response curves of these
systems than the levels of signals impinging on a cell.

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Analysis of the physical targets of Rbf1 point to a richer suite of regulatory mechanisms
for this protein’s output than has been previously indicated. Much attention has been focused on
the role of reversible phosphorylation in regulation of RB activity, and other posttranslational
forms of RB protein family regulation are well known, including proteolysis, methylation, and
acetylation that control abundance, binding of regulatory factors, and nuclear localization. Our
genomic analysis indicates that regulation of the RB pathway may extend to five levels of a
functional hierarchy: through transcriptional control of cyclin and Hippo kinases that modify the
protein and its function, through direct regulation of its own promoter, through regulation of
proteins that work together with Rbf1 in some contexts to effect repression (MMB/dREAM and
l(3)mbt), through regulation of downstream kinases that control meiotic and mitotic cell cycle,
and lastly through regulation of the levels of proteins that can functionally antagonize Rbf1
repression, namely the E(bx)/NURF301 chromatin remodeling factor. These types of regulatory
linkages are unlikely to be restricted to Drosophila, thus the picture that emerges of RB pathways
is one of tightly interwoven connections, where transcriptional links mediated through this
family of proteins are likely to play important roles in adjusting the set points of numerous
signaling pathways.
The almost exclusive genomic binding of Rbf1 very close to transcriptional start sites
indicates that Rbf1 associates with genes in a very different sort of way compared to the binding
of other transcriptional cofactors. Groucho and CtBP corepressor proteins are very broadly
distributed, with no predisposition to localize to the transcriptional start sites (modENCODE). In
contrast, most of the E2F2-containing MMB/dREAM components are tightly linked with basal
promoters (Georlette et al. 2007). We hypothesize that E2F proteins may be short-range
activators that only function when bound close to the basal promoter, similar to Sp1 activator

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proteins. To antagonize them, Rbf1 would be colocalized to these regions. However, many of the
Rbf1-associated promoter regions that we identified lack high-affinity E2F sites, thus other
short-range activators may also be involved. Alternatively, it is possible that the entire Rbf1
regulon consists of genes with compact promoter structures that do not use distantly-acting cis
regulatory elements. Interestingly, in reanalyzing the data developed in Chicas et al. we note that
about 70% of RB and p130 binding interactions are found within 1kb of the transcriptional start
sites of genes, suggesting that promoter proximity is a conserved feature of the RB family of
corepressors (Chicas et al. 2010).
Our bioinformatic analysis of the Rbf1-bound regions clearly indicates that the regions
occupied by Rbf1 are heterogeneous, and that certain combinations of motifs are closely
associated with functionally related genes (Fig. 7). In some cases, these motifs may recruit
proteins that bind adjacent to Rbf1 to provide specialized responses, similar to the way that
modulatory proteins in mammals bind near E2F sites to functionally differentiate subclasses of
these promoters (Jin et al. 2006; Freedman et al. 2009). The Rbf-associated MMB/dREAM
complex, which contains several DNA-binding proteins, provides one example of this context:
the complex binds to about 70% of the genes targeted by Rbf1 in the embryo (Supplementary
Table VIII as in Acharya et al 2012) (Georlette et al. 2007). Interestingly, few of these genes are
involved in signaling pathways, suggesting that alternative Rbf1-containing complexes form on
these promoters. An additional feature of the signaling pathway genes is their lack of highaffinity E2F motifs, or sequences resembling the other three overrepresented motifs found on the
rest of the Rbf1 targets, suggesting that these genes may recruit Rbf1 by interactions with novel
transcription factors. Alternatively, E2F may interact with these promoters via non-canonical
sites (Bieda et al. 2006; Xu et al. 2007). Our functional testing for E2F1 responsiveness

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(Supplementary Figure 7) strongly suggests that at least some of these alternative Rbf1 target
genes are regulated by a distinct promoter grammar. A preliminary bioinformatic analysis of
these signaling pathway promoter regions did not identify motifs common to the whole set,
therefore it is possible that there are subclasses of motifs that are involved in setting a
transcriptional “grammar” for individual pathways.
In summary, our genomic identification of Rbf1 targets in the Drosophila embryo
provides the first view of this important class of corepressor in a whole animal; we find that in
addition to a core of conserved genes related to RB protein function in cell cycle and DNA
replication, the Rbf1 occupied genes are distributed among a diversity of functions. The
complexity of binding regions occupied by Rbf1 among different classes of genes strongly
indicates that this corepressor is involved in gene regulation in very different contexts,
interacting with promoters that are occupied by distinct types of transcription factors. Such
complexity would allow the development of independently-controlled groups of Rbf1 target
genes. Much work remains in deciphering the “promoter grammar” of these regulatory regions.
Most intriguingly, a high degree of enrichment of genes for conserved signaling pathways
suggests that Rbf1 is directly involved in setting levels of components of these systems at
multiple points; such regulation would change the sensitivity of signaling, which may vary from
tissue to tissue. Identifying the functional significance of RB interactions with genes from these
pathways will clarify new pathways of regulation of importance in development and disease.

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Materials and Methods
Fly stocks
Embryos of a Drosophila melanogaster yw67 strain were used for all chromatin
immunoprecipitation assays.
Reporter constructs and luciferase assay
To further analyze target genes bound by Rbf1, upstream promoter regions of InR from 1000 to -1, Mer from -600 to +400, Rab23 from -900 to +100, Hpo from -600 to +61, Dad from 500 to +100, p53-proximal from -204 to +50, Stat92E from -500 to +152, and Act5C from -900
to +100 with respect to the transcriptional initiation sites were PCR amplified and cloned into
XhoI and AscI sites in pAC2T-luciferase vector (Ryu and Arnosti 2003). Each clone contained
the portion of DNA bound by Rbf1 in the embryo. In addition, PCNA-luciferase reporter
(Acharya et al. 2010) was also used as a positive control. Drosophila S2 cells were transfected
using Effectene transfection reagent (Qiagen, Valencia, CA) according to the manufacturer’s
protocol. 1.5 million cells were transfected with 600 ng of one of the luciferase reporters, 250 ng
of pRL-CMV Renilla luciferase reporter (Promega, Madison, WI), and 250 ng of pAX-rbf1
(Acharya et al. 2010) or 20 ng of pIE4-myc-E2F1 (Frolov et al. 2001). Cells were harvested 72h
after transfection and luciferase activity was measured using Dual-Glo Luciferase assay system
(Promega) and quantified using the Veritas microplate luminometer (Turner Biosystems,
Sunnyvale, CA).

Chromatin Immunoprecipitation
Chromatin Immunoprecipitations were conducted using yw Drosophila melanogaster
embryos collected at room temperature and aged as indicated in Figure 1. For Supplementary

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figure 3, embryos were collected from Drosophila melanogaster harboring Flag epitope tagged
rbf1. For preparation of chromatin, embryos were collected, dechorionated with bleach and
placed in a 50-ml tube with 9.4 ml of phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10
mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4). 0.6 ml of freshly prepared 25mM DSP
[dithiobis(succinimidyl propionate)] was added to the embryos in this buffer, and the tube was
shaken vigorously for 30 min at room temperature, centrifuged at 1500 rpm in a Beckman
Allegra 6R clinical centrifuge for 5 min and supernatant was removed. Embryos were fixed for
15 min with vigorous shaking in a 50 ml tube in 9.2 ml crosslinking buffer (50mM HEPES [pH
7.6], 1mM EDTA, 0.5mM EGTA, 100mM NaCl), 0.81 ml of 37% formaldehyde and 30 ml
heptane. The cross-linking reaction was stopped with 25 ml stop buffer (0.125M glycine, 0.01%
Triton X-100 in phosphate-buffered saline [PBS]) while the tube was shaken vigorously for 15
min at room temperature. The supernatant was removed and the embryos were washed in 10 ml
embryo wash buffer (10 mM HEPES[pH 7.6], 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA,
0.01% Triton X-100) for 10 min with vigorous agitation at room temperature. The supernatant
was removed and embryos were resuspended in 5 ml of sonication buffer (10 mM HEPES[pH
7.6], 1mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate), transferred to a 15-ml tube and a
proteinase inhibitor tablet (Roche complete mini, 11-836-153-001) was added. The embryos
were sonicated for 20 s (60% duty cycle) and cooled on ice for 30 s a total of 12 times followed
by three 30 s sonication and 30 s cooling cycles, using a Branson sonicator. Crude chromatin was
centrifuged at 14000 rpm in a microcentrifuge at 4°C and supernatant was transferred to a 15-ml
tube. An equal volume of room temperature 2X radioimmunoprecipitation assay (RIPA) buffer
(2% Triton X-100, 280 mM NaCl, 20 mM Tris-HCl [pH 8.0], 2 mM EDTA) was added. The
chromatin was precleared by adding 10 μl/ml of a 50% slurry containing an equal mixture of

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agarose beads coupled to protein A and protein G (Millipore; equal volume of protein A and G
beads were mixed) previously washed three times with 1 X RIPA buffer and blocked 2 hr at
room temperature with 0.1 mg/ml bovine serum albumin and 0.2 mg/ml salmon sperm DNA. For
immunoprecipitations, 1 ml of precleared chromatin was incubated with 5 μl preimmune, 5 μl
Rbf1 antibody (Keller et al. 2005), 5 μl of Flag antibody (Sigma; F7425) as used in (Acharya et
al. 2010) or 2 μl H3 antibody (Abcam, Cambridge, MA; 0.4 μg/ μl) overnight at 4°C. After
overnight incubation, the samples were centrifuged at 14000 rpm in a microcentrifuge for 10
minutes at 4°C. Supernatant was taken in microcentrifuge tubes and 40 μl blocked beads (as
described above) were added to each sample and incubated for 2 h (with rotating) at 4°C. The
beads were centrifuged in a microcentrifuge at 1000 rpm for 1 min, washed twice with 1ml ice
cold low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl [pH 8.0], 150
mM NaCl), twice with ice cold high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20
mM Tris-Cl [pH 8.0], 500 mM NaCl) and twice with Tris-EDTA (10 mM Tris [pH 8.0], 1 mM
EDTA). Chromatin was eluted at room temperature with 250 μl elution buffer (1% SDS, 0.1 M
NaHCO3) for 15 min without shaking. Beads were centrifuged, the supernatant was transferred
to a microcentrifuge tube, a second elution was performed, and supernatants were combined. 25
μl of 4M NaCl was added, and cross-links were reversed overnight at 65°C. The eluates were
then incubated with 1 μl RNase A (10 mg/ml) at 37°C for 30 min. 10 μl 0.5 M EDTA, 20 μl 1 M
Tris-Cl (pH 6.5), and 1 μl proteinase K (20 mg/ml) were then added and tubes incubated at 42°C
for 1 h. DNA was extracted with phenol-chloroform and precipitated with equal volume of
isopropanol, 3 M sodium acetate (final concentration 0.3M) and GlycoBlue pellet paint by
centrifuging at 14000 rpm at 4°C. The pellets were carefully washed once with 70% ethanol, air
dried and resuspended in 40 μl water. 2 μl of each ChIP sample was used for 31 cycles of PCR

158

for 0-6 hr embryos and 28 cycles of PCR for the DNA from other time points. The
oligonucleotides used for PCR are listed in Supplementary table VIII (as in Acharya et al, 2012).
Sequencing of immunoprecipitated DNA fragments
The double-stranded DNA ends were repaired with T4 DNA polymerase, Klenow
fragment and T4 PNK enzymes. After a second purification step, an adenine-residue was added
-

with Klenow [3’>5’ exo ] enzyme and again purified on Quiaquick columns. Adapters from
Illumina for LM-PCR were then ligated to the end of the DNA molecules. The product of the
reaction was then run on a 2% NuSieve agarose gel and a band corresponding to 200 bp was
extracted and purified. 20 cycles of PCR were performed using Phusion polymerase (Finnzyme
F-530S) and the Illumina oligos, and the products were purified by gel electrophoresis. High
throughput sequencing was performed on an Illumina Genome Analyzer with standard Illumina
36 cycles reaction kit. The DNA libraries generated (two Rbf1 and one preimmune) were
sequenced in one lane each.
Mapping the reads, peak finding and visualization
We obtained 13,909,250, 18,070,094 and 1,247,796 reads for two anti-Rbf1
immunoprecipitation libraries and one control library respectively. The quality-filtered 36-bp
short sequence reads were aligned to Drosophila melanogaster genome (Flybase build r5.28)
using Bowtie 0.12.3 (Langmead et al. 2009) with default parameters except that sequences were
required to map uniquely to the genome (setting –m 1). To identify Rbf1 bound regions, QuEST
software (Valouev et al. 2008) version 2.4 was used with relaxed stringency. A peak was called
if the ChIP to background enrichment was 2.5 fold. The peaks were visualized using the online
version of UCSC genome browser. We identified 2187 and 1337 peaks in two biological antiRbf1 immunoprecipitation replicates; there were 1236 peaks that were present in both replicates,

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which we termed Class A peaks. Additional 951 and 101 non-overlapping peaks are termed
Class B and Class C peaks respectively (Supplementary Table I as in Acharya et al, 2012). Class
A peaks were used for the all analyses except for the signaling pathway components (Figure 3)
where Classes A, B and C peaks were used. Intensities of peaks in first replicate were higher
(from 10 to 656) than in the second replicate (from 10 to 109). The intensities were on average
higher for Class A peaks than for Class B, and higher for Class B than C. We list the higher
intensities obtained from the first replicate experiment in Supplementary Table I (as in Acharya
et al, 2012) for Class A peaks.
Validation of ChIP-seq peaks
To independently assess enrichment of Rbf1 on novel target genes, several genes were
selected and their enrichment in ChIPed chromatin was tested by PCR (Supplementary Figure 2).
The immunoprecipitated material that was also used for Illumina sequencing was used to
validate the ChIP-seq peaks. The oligonucleotides used for PCR are listed in Supplementary
Table VII as in Acharya et al, 2012.
Determination of peak overlap in replicates, denovo motif discovery and motif analysis
Peaks observed in the two Rbf1-immunoprecipitate experiments for which the maximum
points were located within 200 bp of each other were considered to be overlapping peaks. For
each peak region, 100 bp sequences on each side of the peak maximum height location were
extracted using a Perl script (output_genomic_regions_from_calls.pl) that was obtained from
QuEST website (http://www.stanford.edu/~valouev/QuEST). Motif discovery was performed
using MEME suite version 4.3.0 (Bailey and Elkan 1994). The program was set to search for
overrepresented 5 through 15 mer motifs separately. The top four overrepresented motifs
identified for each k-mer were selected and compared against TRANSFAC and JASPAR

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databases using the online version of TOMTOM (http://meme.sdsc.edu). In most cases, similar
motifs were found for different lengths (5 to 15-mers); the motifs shown in Figure 6A represent
the shortest version of the motifs found to be overrepresented. We determined the quality of
individual motifs compared to the defined consensus sequences using MAST (MEME suite
version 4.3.0) on the sequences extracted from peak regions. To determine the significance
threshold that would provide the best discrimination between enriched motifs and chance
sequences shown in Figure 6B, the sequences under the peaks were randomized five times and
MAST was run for each scramble independently. A p <0.0001 was found to provide the biggest
difference between the percentage of randomized promoters containing the overrepresented
motif and the percentage of Rbf1-bound regions. To determine whether the overrepresented
motifs identified were enriched specifically on promoter regions associated with Rbf1 binding
shown in Figure 6C, DNA sequences extending from -100 to -300 from one thousand randomly
selected Drosophila promoters not bound by Rbf1 were used for background analysis using
MAST, and this process was repeated for total of five different sets of one thousand non Rbf1binding promoters.
Gene ontology analysis
Genes with transcriptional start sites (TSS) within 2kb from peak maximum were
considered associated with the peak. 1169 out of 1236 Class A peaks were mapped to TSS of
1890 genes. The enrichment of gene ontology terms was performed using online tool DAVID
(Huang da et al. 2009) and 321 original GO categories were identified. Using visual inspection,
related GO terms such as cell cycle, mitotic cell cycle, meiotic cell cycle etc were pooled into
one broader category Cell Cycle and DNA replication. In this way the GO terms with significant
enrichment were pooled into a total of 10 different categories. Other terms with fewer genes and

161

less significant enrichment were grouped under the “others” category. We report in figure 5 the
p-values for the most populous subcategory of each super category shown on the pie chart.
Cluster analysis
We used Drosophila RNAseq data from modENCODE (http://bit.ly/iSOyEy) from 0 to
24 hours (in two hour windows) and replaced the missing value (NA) with 0. By normalizing
each gene with

xij  xi

ˆ
xij 

, j  1,..., n

n

1
( xij  xi ) 2

n j 1

we obtain the expression change data for each gene. We chose a fuzzy c-means clustering
method (Dembele and Kastner 2003), which creates a fuzzy boundary for each cluster. By
minimizing the objective function

N

C

J m    uij xi  c j ,1  m  
2

m

i 1 j 1

N

uij 
where

1
 xi  c j
 x  c
 i k
k 1

C






2
m1

cj 
,

 uij xi
i 1
N

 uij
i

we obtained the cluster index for each gene. In this study we chose m=1.25, C=8.
162

m

m

Identification of human orthologs to genes bound in Drosophila by Rbf1
The human orthologs of the 1890 genes associated with Class A peaks of Rbf1 in
Drosophila were obtained from FLIGHT (http://flight.icr.ac.uk/). To determine conservation of
RB family binding to conserved signaling pathway genes, the 295 genes listed on Supplementary
Table IX (as in Acharya et al, 2012) were input into FLIGHT. An ortholog was considered
bound in both fly and human if at least one member of an orthologous family was occupied in
each species.

163

Acknowledgements
We thank Titus Brown, Mark Robinson, Shin Han Shiu, Gaurav Moghe, Nitin Raj,
Satyaki Sengupta, Liang Zhang and Arnosti lab members for helpful discussions. We also thank
the MSU Institute for Cyber Enabled Research (iCER) for computational support, He Zhang for
construction of InR-luciferase reporter. PA, RWH and DNA conceived of the project. PA carried
out chromatin immunoprecipitations, cloning, luciferase assays and bioinformatic analysis, and
wrote the paper. NN and KPW conducted the sequencing and assisted with bioinformatic
analysis. JJ assisted with custom python scripts. HW conducted statistical analysis of expression
of Rbf1 target genes compared to total gene expression. YW carried out some of the transfection
assays and qPCR. DNA and RWH directed the project, helped interpret the data, and assisted
with writing. This study was supported by NIH GM079098 to DNA and RWH, and
U01HG004264 from the National Human Genome Research Institute to KPW. PA was
supported by a dissertation continuation fellowship from MSU College of Natural Science and
the Hsiung-Kimball Scholarship from the MSU Department of Microbiology and Molecular
Genetics.

164

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CHAPTER IV
Conclusions and Future Perspectives
The studies that I describe in this dissertation elucidate novel mechanisms through which
the Drosophila Rbf1 is regulated, and uncovers unprecedented sets of Rbf1 target. Here I discuss
three main discoveries that we have made and their significance in the field of retinoblastoma
biology. Firstly, the repression function is closely linked to the turnover of the pocket proteins.
Secondly, Rbf1 appears to target many components of conserved signaling pathways, suggesting
that retinoblastoma family proteins might have broader role in development than just controlling
cell cycle genes. Thirdly, I will also discuss the possibility of E2F independent regulation of
target genes by Rbf1 and the impact on “promoter grammar”.
Evidence of linkage between turnover and repression activity of Rbf1
Several studies have shown that for the transcriptional activity of the activators such as
p53, β-Catenin, Rpn4, Glucocorticoid receptor (GR), c-Jun, Hif1α, VP16, Myc, Gcn4, Ste12,
Androgen receptor (AR), Estrogen receptor, Gal4 and Notch intracellular domain, proteolytic
destruction is required [1-3]. These studies further pointed out that the transcriptional activation
domains and degrons overlap in most of these unstable transcriptional activators [4]. It has been
proposed that when activators interact with general transcriptional machinery, they recruit
ubiquitin ligases to the site of transcription. These ligases then ubiquitylate several factors such
as, the activators, RNA polymerase II and histones, which in turn recruit the 26S proteasome.
This results in turnover of activators and promotion of transcriptional elongation by RNA pol II
[4]. For the first time we reported that this instability-activity link is true for repressors too, as in
the case of Rbf1 discussed in Chapter II [5]. We showed that the instability element (IE) present
in the C-terminal region of Rbf1 is responsible for the activity and stability of the protein. We

172

also showed that this region is responsible for the accumulation of Rbf1 in actively proliferating
tissues and not in the other tissues, suggesting that it has a role in developmental regulation of
Rbf1.
Our study initiates several interesting questions. Is IE an autonomous degron? Future
studies with the IE or IE-inactivating lysine mutations, fused to other protein such as Gal4 would
address this question. An assay with proteasome inhibition would test whether the IE is an
autonomous degron. Additionally, tethering IE or its mutant version to a promoter would test
whether the IE is sufficient for repression. In the same study described in Chapter II, we also
showed that the orthologous region in p107 is also responsible for its accumulation in S2 cells.
Similar studies with the orthologous regions of mammalian pocket proteins would further test
how general is the mechanism of the IE among pRB proteins. This question is especially
important because mutations in C-terminal region of pRB are associated with a variety of
osteosarcomas [6, 7] and mutations in C-terminal region of p130 orthologous to IE are associated
with lung tumors [8]. Furthermore, we have shown that Rbf1 is developmentally regulated and
IE plays a role in it, so are the IEs of mammalian pocket proteins responsible for developmental
regulation? Thus, the studies with IE to answer above mentioned questions would provide further
insights in tumor biology.
We know that Rbf1 is degraded by proteasome mediated turnover and the IE mutants are
resistant to it. However, we showed that certain lysine residues in the IE region are important for
Rbf1’s activity and turnover. We hypothesized that lysines may be ubiquitylation targets of E3
ubiquitin ligases and this process targets Rbf1 to proteasomal degradation. Interestingly, when
we mutated those lysines to arginines conserving the charge, Rbf1 still was active and actively
turned over. So the question is if Rbf1 is turnover by proteasome mediated turnover and IE is

173

also responsible for its turnover, how is IE mediating this regulation even when lysines are
replaced with arginines (which are not ubiquitylated)? Ubiquitylation studies with full length and
IE mutant Rbf1 (and mammalian pocket proteins) and GFP fused to IE or IE mutants can further
elucidate whether the turnover is ubiquitin dependent or independent and whether IE is a target
for ubiquitylation or serves as a docking site for ubiquitin ligases.
Previous studies from our group have shown that the COP9 signalosome (CSN)
physically associates with Rbf2 and knockdown of individual CSN subunits destabilizes Rbf1
and Rbf2, suggesting that COP9 protects Drosophila pocket proteins from degradation [9]. Now
we have two processes to stabilize Rbf1; first is COP9 mediated stabilization and second is IE
mediated destabilization. What is the link between COP9 signalosome’s role in stabilizing Rbf1
and IE? Is COP9 signalosome masking IE from E3 ligases and deletion of IE is sufficient to
provide protection provided by COP9 signalsome? Or is the IE mutant Rbf1, which is stable,
destabilized by CSN knockdowns? These questions can be answered by first finding out the
Rbf1-COP9 interaction domain in Rbf1 followed by generation of CSN-binding deficient and IE
combo mutant Rbf1. This would broaden our understanding of CSN-mediated regulation of Rbf
protein stability.
Targeting of various conserved signaling pathways by Rbf1
One of the most astonishing findings of our study, as described in Chapter III, is
previously unappreciated extensive occupancy of multiple nodes of conserved signaling
pathways. Previous studies that examined the regulons or direct physical targets or functional
targets of pRB proteins have not emphasized this striking aspect of the system [10-14], which
may be partially due to the heavy reliance on cell culture systems of this information. Several
studies have shown the link between retinoblastoma proteins and several conserved signaling

174

pathways [15-24]. As described in Chapter III, we showed possibility of direct regulation of
several components of conserved signaling pathways, which further supports the involvement of
pocket proteins in those pathways as found in studies from different groups. In addition, I
showed that Rbf1 represses InR- (insulin signaling pathway) and Rab23- (hedgehog signaling
pathway) luciferase reporters. This finding strongly suggests that Rbf1 targets these signaling
pathway components and represses them.
Our finding raises an interesting question whether Rbf1 regulates other components of
the conserved signaling pathways. Similar reporter assay in cell culture with promoters of those
genes would answer this question. It might be possible that some of the pathways might require
stimulation in order to show response, because cell culture system might lack those stimulatory
signals. Some of the genes might also be developmentally regulated, and might not show
response in cell culture. These might be reasons this feature of retinoblastoma tumor suppressor
was not underscored in previous studies [10, 11, 13, 14]. In that case a reporter assay in fly
tissues in an rbf1 wild type or mutant background might be helpful to elucidate the function of
the repressor on those genes. In addition, incorporation of Rbf2 in the assays may throw some
light on this feature, as Rbf1 and Rbf2 share some functional redundancy.
Another intriguing finding that may have significant implication is my finding that Rbf1
binds the p53 promoter. Crosstalk between the pRB and p53 pathways has been well studied;
inactivation of retinoblastoma proteins leads to upregulation of p53 [25-30]. My observation of
physical presence of Rbf1 on the p53 promoter raises an important question, namely, does Rbf1
transcriptionally regulate p53? I and Jonas Pedersen (a Master’s degree student in our lab) have
conducted preliminary S2 cell experiments with p53-luciferase reporter, but thus far we have not
seen significant repression of the gene by Rbf1. We may need to first stimulate transcription of

175

p53 using agents such as doxorubicin. Measuring endogenous p53 mRNA levels after Rbf1 and
Rbf2 knockdown would also be helpful in understanding the above question. Furthermore, this
study can be extended in Drosophila by generating rbf1/rbf2 conditional knockout transgenic
flies, knocking out the genes in a tissue specific manner and looking at the endogenous p53
levels. Alternatively, levels of p53-GFP can be visualized in rbf1/rbf2 background tissues.
Finally, extension of these studies in human cell lines would give a bigger picture on how pocket
proteins regulate p53. These studies would help broaden our understanding on crosstalk between
RB and p53 pathways.
E2F-independent regulation of target gene promoters by Rbf1
It is well known that RB family proteins do not bind to DNA directly, but are recruited to
the target gene promoters by the E2F family of transcription factors [31, 32]. In my
bioinformatics analysis described in Chapter III, I found that only a third of Rbf1 binding peaks
had high affinity E2F binding sites. Does Rbf1 bind to the remaining promoters independently of
E2F transcription factors? There are two possibilities, either those regions have poor E2F sites or
maybe Rbf1 is recruited by factors other than E2Fs. Consistent with the latter possibility, we
show that ectopic expression of E2F1 increases expression of a PCNA-luciferase reporter, which
is known to be bound by E2F, but not of an InR-luciferase. This hypothesis would be bolstered
by chromatin immunoprecipitation (ChIP) with Rbf1 and Rbf2 antibodies after E2F1, E2F2 or
DP knockdown. If we can still observe Rbf signal on the promoters lacking strong E2F sites after
E2F or DP knockdown, it would suggest that Rbf1 and/or Rbf2 are recruited to those promoters
in an E2F-independent manner. This approach can identify target genes that are regulated by
Rbf1 in E2F-independent manner, if any. A genomics approach is necessary to identify such
targets globally. The Botchan lab has shown the global targets of dE2F2 [13], however, to my

176

knowledge nobody has reported genome-wide physical targets of dE2F1. Comparison of Rbf1
peaks that we identified with physical targets of dE2F1/2 may provide stronger evidence about
how Rbf1 is recruited to the promoters. These studies may change the current notion that E2Fplays the main role in binding of retinoblastoma proteins.
My genome-wide study of Rbf1 revealed some interesting facts; however, it was done on
12 to 18 h embryos. As we know that a multitude of genes are dynamically expressed throughout
the development of an animal, including Rbf1 and Rbf2. So how do the pocket proteins regulate
the genes at different development stages and different tissues? A developmental ChIP-seq study
with Rbf1 and Rbf2 would provide on the genes that are differentially regulated by these
retinoblastoma proteins.

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APPENDIX A
Nuclear localization of Rbf1 mutants
In Chapter II, we showed that Rbf1 ∆IE mutant loses its activity. We also ruled out the
possibility that the inactivity is not due its inability to localize to nucleus by
immunocytochemistry (Figure 4B, Chapter II). To see if activity of other mutants is affected by
exhibition of aberrant nuclear localization, I performed the same experiment (Chapter II,
Materials and Methods) with other mutants. Except the Rbf1 1-727, a C-terminal mutant, all the
other mutants were exclusively localized to the nucleus (Figure A.1).

182

Figure A.1

183

Figure A.1 cont’d.

184

Figure A.1 cont’d.

185

Figure A.1 cont’d.

186

Figure A.1 cont’d.
Figure A.1. Nuclear localization of Rbf1 mutants. Drosophila S2 cells were transfected with
flag-tagged Rbf1 (wild type or mutants) or RB or p107 and cultured for three days. Cells were
then fixed and the intracellular localization of Rbf1 proteins was assayed by indirect
immunofluorescence using the mouse monoclonal M2 anti-flag antibody followed by the antimouse IgG-FITC (red). Cells were counterstained with DAPI in order to visualize DNA (green).
Merged images are shown on the right column. 1-845 is Rbf1 wild type; 1-727 is Rbf1 ∆Cterminus; ∆728-786 is Rbf1 ∆IE; ∆728-747, ∆748-767 and ∆768-786 are three small deletions
within IE; 3K  A is K732A, K739A and K740A; 4K  A is K732A, K739A, K740A and
K754A; 6K A is K732A, K739A, K740A, K754A, K774A and K782A; 3K  R is K732R,
K739R and K740R; 4K  R is K732R, K739R, K740R and K754R; 6K R is K732R, K739R,
K740R, K754R, K774R and K782R; RB, human pRB; p107, human p107; Rbf10 is S728A,
S760A and S771A; Rbf30 is T715A, S728A, S760A and S771A; and Rbf280 is T356A, S728A,
S760A and S771A. Rbf10, Rbf30 and Rbf280 are mutants reported in Xin et al[1]. For
interpretation of the references to color in this and all other figures, the reader is referred to the
electronic version of this dissertation.

187

References

188

References
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Xin, S., et al., The role of RBF in developmentally regulated cell proliferation in the eye
disc and in Cyclin D/Cdk4 induced cellular growth. Development, 2002. 129(6): p. 134556.

189

APPENDIX B
Generation of epitope tagged CNS genomic rescue constructs
We showed that COP9 signalosome associates with Rbf1 and Rbf2 and protects them
from proteasomal mediated degradation [1] and that CSN4, a subunit of COP9 signalosome, cooccupies Rbf target genes together with Rbf1 and Rbf2. CSN4, CSN5 and CSN7 are found as
monomers [2, 3], it might be possible that just CSN4 be present at Rbf target promoters or the
whole complex is present. We hypothesized that “Entire COP9 complex co-occupies Rbf target
gene promoters with Rbf.” Since chromatin immunoprecipitation (ChIP) is considered a gold
standard assay for investing promoter occupancy by any protein, I sought to perform ChIPs with
COP9 subunits and Rbf1. In addition we also sought to map the interacting COP9 subunit with
Rbf1 or Rbf2. To pursue above experiments, we a handle on the proteins would be handy, thus I
constructed flag epitope tagged CSN2, CSN3, CSN4, CSN5, CSN6 and CSN8 genomic rescue
contructs. Further, I generated transgenic flies for these subunits using P-element mediated
germline transformation of yw flies.
Materials and Methods
Expression Constructs and Transgenic Lines
To express CSN proteins under control of the endogenous regulatory sequences, a 6 kb
genomic locus of CSN2 was cloned, extending from 2.1 kb upstream of first exon to 2.1 kb
downstream stop (2.1 kb downstream end of the last exon) into pCaSpeR[4] between KpnI and
XhoI sites in two steps using PCR amplification of genomic DNA. A 5.5 kb locus of CSN3 (1.8
kb upstream of first exon to 1.4 kb downstream of the last exon), 4.5 kb locus of CSN6 (2 kb
upstream of first exon to 1.3 kb downstream of the last exon), and 4.0 kb locus of CSN8 (1.8 kb
upstream of first exon to 1.4 kb downstream of the last exon) were cloned into pCaSpeR between

190

KpnI and XhoI sites in two steps using PCR amplification of genomic DNA. Similarly a 5.0 kb
locus of CSN4 (1.7 kb upstream of first exon to 1.6 kb downstream of the last exon) was cloned
into pCaSpeR between KpnI and HindIII sites and a 4.5 kb locus of CSN5 (1.0 kb upstream of
first exon to 2.2 kb downstream of the last exon), were cloned into pCaSpeR between EcoRI and
BamHI sites in two steps using PCR amplification of genomic DNA. Two Flag epitope tags were
inserted 5’ of the stop codon. The plasmids were used to generate transgenic flies by P-element
mediated germline transformation of yw flies. The transgenic flies were then balanced with SM2
CyO or TM3 Sb balancers.

191

References

192

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Ullah, Z., et al., Retinoblastoma protein regulation by the COP9 signalosome. Mol Biol
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2.

Freilich, S., et al., The COP9 signalosome is essential for development of Drosophila
melanogaster. Curr Biol, 1999. 9(20): p. 1187-90.

3.

Oron, E., et al., COP9 signalosome subunits 4 and 5 regulate multiple pleiotropic
pathways in Drosophila melanogaster. Development, 2002. 129(19): p. 4399-409.

4.

Schejter, E.D. and B.Z. Shilo, The Drosophila EGF receptor homolog (DER) gene is
allelic to faint little ball, a locus essential for embryonic development. Cell, 1989. 56(6):
p. 1093-104.

193