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31293 01820 0224

This is to certify that the

dissertation entitled

Structural and Functional Dissection of the
Activation Domains of the Yeast Transcriptional
Activator HAP4

presented by
John L. Stebbins

has been accepted towards fulfillment
of the requirements for

Ph.D . degree in Genetics

 

 

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1M chlRC/D‘qupfiG—au

STRUCTURAL AND FUNCTIONAL DISSECT ION OF THE ACTIVATION
DOMAINS OF THE YEAST TRANSCRIPTIONAL ACTIVATOR HAP4

By

John L. Stebbins

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Genetics Program
&
Cell and Molecular Biology Program

1 999

ABSTRACT

STRUCTURAL AND FUNCTIONAL DISSECT ION OF THE ACTIVATION
DOMAINS OF THE YEAST TRANSCRIPTIONAL ACT IVATOR HAP4

By

John L. Stebbins

The yeast protein Hap4p activates the transcription of genes required for growth
on nonferrnentable carbon sources. Like other transcriptional activators, Hap4p when
overexpressed causes toxicity. Unlike other activators, this toxicity is not relieved by
mutations in ADA2, ADA3, or GCN5, nor is the transcriptional activity of Hap4p reduced
in strains lacking ADA2, ADA3, or GCN5. Moreover, Gal4 fusion proteins bearing the
activation domain of Hap4 (but not those of VP16 or GCN4) were able to function in
ada2 or 3015 mutant strains. These results suggest that the HAP4 activation domain is
intrinsically ADA2, ADA3, and GCNS independent. Our goal is to define the essential
features and mechanism of action of Hap4p as a prototypical ADA-independent activator.

The activation domain of Hap4p has been previously mapped to amino acids 330-
554 (C-domain). In this work the boundaries of this C-terminal activation domain has
been further defined by deletion mutational analysis. The ADA-independent activation
function has been mapped to the 359-476 region of Hap4p. Within this minimal C-
domain we have identified several clusters of hydrophobic amino acids that are critical
for the trans-activation function of this domain. The simultaneous mutation to serine of
He390/I‘rp39l/Lys393/Ieu394, Phe456/Tyr458, or Leu466/Met467 resulted in a greater

than 10-fold fold reduction in activation potential of the 359-476 region of Hap4.

Contrary to previous results, we found that Hap4p 1-330 (N -domain) is able to
support growth of yeast on lactate medium. Moreover, when tethered to lexA, the Hap4p
N-domain can activate a reporter gene with lexA binding sites upstream. These results
imply that some region within amino acids 1-330 of Hap4p also possesses the ability to
activate transcription. This function has been mapped by deletion mutational analysis to
the 124-329 region of Hap4p. Transcriptional activation by this region is ADA-
depcndent and is less efficient than the C-domain (aa 359-476) of Hap4p. The activation
potential of the 124-329 region of Hap4 is dependent on Phel48/Leul49/Phe151 as the
simultaneous mutation of these residues to serine resulted in a construct that is devoid of
trans-activation potential.

To identify mechanistic targets of the Hap4C subdomain, we designed genetic
strategies based on screening or selecting for loss of lexA—Hap4 (aa330-554) activation
function. While we have been unable to identify any such targets as a result of these
genetic strategies, our attempts have resulted in the development of a new and promising
strategy. We will attempt to suppress point mutations that reduce the trans-activation
potential of the 359-476 region of Hap4 with a high copy yeast genomic library. We
hypothesize that we will be able to identify a mechanistic target of the 359-476 region in
this way.

The genetic screens described to this point represent one strategy for identifying
putative targets of the Hap4 activation domain. An alternative strategy is to test for
genetic interactions between Hap4 and genes already implicated in co-activator
mechanisms from other studies. We therefore tested the ability of Hap4 330-554 to

activate transcription in yeast strains that lacked SPD, SPT7, SPT8, or SPTZO and found

that lexA-Hap4 (aa330-554) is dependent on SPT7 and SP720 for its ability to function as

a transcriptional activator.

Copyright by
.HDPHQIiEYTEIHBHVS

1 999

To my family, and to Kirsten with love.
You are the most important people in my life!

Meet triumph and disaster
And treat those two imposters just the same

Paraphrased from Rudyard Kipling’s “If"

vi

ACKNOWLEDGMENTS

I am deeply indebted to Steve Triezenberg, as he has been both patient and stem
with me as needed. In large part due to his efforts and examples I have been able to
develop and grow as a scientist. I thank the members of my committee: Mike
Thomashow, Dennis Thiele, Helmut “Bert” Bertrand, and Lenny Robbins. They have
been singularly and collectively great. Their enthusiasm and effort has really helped the
evolution of not only this project but my scientific-self as well. I would especially like to
thank Dennis as he has always been an eager participant even though it meant that he had
to drive to East Lansing from Ann Arbor.

I thank members of the Triezenberg Lab past and present (Jeff, Eugene, Rath -
the original nut case, Peter — the original grump, The Big Danish (SJ 0), Crazy-Mao,
Greeny (DG), Marty, Lee, Susan, Kostas, Yuri, and Fan) for their companionship,
friendship, help, and advice. I learned a lot from each one of you. THANKS!

I have made many life-long friends during my time here and thank them for their
role in helping me through. This list includes among others: Tubby, Tuffy, Tim, Mark
the “Dr. Dr.”, Luc, Odette, and especially my local—H: Spencaaaaaahh.

I would also like to thank the members of the greater yeast community. Without
the generous help of people like Fred Winston and members of the Winston Lab, Shelley
Berger, Reyes Candau, Craig Peterson, Dan Gottschling, Kevin Morano, Eric Andrulis,
Jeff Smith, Susanne Kleff, and especially Dave McNabb (he has been extremely patient
and generous as he has fielded a majority of my inquires) none of this would be possible.

I thank my family for their support, encouragement, and cease-fire on the “When

are you going to graduate?” questions. And lastly, I thank Kirsten. I owe her a great deal

vii

for all that she has done to help me attain this goal. She has spent far too much time

alone while I have been in the lab. Thanks and I love you!

viii

TABLE OF CONTENTS

 

List of Figures xi
List of Tables xiii
List of Abbreviations xiv
Chapter One
Literature Review 1-29
Overview 1
Promoter Architecture 2
Promoter Context 7
RNA Polymerase II and Associated Factors ll
Chromatin Remodeling Complexes 24
Hap2/3/4/5 27
Chapter Two
Identification and Characterization of the Hap4 Activation Domains 30-72
Introduction 30
Experimental Methods 34
Deletion mutagenesis of Hap4 aa330-554 35
Deletion mutagenesis of Hap4 aa1-33O 37
B-Galactosidase Assays 37
Immunoblot Assays 37
Point mutant construction 38
Growth curve 39
Results 45
Discussion 64
Chapter Three
Identification of Hag activation domain targetm 73-100
Introduction 73
Experimental Methods 77
EMS mutagenesis 77
Mutant isolation using S-Floroorotic Acid 78
Yeast strain production 79

High copy suppression of Hap4 point mutants 79

ix

Results 82

L40/AMR70 82
BCYOS 84
JSYO3 86
High Copy Suppression 89
SPT3/7/8/20 94
Discussion 98
Chapter Four
Future direction and mmfitives 101-106

Bibliography 107- l 31

LIST OF FIGURES

Figure 1. Specific activities of deletion mutants of the Hap4 C-terminal domain

(aa-330-554) ............................................................................................................... 47
Figure 2. Immunoblot analysis of yeast cells expressing deletion mutants of

HAP4 gene fused to lexA. .......................................................................................... 49
Figure 3. The amino acid sequence of the Hap4 359—476 region .............................. 51
Figure 4. The contribution of hydrophobic residues to the activation potential

of the Hap4 359-476 region ........................................................................................ 53
Figure 5. Immunoblot analysis of yeast cells expressing point mutants of the

HAP4 gene fused to lexA ......................................................................................... 54
Figure 6. Hap4 (aa1-330) is capable of supporting growth in lactate media

in the yeast strain DMYl48 ........................................................................................ 56
Figure 7. Specific activities of deletion mutants of Hap4 N-terminal domain
(aal-330) .................................................................................................................... 57
Figure 8. The amino acid sequence of the Hap4 124-160 region .............................. 59

Figure 9. The contribution of hydrophobic residues to the activation potential
of the Hap4 124-329 region ........................................................................................ 60

Figure 10. Effect of deletion of gcn5 on the ability of Hap4 derivatives to
activate transcription of a lacZ gene ........................................................................... 62

Figure 11. Hap4 (aal-330) fails to support growth in lactate media in
gcn5 yeast. .................................................................................................................. 63

Figure 12. Sequence comparison of portions of the transcriptional activation
domains of VP16, GCN4, and Hap4 .......................................................................... 70

Figure 13. Schematic representation of the evolutionarily conserved sequences in
KlHAP4 and ScHAP4 ......................................................................... 72

Figure 14. The ability of a spontaneously occun‘ing JSYO3 mutant to
support trans-activation by lexA-Hap4 (aa330-554) .................................................. 91

Figure 15. The ability of a spontaneously occurring JSYO3 mutant to
support trans-activation by lexA-Hap4 or lexA-GCN4. ............................................ 92

xi

Figure 16. The effect of deletion on either SPT3, SP77, SPT8, or
SP720 on the ability of lexA-Hap4 (aa330-554) to activate transcription ................. 96

Figure 17. Immunoblot analysis of Hap4 (aa330-476) fused to lexA in
either wild type, Aspt7 or Aspt20 yeast cells ............................................................. 97

xii

LIST OF TABLES

Table l. Oligonucleotides used to construct Hap4 deletion mutants ........................ 40
Table 2. Mutagenic Oligonucleotides ........................................................................ 42
Table 3. Table of yeast strain genotypes ................................................................... 44
Table 4. Table of yeast strain genotypes .................................................................... 81

xiii

EMS

FOA

GTF

HAP4C

HAP4N

OD

PAGE

PBS

PCR

PIC

SDS-PAGE

TAF

TBP

LIST OF ABBREVIATIONS

amino acid

activation domain

3-amino- 1 ,2,4-triazole

base pair

C-terminal domain of RNA polymerase H
ethyl methane sulfonate

S-fluoroorotic acid

general transcription factor

heme activated protein

carboxyl terminal domain of HAP4
amino terminal domain of HAP4
kiloDaltons

RNA polymerase

optical density

polyacrylamide gel electrophoresis
phosphate buffered saline

polymerase chain reaction

pre-initiation complex
sodium-dodecyl-sulfate polyacrylamide gel electrophoresis
TBP associated facter

TATA-binding protein

xiv

TFII transcription factor, RNA polymerase H
UAS upstream activating sequence
URS upstream repressing sequence

VP16 virion protein 16 of herpes simplex virus

XV

W

W91

OVERVIEW

The appropriate and timely response to environmental stimuli and the control and
coordination of developmental processes and pathways are examples of how the accurate
control of gene expression is required for the survival and growth of an organism. Gene
expression is regulated through transcriptional and post-transcriptional mechanisms.
While the decision whether or not to transcribe a given gene at any given moment is an
obvious level of regulation, many other regulatory mechanisms exist such as mRNA
stability, mRNA export, differential splicing, as well as post-translational protein
modifications such as phosphorylation. Perturbation of the control of gene expression
can lead to various diseases such as diabetes and cancer. Thus the understanding of how
organisms maintain accurate control of gene expression is a topic of intense scientific
investigation.

In eukaryotes, transcription of the nuclear genome is the responsibility of three
different RNA polymerases. RNA polymerase I synthesizes the large ribosomal RNAs.
RNA polymerase II (RNAPII) is responsible for transcribing protein-coding genes and
the production of some small nuclear RNAs. RNA polymerase III synthesizes the
remaining small nuclear RNAs, as well as 5S ribosomal RNA and transfer RNAs. All
three polymerases depend on auxiliary factors called general transcription factors (GTFs)
for core promoter recognition. The GTFs, also referred to as basal factors, that are

required for recruitment of RNAPII to its cognate promoters include TFIIA, TFIIB,

TFIID, TFIIE, TFIIF, and TFIIH (where TFII stands for transcription factor for RNA
polymerase fl). Together the GTFs and RNAPH and promoter DNA make up the pre-
initiation complex (PIC).

The typical core promoter elements can include a TATA-like sequence, located
upstream of the transcription initiation site, and an initiator sequence (Inr) that
encompasses the transcriptional start site. The core promoter directs the accurate
initiation site, PIC complex formation site, and contributes to the overall level of
transcription. Regulatory elements include both upstream elements such as upstream
activating sequences (UAS) and upstream repressing sequences (URS) and bind activator
or repressor proteins respectively. These regulatory elements direct the spatial and
temporal patterns of gene-selective transcription.

Since the focus of my work is the control of expression of protein-encoding genes
in the budding yeast Saccharomyces cerevisiae, I focus on the details of RN APH function
in this organism for the remainder of this review. I will emphasize the role of yeast

genetics in establishing current models and in defining the roles of the known factors.

PROMOTER ARCHITECTURE

RN APII dependent promoters in Saccharomyces cerevisiae are typically
composite in nature containing combinations of five elements: upstream activating
sequences (UAS), upstream repressing sequences (URS), TATA elements, sites of

transcriptional initiation (Inr element), and homopolymeric (dA-dT) elements.

UAS

UAS elements are the sites for binding gene-specific activator proteins. Activator
binding results in promoter stimulation in response to environmental stimuli. UAS
elements are analogous to metazoan enhancer elements. However, while enhancers are
able to function when located either 5’ or 3’ of the TATA element, yeast UAS elements
are functional only when located 5’ of the TATA element in the few cases tested thus far
(Guarente, et al., 1984; Struhl, 1984). Once associated with its cognate UAS element, a
transcriptional activator protein facilitates an increase in mRN A production by RNAPII.
Mechanisms employed by transcriptional activators will be discussed in detail in sections

to follow.

URS

URS elements are the sites of binding for gene-specific transcriptional repressor
proteins. Repressor production is a cellular response to environmental stimuli.
Association of a repressor with its cognate URS binding site results in a reduction of
mRNA production by RNAPH. Several different mechanisms for repression exist.
Repressor proteins bound to URS elements can recruit co-repressor complexes such as
Ssn6-Tupl. Ssn6Tupl appears to mediate repression by modifying promoter-proximal
chromatin structures (Edmondson, et al., 1996; Gavin and Simpson, 1997; Huang, et al.,
1997; Roth, 1995). When the repressor Ume6 binds to its cognate URS element it
recruits the Sin3-de3 histone deacetylase complex (Kadosh and Struhl, 1997) which in
turn removes acetyl groups from histone H4 and lysine 5 and 12 within histone H3

(Kadosh and Struhl, 1998). This alteration of histones H3 and H4 results in a repressive

chromatin structure. Other known repression mechanisms include inhibition of activator
UAS association, such as the mechanism used by the YB-l protein, which binds to
single-stranded regions within the promoter, thus preventing loading and/or function of
other DNA-specific transactivating factors (MacDonald, et al., 1995). Direct inhibition,
or quenching, of activator function as seen with the Kruppel protein (Licht, et al., 1993).
Direct GTF interaction such as Drl-DRAPI inhibition of the association of TBP with

TFIIA and TFIIB (Inostroza, et al., 1992; Johnson, 1995; Meisteremst and Roeder, 1991).

TATA

TATA elements are the sites of binding for the TATA-binding protein (TBP).
TBP-TATA association nucleates the assembly of the pre-initiation complex. In S.
cerevisiae the TATA element is located within a window 30 to 120 bp 5’ of the site of
transcriptional initiation. This is in contrast to most other eukaryotes where the TATA
element is located, without exception, 25-30 bp 5’ of the transcriptional initiation site
(Struhl, 1995). While many sequence variations of the TATA-element exist, “TATAAA”
has been defined as the optimal sequence for the TATA element in yeast through
saturation mutagenesis (Chen and Struhl, 1988; Singer, et al., 1990; Wobbe and Struhl,
1990). The importance of TBP-TATA element association is underscored by the
discovery of altered specificity TBP molecules, TBP“, that bind to the TATA derivative
TGTAAA (Strubin and Struhl, 1992). TGTAAA in the absence of TBP’“3 does not
support efficient transcriptional activity.

Promoters lacking canonical TATA elements (TATA-less promoters) have also

been identified. The name “TATA-less” denotes weak TBP-DN A affinity rather than a

qualitatively distinct promoter element. Thus while TATA-less promoters are still
dependent on TBP binding, TBP association with the promoter is unlikely to be the rate-
limiting step in PIC formation. TATA-less promoters likely depend on other factors,

such as TAFns, to recognize the promoter and nucleate PIC formation.

INR

The Inr element is a transcriptional regulatory sequence that encompasses the
transcriptional start site. The Inr was defined as the promoter element present in the
terminal-deoxynucleotidetransferase gene, distinct from TATA, that can nucleate PIC
assembly (Smale and Baltimore, 1989). The term ‘initiator’ was first used to describe the
60bp region spanning the transcription start site of the TATA containing sea urchin H2A
gene (Grosschedl and Bimstiel, 1980). Deletion of this 60bp region resulted in
transcription initiation 25bp downstream from the TATA element, but promoter strength
was reduced 4-fold. However, the variable distance between the TATA element and the
Inr in yeast implies the existence of specific Inr sequences that direct proper
transcriptional initiation in addition to stimulating mRN A production. TATA elements
apparently establish the window within which initiation can occur but Inr elements are
defined by specific sequences within that window (Hahn, et al., 1985; Healy, etal., 1987;
Li and Sherman, 1991; Nagawa and Fink, 1985; Rudolph and Hinnen, 1987). Mutational
analysis revealed a loose, but consistent Inr sequence of Py Py A N T/A Py Py, where Py
= pyrimidine and underline marks the first nucleotide of the transcript (J avahery, et al.,
1994). Inr elements are core promoter elements able to nucleate PIC assembly and direct

accurate transcription initiation in a TATA independent manner in higher eukaryotes

(W eis and Reinberg, 1992). Proteins that bind INR elements in higher eukaryotes
include CIF (Kaufmann, et al., 1996), YYl (Usheva and Shenk, 1994), E2F (Means, et
al., 1992), TFII-I, and USF (Roy, et al., 1997; Roy, et al., 1993; Sadovsky, et al., 1995),
as well as RNAPII (Aso, et al., 1994; Carcamo, etal., 1991). Whether Inr elements are
capable of functioning as independent promoter elements in yeast is unclear. However,
experiments focusing on the GAL80 promoter have provided some intriguing results.
The GAL80 promoter contains both TATA and Inr elements, with these two elements
directing initiation at distinct sites in response to different environmental stimuli. Thus
two independent pathways lead to GAL80 transcription, one INR dependent and one
TATA dependent (Sakurai, et al., 1994). A nuclear protein that binds to the GAL80 INR

has also been detected (Sakurai, et al., 1994).

Poly (dA-dT) Elements

Homopolymeric dA-dT sequences are commonly found in yeast promoters and in
several cases have been shown to be necessary for wild-type levels of transcription in
viva (Struhl, 1985). Such sequences posses unique structural characteristics which
include an unusually short helical repeat (10.0 bp instead of 10.6 bp typical of B-DNA)
(Peck and Wang, 1981; Rhodes and King, 1981), a distinctively narrow minor groove
(Alexeev, et al., 1987), and structural rigidity (Nelson, et al., 1987). Poly dA-dT
sequences do not favor the assembly and stability of nucleosomes in vitro (Kunkel and
Martinson, 1981; Prunell, 1982). Thus, poly dA-dT elements function as promoter
elements based on their intrinsic structure (Iyer and Struhl, 1995). Poly dA-dT tracts are

abundant within the S. cerevisiae genome and occur predominantly at unit nucleosomal

length both upstream and downstream of open reading frames (Raghavan, et al., 1997).
However, the idea that poly dA-dT elements are simply architectural elements is
contradicted by the finding that a related oligonucleotide was able to squelch the positive
effect of a poly dA-dT element had on transcription (Lue, et al., 1989). This result argues
for a specific polydA-dT binding protein. Indeed, such a protein, datin, has been
identified (Winter and Varshavsky, 1989). However, the exact role that datin plays in

transcriptional activation is still unclear.

PROMOTER CONTEXT

The previously described promoter elements exist in the nucleus in the context of
chromatin, a structure of varying complexity that plays a regulatory role in gene
expression. Chromatin simply defined is a complex with a 2:1 mass ratio of protein to
DNA. The structure of chromatin varies from a decondensed and unfolded state to a
highly ordered and compacted state. It follows that in the decondensed state the DNA is
more accessible to transcription factors and thus transcriptionally active whereas in the
highly ordered and compacted state it is inaccessible to transcription factors and is thus
transcriptionally silent.

In its simplest form chromatin consists of 145-147bp of DNA coiled around a
histone octamer that consists of two copies of each histone protein, H2A, H2B, H3, and
H4. This octamer structure is also known as a nucleosome (Luger, et al., 1997). The
nucleosome structure is repeated in yeast about every 165bp (Thomas and Furber, 1976)
leaving short segments of linker DNA. The extended chromatin fiber is a 10nm diameter

filament with an appearance that resembles “beads on a string”.

In higher eukaryotes the linker histone H1 binds to the linker DNA and facilitates
the folding of the 10nm filament into the 30nm chromatin fiber and stabilizes the
compacted structure (Butler and Thiele, 1991; Finch and King, 1976; Renz, et al., 1977;
Thoma, et al., 1979). No functional homolog of histone H1 exists in yeast, a finding that
is not so surprising given the small amount of linker DNA between nucleosomes in yeast,
and likewise there is a paucity of 30nm chromatin in yeast.

Covalent modifications of the histones affect the packaging of nucleosomal DNA.
For example, acetylation of the lysine residues at the N-terminal tails is believed to
loosen the nucleosomal structure and expose the DNA sequence, while deacetylation of
these residues is believed to tighten the nucleosome and make DNA less accessible
(Hampsey, 1997 ; Pazin and Kadonaga, 1997). Recently many different chromatin
remodeling complexes have been described - complexes that act at the level of
nucleosome rearrangement. These complexes will be discussed in detail below.

Mutations in HTAI-HTBI, one of two gene pairs encoding histones H2A and
H2B have phenotypes similar to mutations in SPT4, SPT5, and SPT6, genes that encode
proteins that affect chromatin structure. Since htaI-htbl mediated suppression occurs by
altering chromatin structure (Hirschhom, et al., 1992) and Spt5, Spt6, and Spt7 have been
implicated in transcriptional regulation (Winston and Carlson, 1992) a link between
chromatin and transcriptional repression is established.

Non-histone chromatin proteins that serve as both negative and positive regulators
of gene expression exist in yeast. These proteins are often referred to as “architectural”
proteins because of their structural role in bending or kinking DNA strands into

conformations that either facilitate or hinder interactions of other proteins with DNA.

The association of the nonhistone chromosomal proteins with DNA thus results in a
higher order of chromatin structure that has both negative and positive effects on gene
expression, depending on the locus.

The recently described “CP” complex has been shown to be required for global
gene activation and/or for chromatin-mediated repression (Brewster, et al., 1998). The
CP complex in yeast is an abundant nuclear dimer consisting of Cdc68 and Pob3
proteins. A cdc68(Ts) impairs gene expression at the restrictive temperature as a result of
an alteration in Cdc68 protein structure (Rowley, et al., 1991). Other cdc68 mutations
result in derepression of SUC2 and GALl when the UAS sequences for SUC2 and GALl
are deleted (Lycan, et al., 1994; Malone, et al., 1991; Prelich and Winston, 1993). The
CP complex maintains the H0 gene in a transcriptionally inactive state in the absence of
the Swi4-Swi6 transcriptional activator (Lycan, ct al., 1994).

The high mobility group (HMG) proteins are another set of nonhistone
chromosomal proteins. HMG] and HMG2 are related proteins that affect the assembly of
nucleosomes and the organization of chromatin structure in vitro (Bonne Andrea, etal.,
1984; Nightingale, et al., 1996). Stimulatory effects of HMGl/2 on transcription in
eukaryotic cell extracts have been reported (Paull, et al., 1996; Shykind, et al., 1995;
Singh and Dixon, 1990; Tremethick and Molloy, 1988). HMGl/2 do not, however,
function directly as transcriptional activators (Landsman and Bustin, 1991). In contrast,
other reports have shown that HMGl/2 proteins act as repressors of RNAPH mediated
transcription in vitro (Ge and Roeder, 1994; Stelzer, et al., 1994). Other HMG proteins
with functions less well defined included HMG14/ 17 and HMG I/Y. HMG14/17 are

small, highly charged proteins shown to be modestly enriched in an actively transcribing

gene relative to inactive genes (Postnikov, et al., 1991). HMG W are small, highly
related proteins known to interact with the PRDII promoter element and to stimulate the
simultaneous binding of NF-KB to this element (Thanos and Maniatis, 1992). HMG W
has a similar affect on NF-KB binding to the nitric-oxide synthase promoter/enhancer
(Perrella, et al., 1999).

The condensation of chromosomes into heterochromatin and heterochromatin-like
structures represents one extreme of the spectrum of chromatin structures.
Heterchromatin is refractory to the binding and influence of transcriptional activator
proteins. This was elegantly demonstrated by the loss of ADEZ gene expression as a
result of local chromatin structures - an effect known as position effect variagation
(Gottschling, et al., 1990). Heterochromatin regions appear to be condensed since they
prevent access to DNA-altering enzymes, are late replicating in S phase, and are
associated in foci that appear to be at the nuclear periphery (Gotta and Gasser, 1996). S.
cerevisiae heterochromatin contains histone H4 that is uniquely hypoacetylated at lysines
K5, K8, and K16 but not at K12 (references in (Lowell and Pillus, 1998)). In yeast such
regions are found at the telomeres as well as at the silent (HM) mating and ribosomal
DNA (rDNA) loci. The transcriptional repression witnessed at these loci is mediated by
the SIRI-4 (silent information regulator) genes. Repression at the HM loci and telomeres
also requires other factors including Raplp (reviewed in (Shore, 1994)), as well as
histones H3 and H4 (reviewed in (Loo and Rine, 1995)). The SIR genes are variably
required for silencing at different loci. rDNA silencing depends only on SIR2 (Smith and

Boeke, 1997). In contrast, all four SIR genes contribute to silencing the HM loci, whereas

10

telomeres only require SIR2, SIR3, and SIR4 (Aparicio, et al., 1991; Haber and George,

1979; Klar, et al., 1979; Rine and Herskowitz, 1987; Rine, et al., 1979).

RNA POLYMERASE 11 AND ASSOCIATED FACTORS

RNA Polymerase II

Yeast RNAPII is a complex enzyme comprising twelve polypeptides encoded by
RPBI through RPBIZ (W oychik and Young, 1994). Extensive structural conservation
exists among the RNAPII subunits from diverse eukaryotic organisms; six of the yeast
RNAPII subunits can be functionally replaced by their human counterparts (McKune, et
al., 1995). RNAPII is composed of common as well as class-specific subunits. Five
subunits, pr5, pr6, pr8, pr10, and pr12 are essential components of all three
eukaryotic polymerases. RNAPII subunits prl, pr2, pr3, and pr11 are
homologous to RNA polymerase I and RNAPII] subunits. Only pr4, pr7, and pr9
are unique to RNAPH. With the exception of RPB4 and RPB9, all of the genes that
encode the yeast RNAPH subunits are essential.

Yeast genetics has provided some insight into the in viva functions of some of the
individual RNAPH subunits. RPBI and RPBZ mutations have been isolated that
influence the accuracy of transcriptional initiation, implicating these two subunits in start
site selection (Amdt, et al., 1989; Berroteran, et al., 1994; Hekmatpanah and Young,
1991). 6-azauracil (6-AU) sensitivity is a phenotype of other RPBI and RPBZ mutations,
a phenotype associated with transcriptional elongation defects. This finding suggests that

both pr1 and pr2 play a role in transcriptional elongation (Archambault, etal., 1992;

ll

Powell and Reines, 1996). Sequence similarity of RPBI and RPBZ to Band B’ of the
prokaryotic RNA polymerase also indicates that RPBI and RPBZ likely encode the
catalytic subunits of RNAPII (Markovtsov, et al., 1996; Mustaev, et al., 1997; Severinov,
et al., 1996; Zaychikov, et al., 1996). pr3 is involved in assembly of the RNAPII
complex (Kolodziej and Young, 1989; Kolodziej, et al., 1990; Kolodziej and Young,
1991). pr9 has a zinc finger motif, mutations in which affect start site selection (Furter
Graves, etal., 1994; Gadbois, etal., 1997; Hull, et al., 1995; Sun and Hampsey, 1996).
An rpb9 allele that suppresses a TFIIB mutation that affects start site selection has been
identified (Sun, et al., 1996). This finding leads to the supposition that pr9 affects start
site selection in concert with TFIIB. A form of yeast RNAPII lacking the pr4 and
pr7 subunits has been identified. Indeed biochemical analysis revealed that pr4 and
pr7 are loosely associated with RNAPH. While yeast RNAPII lacking pr4 and pr7
is capable of elongation in vitro it is deficient in accurate initiation in vitro (Edwards, et
al., 1991). pr4 has been implicated as playing a role in tolerance of RNAPII to stress.
rpb4 mutants display substantially impaired growth rates at elevated temperature or under
conditions of nutritional deprivation (Choder and Young, 1993).

The largest subunit of the RNAPII complex, pr1, has a unique tandemly
repeated heptapeptide sequence at its carboxy-terminus. This carboxy-terminal domain
(CTD) is highly conserved among species and has a consensus sequence Tyr-Ser-Pro-
Thr-Ser-Pro-Ser. The number of repeats appears to increase with the complexity of the
organism, ranging from 26-27 in yeast to 52 in human cells (Young, 1991). The CTD is
essential for viability, yet its function is not yet clear. Yeast that contains RNAPH

lacking all but 8 to 10 repeats is viable (Nonet and Young, 1989; West and Corden,

l2

1995). However, strains with only 8 to 10 CTD repeats display a cold-sensitive growth
defect, a phenotype that was exploited to isolate extragenic suppressors of CTD
truncations and resulted in the discovery of the SRB genes (Nonet and Young, 1989).
RNAPII with a truncated CTD is also unable to initiate transcription at promoters that
lack a TATA element (Akoulitchev, et al., 1995; Buermeyer, et al., 1995).

Two forms of RNAPH exist in vivo, RNA polymerase 110, which is
hyperphosporylated at the CT D, and RNA polymerase IIA, which is not phosphorylated.
RNA polymerase IIA preferentially enters into the PIC, whereas RNA polymerase 110 is
found in the elongating complex (Dahmus, 1996). Conversion of RNA polymerase 11A to
110 correlates with the transition from initiation to elongation (Lu, et al., 1991; O’Brien, et
al., 1994). These results implicate the CTD as having a central role in the conversion of
RNAPII from a molecule intent on promoter recognition to one that is elongation competent.
CTD kinase candidates are numerous and include TFIIH (Feaver, et al., 1991; Lu, et al.,
1992; Serizawa, et al., 1992), P-TEFb (Marshall, et al., 1996), SrblO/ll (Liao, et al., 1995),
Cdc2 (Cisek and Corden, 1989), and Ctkl (Lee and Greenleaf, 1991). Whether these CTD
kinases are gene specific or if they affect different steps during the transition from promoter
recognition to elongation is still unclear. Just as RNAPII is converted from the HA form to
the 110 form by a kinase it is recycled back to the HA by a phosphatase (Chambers and
Dahmus, 1994). CTD phosphatase activity is regulated by TFIIB and TFIIF (Chambers, et
al., 1995). It has been proposed that since RNA polymerase IIA preferentially enters the PIC

that TFIIB and TFIIF are responsible for RNAPH recycling (Lei, et al., 1998).

General Transcription Factors

l3

As mentioned previously the general transcription factors (GTFs) include TFIIA,
TFHB, TFIID, TFIIB, TFIIF, and TFIIH. The GTFs were identified in order-of-addition
experiments as the protein fractions necessary for accurate transcription initiation by RNAPH
from double stranded templates in vitro (Sayre, et al., 1992). These factors plus RNAPII

comprise the PIC.

TFIID

TFIID is a multisubunit complex that consists of TATA binding protein (TBP)
and TBP-associated factors (TAFus) (reviewed in (Burley and Roeder, 1996; Hernandez,
1993). In vitro results indicate that, depending on the promoter, either TBP or TFIID is
involved in promoter recognition and thus serves to nucleate PIC formation. At promoters
that contain a TATA element TBP is sufficient whereas TAFns are required at TATA-less
promoters (Aso, et al., 1994; Kaufmann and Smale, 1994; Martinez, et al., 1994; Smale, et
al., 1990; Verrijzer, et al., 1995). Immunodepletion of yTFIID by TAP-specific antibodies
blocks in vitro transcription by RNAPII and not RNAPI or RNAPIII, this indicates that

TFIID is a RNAPII specific complex (Klebanow, et al., 1997).

TBP is a universal transcription factor, required for initiation by all three eukaryotic
RNA polymerases (Hernandez, 1993). TBP is a subunit of the 750kDa TFIID complex
composed of TBP and TBP-associated factors (T AFs). Whereas TBP functions in basal level
transcription, TFIID is required for response to transcriptional activators in metazoan in vitro
transcription systems (Burley and Roeder, 1996; Pugh and Tjian, 1992; Verrijzer and Tjian,

1996).

14

Yeast TBP is a monomer of 27 kDa and can be divided into two structural domains,
the less well conserved N-terminal domain and the highly conserved C-terminal domain.
The conserved C-terminal domain of TBP consists of two direct repeats, suggesting that TBP
may have been the result of a gene fusion event of two monomers. The C-terminal region of
TBP is both necessary and sufficient for TATA binding as well as transcription initiation
(Zhou, et al., 1993). The crystal structure of the C-terminal TBP domain from yeast has been
solved (Kim, et al., 1993) and reveals a “molecular saddle” type structure made up of two
roughly symmetrical halves. The direct repeats of the TBP molecule display the expected
twofold symmetry. Each repeat folds into a twisted beta-sheet, topped by a long C-terminal
alpha helix that is available for interaction with other factors. TBP has a convex upper
surface as well as a concave lower surface capable of DNA minor groove binding via a
curved eight-stranded antiparallel beta-sheet on the inner surface of the saddle. TBP-DNA
interaction results in a kink in the DNA that bends the DNA about 80° toward the major
groove. The DNA bending brings proteins bound on either side of TBP, such as TFHA and
TFIIB, into close physical proximity. The notion that GTF function is conserved among
species is bolstered by the fact that yeast TBP is functionally interchangeable with
mammalian TBP in in vitro transcription experiments (Buratowski, et al., 1988; Cavallini, et
al., 1989; Hahn, et al., 1989).

The yeast TBP gene is known as SPTIS. SPT15 was identified in a genetic selection

for suppressors of a Ty insertion in the HIS4 promoter (his4-9I 7d) (Eisenmann, et al., 1989).
In the his4-9l 75 mutant, transcription initiates within the Ty (Selement resulting in non-
functional transcripts. Mutations within SPT15 suppressed his4-9175 by shifting initiation

from within the 5element to the his4 promoter, resulting in a His+ phenotype. SPT15 is an

15

essential gene and spt15 mutants have pleiotropic effects, further demonstrating the essential

nature of this transcription factor (Eisenmann, et al., 1989).

TAFIIS

Two observations lead to the speculation that yeast TBP existed in a complex with
other proteins. First, mutations in several SPT genes resulted in a similar set of pleiotropic
phenotypes including SPTI5 (TBP). Secondly, the SPT3 gene product was shown to
physically interact with TBP (Edwards, et al., 1991). Indeed, 12 TAF subunits have now
been identified in yeast ranging in size from 17 to 155 kDa (Klebanow, et al., 1997;
Klebanow, et al., 1996; Moqtaderi, et al., 1996; Poon, et al., 1995; Reese, 1994; Walker, et
al., 1996). All of the genes encoding the yTAFus are essential except the gene that encodes
yTAFn30. Furthermore, significant similarity exists between TAFus from various species.
The only metazoan TAFu that does not have a homolog in yeast is TAFn110, which is
required for activation by the glutamine-rich activator Spl. It is interesting to note that
glutarnine-rich activators are not known exist in yeast and that metazoan glutamine-rich
activators function poorly, if at all, in yeast. The lack of a TAFu110 homolog may be the
reason that glutamine-rich activators do not exist in yeast (Moqtaderi, et al., 1996).

Yeast TAFnl45, also known as y TAFnl30, binds to TBP and is believed to serve as
a scaffold for the assembly of the other TAFns into TFIID. Biochemical and crystallographic
analysis has indicated that a histone octamer-like structure may exist within TFIID. TAFns
that possess domain structures reminiscent of those found in histone H3-, H4-, and H2B have
been identified in human, Drosophila, and yeast TFIID complexes (Burley and Roeder, 1996;
Nakatani, et al., 1996; Xie, et al., 1996). In the case of yeast TFIID, a putative yTAFn

histone octamer-like structure would contain a heterodimer of yTAFnl7 (H3-1ike) and

16

yTAFn60 (H4-like) sandwiched between two yTAFn61 (H2B-like) homodimers. No H2A-
like TAFn has been identified in any species. This has lead to the proposal that this octamer
like structure may wrap DNA on the surface of TFIID. However, the recent nucleosome
crystal structure indicates that arginine side-chains in histones that interact with DNA are not
conserved in histone-like TAFus (Luger, et al., 1997). Thus if the histone-like TAFus do
bind DNA, they likely do so differently than the histones that comprise a nucleosome.

The essential nature of the yTAFn genes clearly points to a critical role for them in
viva. However, their exact role remains undefined. Yeast TAFus were demonstrated to
function as co-activators of transcription in vitro (Sauer, et al., 1995; Sauer, et al., 1996;
Verrijzer and Tjian, 1996) which lead to the presumption that this was a general
phenomenon. However, this presumption was undermined when depletion or inactivation of
several yTAFns, including yTAFul45/yTAFnl30, did not compromise transcriptional
activation in viva (Moqtaderi, et al., 1996; Walker, et al., 1996). In the case where the
yTAFus were required for response to activators, the requirement mapped to the core
promoter element instead of the elements bound by activators — the promoter lacked a
TATA-element and thus the requirement for the yTAFns (Moqtaderi, et al., 1996). This
result does however implicate yTAFnl45/ yTAFul 30 as a promoter selectivity factor.
Indeed, yTAFul45/ yTAFul30 mediates the process of activation at promoters lacking a
TATA element (Moqtaderi, et al., 1996). These conclusions were later tempered when it was
demonstrated that the histone-like yTAFns were indeed required for the expression of a
subset of yeast genes (Apone, et al., 1998; Michel, et al., 1998; Moqtaderi, et al., 1998).

It is possible that yTAFus are activator cofactors, but only for a subset of genes -

specifically for genes involved in cell cycle control. A taf90(Ts) mutant fails to progress

17

through the Gle phase of the cell cycle at the restrictive temperature (Apone, et al., 1996).
Another example is a tafl45(Ts) mutation that blocks transcription of G1/S cyclin genes at

the restrictive temperature (Walker, et al., 1997).

TFIIB

Yeast TFIIB is a monomer of 38kDa encoded by the S UA7 gene. S UA7 was identified
in a genetic screen where suppressors of a translational defect at the CYCI locus were sought.
A recessive mutation at the sua7 locus that shifted the transcription start site downstream of
the normal start site such that the translational impediment was eliminated from the cycI
transcript was isolated (Pinto, et al., 1992). This result implicates TFIIB as being involved in

start site selection in yeast (Li, et al., 1994; Pinto, et al., 1992).

TFIIB enters the PIC after TBP binding to the TATA element and recruits RNAPII.
TFIIB also interacts with other GTFs, including the TFIIF subunits RAP30 and RAP74
(Buratowski, et al., 1989). However, TFIIB associated with RAP74 is unable to associate
with RAP30, perhaps because of some dynamic interaction between TFIIF and TFIIB during
PIC assembly (Fang and Burton, 1996). TFIIB has been implicated as the direct target of
many gene-specific activators, leading to the proposal that certain activators stimulate
transcription by TFIIB recruitment (Lin and Green, 1991; Roberts, et al., 1993). TFIIB has

been shown to interact with TAFn40 as well (Goodrich, et al., 1993).

TFIIB consists of two domains, an N-terminal cysteine rich region that includes a
zinc binding motif and a C—terminal protease resistant region that contains two imperfect 75

amino acid repeats (Ha, et al., 1991; Malik, et al., 1991; Pinto, et al., 1992). The C-terminal

18

region (TFIIBc) is capable of binding TBP but not RNAPII or TFIIF and is thus unable to
support transcription (Barberis, et al., 1993; Buratowski and Zhou, 1993; Ha, et al., 1991;
Yamashita, et al., 1993). In the absence of transcriptional activators the N- and C-terminal
domains of TFIIB are involved in an intramolecular interaction that preclude interaction with
other PIC members. In the presence of a transcriptional activator TFIIB undergoes a
conformational change that allows for PIC assembly (Roberts, 1994). This has lead to the

proposal that TFIIB is a target of transcriptional activators.

The three dimensional structure of a TFIIBc-TBP-DNA complex has been solved
(Nikolov, et al., 1995). In this structure TFIIB binds directly to TBP, and also contacts DNA
immediately upstream and downstream of the TATA element. These TFIleDNA contacts
are made possible by the bend put into the DNA as a result of TBP binding. The binding of
TFIIB thus stabilizes the TBP-TATA interaction. Like TBP, TFIIB recognizes specific
sequences found in certain promoters (Lagrange, et al., 1998). The cysteine rich N-terminal
zinc binding domain is absent in the crystal structure, but it is thought to bind DNA in the

vicinity of the transcription start site and to stabilize melting of the promoter.

TFIIF

Yeast TFIIF is composed of three subunits with apparent molecular masses of 105,
54, and 30 kDa encoded by the genes TFGI, TFGZ, and TF G3 respectively (Henry, et al.,
1992). TF GI and TFGZ are both about 50% similar to RAP30 and RAP74, the two subunits
that comprise human TFIIF, respectively (Henry, et al., 1994). TFIIF binds tightly to

RNAPII and suppresses nonspecific binding of RNAPH to DNA (Conaway and Conaway,

l9

1993). Stabilization of the PIC is another function of TFHF (Conaway and Conaway, 1993;
Greenblatt, 1991).

Photo-crosslinking studies have shown that the two largest subunits of TFIIF bind
promoter DNA between the TATA element and start site (Coulombe, et al., 1994; Kim, et al.,
1997; Robert, et al., 1996). The largest subunit of TFIIF also binds DNA upstream of the
TATA element and induces a conformational change that affects the position of RNAPII
relative to the DNA template (Forget, et al., 1997). TFIIF also plays a role in transcriptional
elongation by suppressing the transient pausing of RNAPH (Bengal, et al., 1991; Bradsher, et
al., 1993; Flores, et al., 1989; Izban and Luse, 1992; Price, et al., 1989).

TF GI is the only gene of any of the TFIIF components to be identified in a genetic
selection for transcription factors. SSU 71 was identified as a suppressor of a TFIIB defect
that resulted in spurious start site selection (Sun and Hampsey, 1995). Sequence analysis
revealed that Ssu71 is identical to ng1 (Henry, et al., 1994; Sun and Hampsey, 1995). Thus,

TFIIB functionally interacts with TFIIF.

TFIIE

Yeast TFIIE is composed of a 56 kDa subunit (TFIIB-0t) and a 34 kDa subunit
(TFIIEB). The OLB heterodimer structure of the yeast TFIIB is in contrast with the 012152
heterotetramer form of TFIIE found in human cells (Leuther, et al., 1996). Order of addition
experiments show that TFIIE enters the PIC after RNAPII and TFIIF and that TFIIB recruits
TFIIH (Buratowski, et al., 1989; Flores, et al., 1992). TFIIB interacts with the
unphosphorylated form of RNAPH, with both subunits of TFIIF, and with TFIIH (Flores, et

al., 1989; Maxon and Tjian, 1994). The functional importance of the TFIIB-TFIIH

20

interaction was demonstrated by the inability of S. cerevisiae TFIIB to functionally replace
the S. pambe TFIIE unless TFIIH from S. cerevisiae replaced its counterpart from S. pambe
as well (Li, et al., 1994). TFIIB influences TFIIH in the following ways: TFIIE recruits
TFIIH to the PIC, TFIIB stimulates TFIIH dependent phosphorylation of RNAPII, and
TFIIB stimulates TFIIH-dependent ATP hydrolysis (Lu, et al., 1992; Ohkuma, et al., 1995;
Ohkuma and Roeder, 1994). Another possible role for TFIIE is in the formation or
stabilization of the melted promoter region based on the fact yTFIIE binds single-stranded
DNA (Kuldell and Buratowski, 1997). TFHE has also been implicated as the direct target of

some transcriptional activators (Sauer, et al., 1995; Zhu and Kuziora, 1996).

TFIIH

Upon TFIIH entry the formation of the PIC is complete (Flores, et al., 1992). Yeast
TFIIH is a nine subunit complex with a total mass of approximately 500 kDa. TFIIH is the
only GTF that has known enzymatic properties, which include DNA-dependent ATPase
activity (Conaway and Conaway, 1989; Roy, et al., 1994), ATP-dependent DNA helicase
activity (Schaeffer, et al., 1993; Serizawa, et al., 1993), and CTD kinase activity (Feaver, et
al., 1991; Lu, et al., 1992; Serizawa, et al., 1992).

TFIIH plays a central role in forming an open transcription complex. The TFIIH
component Rad25, an ATP-dependent DNA helicase, is believed to be necessary to form the
open promoter complex (J iang, et al., 1994; Wang, et al., 1992). If the DNA template is
premelted or supercoiled, a structure that inherently favors unwinding, the requirement for
TFIIH, TFIIB, and ATP can be bypassed (Holstege, et al., 1995; Pan and Greenblatt, 1994;

Parvin and Sharp, 1993; Parvin, et al., 1994; Tantin and Carey, 1994; Tyree, et al., 1993).

21

The CTD phosphorylation function of TFIIH is implicated in the transition of the
initiation competent form of RNAPII to the elongation competent form of RNAPII.
Presumably, phosphorylation of the CTD causes a conformational change in the PIC,
disrupting the interaction between CTD and TBP thus leading to promoter clearance
(Dahmus, 1994; O’Brien, et al., 1994). TFIIH also promotes the transition from very early

elongation complexes to stable elongation complexes (Dvir, et al., 1997).

TFIIA
Yeast TFIIA is composed of two polypeptides with apparent molecular masses of

32 and 13.5 kDa encoded by the essential genes TOAI and TOA2 respectively
(Radhakrishnan, et al., 1997; Ranish and Hahn, 1991; Ranish, et al., 1992). TFIIA associates
with the PIC through interaction with TBP and stabilizes TBP-TATA element binding
(Buratowski, et al., 1989; Irnbalzano, et al., 1994). The fact that TFIIA is dispensable for
accurate initiation in a purified system direct by TBP calls its classification as a GTF into
question (Cortes, et al., 1992; DeJong, et al., 1995; Hansen and Tjian, 1995; Ozer, et al.,
1994; Sayre, et al., 1992; Sun, et al., 1994; Yokomori, etal., 1993). TFIIA does, however,
counteract negative factors that associate with the TFHD complex such as Drl-DRAPl/NCZ,
PC3/Dr2, HMG], and Motl , and thus TFIIA is necessary for TFIID directed transcription
(Auble, 1994; Ge and Roeder, 1994; Inostroza, et al., 1992; Meisteremst, et al., 1991;
Merino, et al., 1993).

Co-overexpression of TOAI and TOA2 suppresses the phenotypes of TBP mutants
identified as having reduced TATA element affinity. This finding suggests that TFIIA is

important for transcriptional activation, and the degree to which different promoters require

22

TFIIA may vary (K. Amdt, personal communication). Yeast TFIIA is known to interact with
specific transcriptional activators such as Zta (Ozer, et al., 1994; Yokomori, et al., 1994), the
coactivators PC4 and HMG2 (Ge, et al., 1994; Shykind, et al., 1995), and VP16 (Kobayashi,

etaL,1998)

Holoenzymes

The fact that several GTFs were known to associate with RNAPII in the absence of
DNA implied the existence of a holoenzyme complex (Conaway and Conaway, 1993).
Indeed such complexes have been identified in yeast and in mammalian cells. Essentially the
same yeast holoenzyme was independently identified in both the Young and Kornberg labs.
Using antibodies directed against the SRB proteins, Young and coworkers isolated and
characterized a version of the holoenzyme (Koleske and Young, 1994). Simultaneously
Kornberg and coworkers identified a high molecular wieght complex associated with the
CTD that they called “mediator” or MED (Kim, et al., 1994). The MED complex contains
the SRB proteins (reviewed (Bjorklund and Kim, 1996)). The yeast RNAPII holoenzyme
includes all twelve pr molecules, SRB/MED proteins, TFIIB, TFIIF, and TFIIB (Koleske
and Young, 1994). A distinct form of yeast holoenzyme was discovered using a strategy
based on an RNAPII affinity column immobilized through the CTD (Shi, et al., 1997).
Proteins common to both versions of holoenzyme include TFIIB, TFIIS, TFIIF, and Gall 1,
but not SRB/MED proteins (Wade, et al., 1996). Novel holoenzyme proteins include Paf 1,
Cdc73, Ccr4, and Hprl (Chang and Jaehning, 1997; Shi, et al., 1997; Wade, et al., 1996).
The second holoenzyme affects the expression of a different spectrum of genes than the

SRB/MED containing holoenzyme and is thus unique (Shi, et al., 1996).

23

Quantitation by comparative Western blotting of RNAPII and SRB proteins has
determined that only a portion of RNAPH is present in SRB/MED holoenzyme complexes
(Koleske and Young, 1994). The percentage of total RNAPH present in the Pafl-Cdc73-
Ccr4-Hpr1 holoenzyme has yet to be determined, although crude analysis indicates that this
holoenzyme is less abundant than the SRB/MED holoenzyme (Hampsey, 1998).

The identification of holoenzyme has challenged the assumption that RNAPII and the
GTFs assemble on the promoter in a stepwise fashion. It is possible that a subset of RNAPII
is present in a preassembled complex lacking only TFIID and TFIIA. Thus assembly of the
PIC at a promoter may occur in only a two step process with TFIII) promoter recognition

followed by holoenzyme association with THE).
CHROMATIN REMODELING COMPLEXES

SWIISNF

The SWI genes were originally identified in a genetic screen for yeast mutants
defective in mating type switching (Stern, et al., 1984), whereas the SNF genes were
identified based on an inability to express the S UCZ gene and therefore unable to ferment
sucrose (Neigeborn and Carlson, 1984). SNFZ and SW12 were later found to be identical, and
thus the connection between the two systems was made. Suppressors of snf and swi
mutations were found in the ssn and sin genes, respectively. SSN20 and SIN2 turned out to
be identical to SPT6 (Clark Adams and Winston, 1987; Neigebom, et al., 1987) and HHTI
(Kruger, et al., 1995), respectively. As previously described, SPT6 is a member of a class of

SPT genes that either encode histones or are linked to chromatin function. HHTI encodes

24

histone H3. Thus the link between the S WI/SNF gene products and chromatin was
established. The mOdel that evolved maintains that the SPT/SIN gene products are involved
in the establishment of inactive chromatin whereas the SWIISNF genes are involved in
overcoming the repressive effects of chromatin (Winston and Carlson, 1992). Indeed, this
model received support from the finding that the chromatin structure at the SUC2 promoter is
altered in swi2/snf2 and snf5 mutants, and this chromatin defect is suppressed by A(htal-
htbl) (Hirschhom, et al., 1992).

The SWI/SNF complex in yeast is an ll-subunit complex (Cairns, et al., 1994; Cote,
et al., 1994). SWIISNF subunits include Swil, SwiZ/Snf2, Swi3, Snf5, Snf6, Snfl l, SWp29,
Swp56, SWp61, Swp73, and Swp82 (Burns and Peterson, 1997). The only enzymatic activity
known to reside in the complex is the DNA-dependent ATPase activity of Swi2/Snf2. A
functional link to the PIC is made through SWp29, which is identical to both the ng3 subunit
of TFIIF and the TAFn30 subunit of TFIID (Cairns, et al., 1996). SWIISNF was reported to
be a component of the SRB/mediator holoenzyme, but some methods of purifying
holoenzyme do not yield SWIISNF (Cairns, et al., 1996; Li, et al., 1996; Myers, et al., 1998;
Wilson, et al., 1996). The SWIISNF complex binds a DNA structure that mimics the
structure of DNA as it enters and exits the nucleosome (Quinn, et al., 1996). This property
raises the possibility that SWI/SNF binds specifically to nucleosomal DNA and subsequently
is able to disrupt nucleosome structure by modulating the DNA structure and topology. The
SWI/SNF complex is only required for the expression of 6% of yeast genes when tested in
glucose rich medium, including [-10, SUC2, Ty, ADHI, ADHZ, 1N0], STA] as determined by
the relative abundance of mRNAs in yeast strains with and without Swi2/Snf2 (Holstege, et

al., 1998).

25

RSC

The RSC (remodels the structure of chromatin) complex was isolated from yeast
based on homology to components of the SWIISNF complex (Cairns, et al., 1996). RSC is a
15-subunit complex that includes several SWIISNF related subunits as well as a DNA-
dependent ATPase (Cairns, et al., 1996; Cao, et al., 1997). However, RSC is approximately
10-fold more abundant that SWIISNF and unlike any genes encoding SWIISNF subunits
certain genes encoding RSC subunits are essential. No current evidence links RSC to
transcription. RSC does not play a role in either SUC2 or GALIO expression nor have any
histone mutants been found that suppress RSC defects (Hampsey, 1998). RSC mutants cause
cell cycle arrest in the Gle phase of the cell cycle (Cao, et al., 1997). It thus appears that

SWIISNF and RSC are functionally distinct.

I-Iistone Acetyltransferases

When the transcriptional activation domain (AD) of VP16 was fused to the DNA
binding domain of Gal4 and overexpressed in viva in yeast, cell growth was inhibited
(Berger, et al., 1992; Gill and Ptashne, 1988). Isolation of yeast mutants that relieved this
toxicity led to the identification of ADA I, ADA2, ADA3, ADAS/SPTZO, and GCN5, genes that
encode putative adaptor proteins (Berger, et al., 1992; Horiuchi, et al., 1997; Marcus, et al.,
1996; Marcus, et al., 1994; Pina, etal., 1993; Roberts and Winston, 1996). ADA2, ADA3,
and GCN5 were necessary for maximal activation by VP16 and Gcn4 (Berger, et al., 1992;
Georgakopoulos, et al., 1995; Georgakopoulos and Thireos, 1992; Pina, et al., 1993). Ada2

binds to the activation domains of both of these activators as well (Barlev, et al., 1995;

26

Silverrnan, et al., 1994). The discovery that GCN5 encoded a histone acetyltransferase
(HAT) allowed a specific function to be assigned to this protein and provided a direct link
between histone acetylation and transcriptional activation (Brownell and Allis, 1996;
Brownell, et al., 1996) - a link that had been presumed for 30 years (Allfrey, et al., 1964).
Acetylation of lysine residues at the N-terminal tails of histones neutralizes their positive
charge and presumably reduces the interaction of the core histone tails with DNA, which is
postulated to result in the activation of gene expression. However, it remained troubling that
while Gcn5 was able to acetylate free histones it was unable to use histones assembled into
nucleosomes as a substrate (Kuo, et al., 1996; Yang, et al., 1996). Recently, the issue has
been settled as a result of the identification of a complex that includes Gcn5 and is able to use
histones assembled into nucleosomes as substrates. The 1.8 MDa SAGA complex (Spt-Ada-
Gcn5-Acetyltransferase) contains Gcn5, Adal , Ada2, Ada3, Spt3, Spt7, Spt8, Spt20, Tral ,
and several yTAFu subunits (Grant, et al., 1997; Grant, et al., 1998). Other nucleosomal
HAT complexes have been identified, some that contain Gcn5 and are distinct from SAGA
(RuizGarcia, et al., 1997; Saleh, et al., 1997). It was also determined that under optimal

solution conditions GCN5 alone is indeed able to acetylate nucleosomes (Tse, et al., 1998).

HAP fl3l4l5

The Hap2/3/4/5 protein complex positively regulates many of the genes involved
in the TCA cycle and oxidative phosphorylation in the budding yeast Saccharamyces
cerevisiae (Forsburg and Guarente, 1989; Hahn and Guarente, 1988; Pinkham and
Guarente, 1985; Pinkham, et al., 1987). The Hap2/3/5 polypeptides form the DNA

binding component of the complex (McNabb, et al., 1995 ; Olesen, et al., 1987; Xing, et

27

al., 1993) specific for the CCAAT-related cis element of UASZ found upstream of the
CYCI gene and other Hap2/3/4/5 regulated genes. Hap2/3/5 are quite similar to proteins
known to contain a histone fold and likely employ this structure to associate with DNA
(Coustry, etal., 1996). Hap4 is responsible for the transcriptional activation capability
of the Hap2/3/4/5 complex (Forsburg and Guarente, 1989). HAP4 expression is
repressed in the presence of glucose, presumably by Migl which is known to bind
upstream of HAP4 (Ronne, 1995), and stimulated by an undefined factor (D. McNabb
personal communication) in the presence of non-fermentable carbon sources (Guarente,
et al., 1984). Hap4 is not required for the DNA-binding activity of the Hap2/3/4/5
complex (Olesen and Guarente, 1990; Xing, et al., 1993). In this respect Hap4 is
analogous to the VP16 trans-activator of herpes simplex virus, which also contains a
potent transcriptional activation domain but associates with DNA cis regulatory elements
only through a complex containing two cellular proteins, Oct-l (Abate, et al., 1990;
apRhys, et al., 1989; Kristie and Roizman, 1988; O’Hare and Goding, 1988; Preston, et
al., 1988) and HCF (Wilson, et al., 1993).

In some respects, the mechanisms employed for transcriptional activation by
VP16 and Hap4 appear to be fundamentally different. Whereas mutations in ADA2,
ADA3, or GCN5 significantly reduce activation by VP16 and Gcn4, they have a much
smaller effect on activation by Hap4 (Berger, et al., 1992; Pina, et al., 1993) (personal
observation). Furthermore, Wang et a1. (Wang, et al., 1995) demonstrated that VP16 and
HAP4 have different co-factor requirements. However, they were unable to clone a

HAP4 specific adaptor.

28

CBF-A, CBF-B, and CBF-C appear to be the human homologues of Hap2,
Hap3, and Hap5 respectively. CBF-A/B/C are the subunits of CBF (also known as NF-Y
and CH) the heterotrimeric ubiquitous DNA binding protein that binds to CCAAT
motifs in promoters of numerous eukaryotic genes (Chodosh, et al., 1988; Dom, et al.,
1987; Maire, et al., 1989; Maity, et al., 1988). In the case of CBF, CBF-B and CBF-C
provide the trans-activation potential (Coustry, et al., 1996), and no human homolg of
HAP4 has been identified.

Recently, a functional homologue of Hap4 was identified in the respiratory yeast
Kluyveramyces lactis (Bourgarel and Nguyen, 1999) that exhibits two small domains (11
and 16 amino acids respectively) highly homologous to corresponding domains of
Saccharamyces cerevisiae Hap4, while overall similarity is rather weak. The K. lactis
homolog of Hap4 was identified by heterologous complementation of a Ahap4
Saccharamyces cerevisiae mutant. This is the first example of a Hap4 homologue in any
organism other than Saccharamyces cerevisiae. Hap2 and Hap3 homologues have also

been identified in K. lactis (Mulder, et al., 1994; Nguyen, et al., 1995).

29

W

Identification and Characterization of the Hag Activation
Domainj s)

INTRODUCTION

The Hap2/3/4/5 protein complex positively regulates many of the genes involved
in the TCA cycle and oxidative phosphorylation in the budding yeast Saccharamyces
cerevisiae (Forsburg and Guarente, 1989; Hahn and Guarente, 1988; Pinkham and
Guarente, 1985; Pinkham, et al., 1987). Hap4 is responsible for the transcriptional
activation capability of the Hap2/3/4/5 complex (Forsburg and Guarente, 1989), and is
not required for the DNA-binding activity (McNabb, et al., 1995; Olesen, et al., 1987;
Xing, et al., 1993). In this respect, Hap4 is analogous to the VP16 trans-activator of
herpes simplex virus, which also bears a potent transcriptional activation domain but
associates with DNA cis regulatory elements only through a complex containing two
cellular proteins, Oct-1 (apRhys, et al., 1989; Kristie and Roizman, 1988; O’Hare and
Goding, 1988; Preston, et al., 1988) and HCF (Wilson, et al., 1993). Hap2/3/5 are ’
constitutively expressed whereas Hap4 expression is strongly induced when any non-
ferrnentable sugar is the sole carbon source (Forsburg and Guarente, 1989; Hahn and
Guarente, 1988; McNabb, et al., 1995; Pinkham and Guarente, 1985). Therefore, the
activity of this complex transcriptional activator is regulated by production of the
activation subunit.

Transcriptional activation proteins are typically bipartite in structure, with

separate domains for DNA binding and trans-activation (Mitchell and Tjian, 1989;

30

Triezenberg, 1995). Activation domains have traditionally been classified according to
amino acid composition. Thus activators have been classified as acidic, glutamine-rich,
proline-rich, serine/threonine-rich, isolucine-rich, or basic (Attardi and Tjian, 1993;
Estruch, et al., 1994; Johnson, et al., 1993). However, mutational analyses of Gal4
(Leuther, et al., 1993), RelA (Blair, et al., 1994), p53 (Lin, et al., 1994), VP16 (Cress and
Triezenberg, 1991; Regier, et al., 1993; Sullivan, etal., 1998), Gcn4 (Drysdale, et al.,
1995), the glucocorticoid receptor (Almlof, et al., 1997; Iniguez-Lluhi, et al., 1997), C 1
(Sainz, et al., 1997), NRF—l and NRF—2 (Gugneja, et al., 1996), and Spl (Gill, et al.,
1994) have demonstrated that specific patterns of bulky hydrophobic and aromatic amino
acids are more critical for activation domain function than the residues that are most
abundant.

As shown in the cases of VP16 (Triezenberg, et al., 1988), p53 (Horikoshi, et al.,
1995) and Gcn4 (Drysdale, et al., 1995), activation domains can often be further divided
into regions each capable of activating transcription independently and all of which are
dependent on hydrophobic residues. The VP16 activation domain can be divided into
small definable regions, VP16 N (aa410-546) and VP16 C (33456-490), each of which
relies on a central cluster of hydrophobic residues (Cress and Triezenberg, 1991; Regier,
et al., 1993; Sullivan, et al., 1998). In contrast, Gcn4 relies on multiple overlapping
regions containing seven different clusters of hydrophobic residues (Jackson, et al.,
1996). Collectively, these results suggest that activation domains are composite in
nature, containing multiple hydrophobic motifs.

One reason that transcriptional activators often possess multiple activation

domains may be that different segments of the activation domain interact with different

31

proteins in the PIC. The ability to interact with numerous targets is required because
transcriptional activators act at a variety of promoters that may have different rate
limiting steps. For instance, VP16 is known to interact in vitro with TBP (Ingles, et al.,
1991; Stringer, et al., 1990), TFIIB (Labow, et al., 1990; Lin and Green, 1991; Lin, et al.,
1991; Roberts, et al., 1993), TFIIA (Kobayashi, et al., 1998), TFIIH (Xiao, et al., 1994),
and TAFn40 (Goodrich, et al., 1993). Transcriptional activators have evolved able to
interact with numerous targets and therefore able to act at a variety of promoters no
matter what the local constraints to activation.

Despite the similar requirement for hydrophobic residues, no consensus motif
characteristic of activation domains has been defined. This has lead to the proposal that
the critical structural features for activation domains are somewhat flexible. The
activation domains of VP16, Gal4, Gcn4, RelA, and glucocorticoid receptor have been
demonstrated to be unstructured in aqueous solution under neutral pH (Dahlman-Wright,
et al., 1995; Donaldson and Capone, 1992; O’Hare and Williams, 1992; Schmitz, et al.,
1994; Van Hoy, et al., 1993). However, activation domains can adopt specific structures
in different solvents (Dahlman-Wright, et al., 1995; Donaldson and Capone, 1992;
O’Hare and Williams, 1992; Schmitz, et al., 1994; Van Hoy, et al., 1993). This lead to
the speculation that activation domains are able to adopt higher-order structure upon
contacting their target molecules by an “induced fit” mechanism. Indeed, fluorescence
studies later demonstrated the existence of an induced conformation for the VP16
activation domain upon interaction with TBP, and to a lesser extent, TFIIB (Shen, et al.,
1996). Other examples of induced fits include p53/MDM2 (Blommers, et al., 1997) and

VP16/hTAF1132 (Goodrich, et al., 1993).

32

Hap4 and VP16 are both members of the acidic class of transcriptional activators.
However, a fundamental difference in the two activation domains is that in yeast, Gal4-
VP16 is unable to stimulate the expression of a lacZ reporter gene in the absence of
Ada2, Ada3, or Gcn5 while Gal4-Hap4 is able to stimulate expression of the same lacZ
reporter under such conditions (Horiuchi, et al., 1995). This suggests that a qualitative
difference exist between the VP16 activation domain and the Hap4 activation domain.
Hap4 was also shown to be capable of activating transcription in the absence of Ada2
when acting in concert with Hap2/3/5 at a natural promoter (Berger, et al., 1992).

The observation that Hap4 (aa330-554) is able to function independent of Ada2,
Ada3, and Gcn5 while VP16 tested in the same context is dependent on Ada2, Ada3, and
Gcn5 indicates that the Hap4 activation domain is intrinsically Ada2, Ada3, and Gcn5
independent. To define such an ADA independent activation domain, identification and
characterization of the Hap4 minimal activation domain(s) was undertaken. This work
thus represents the only known dissection of an ADA-independent activator.

I constructed an extensive set of deletion mutants of the Hap4 330-554 region,
fused to the lexA DNA binding domain present within amino acids 1-202, and tested
their ability to activate transcription of a lacZ reporter gene. This approach resulted in
the identification of the Hap4 359-476 region as a potent Ada2, Ada3, and Gcn5
independent activation domain. Resident within the 359-476 region of Hap4 are seven
clusters of hydrophobic amino acids with 2-4 hydrophobic amino acids present per
cluster. I constructed Hap4 activation mutants in which serine was substituted for all of
the hydrophobic residues within any given cluster to serines and tested the effect on

trans-activation potential. I found that L36SS/L366S and I37OS/L37IS mutations had no

33

effect on the activation potential of Hap4 359-476 and I4508/L452S had only a moderate
negative effect. Hap4 359-476 Y427S/L428S/F429S/L43OS resulted in a 5-fold
reduction in activation potential. In contrast, I39OS/W39lS/Y393S/L394S,
F456S/Y4588, and L466S/M467S resulted in a greater than 10-fold reduction in
activation potential.

The original work on Hap4 was carried out in the yeast strain BWGl-7a
(Forsburg and Guarente, 1989). In BWGl-7a the 1-330 region of Hap4 was unable to
support growth on a non-fermentable carbon source. However, when tested in the more
robust yeast strain DMYl48, Hap4 1-330 was able to support growth on a non-
fermentable carbon source implying the existence of an activation domain within this
region. The sequences needed for efficient transcriptional activation when tethered to
lexA extended from amino acid 124 to amino acid 270 and those sequences needed for
maximal activation potential extended from amino acid 124 to amino acid 329.
However, both segments require GCN5 for efficient activation. The 124-329 region
depends on hydrophobic residues for it trans-activation function. An
F148S/L149S/F1518 mutant was unable to direct the activated transcription on an

integrated lacZ reporter gene.

EXPERIMENTAL METHODS

Yeast Strains

The yeast strains used for the experiments detailed in this chapter are described in

Table 3.

34

Deletion Mutagenesis of Hap4 aa330-554

Deletion mutants of the HAP4 gene that encodes the domain of Hap4 from amino
acid 330-554 were constructed to identify a minimal activation domain. The initial
constructs were fused to the Gal4 DNA binding domain (aa1-147). Hap4 deletion
mutants were also studied as fusion proteins joined to lexA.

The plasmid pJLSSS is an ARS/CEN recipient plasmid for mutants of the HAP4
gene fragment encoding amino acids 330-554. pJLSSS provides the Gal4 DNA-binding
domain (aa1-147). This plasmid was constructed by subcloning the SacI-BamHI
fragment from pJLS34, which contains the Gal4-Hap4 fragment with the ADHI promoter
upstream, into the SacI and BamHI sites of pRS315. 1.

Amino terminal deletions of Hap4 (aa330-554) were obtained by linearizing
pJLSSS with XhaI followed by ExaIII/Sl treatment for various amounts of time.
Following the EonII/Sl treatment the DNA was cut with SacI and then subcloned into a
vector obtained by digesting pJLSSS with Sacl and SmaI. Deletion endpoints and reading
frames were confirmed by dideoxy sequencing. Three HAP4 deletion mutants were
selected from this set, with amino terminal truncations at codons 359, 400, and 424.

Carboxy terminal deletions were obtained by employing unique XbaI and Bng
sites present within HAP4 and combining them with the above described amino terminus
deletion endpoints. HAP4 DNA that already encoded the amino terminal truncations
previously described was digested with either XbaI or Bng followed by a fill-in reaction
using the Klenaw fragment of E. cali DNA polymerase I followed by digestion with

XhaI. The resulting fragment was subcloned into XhaI-Smal treated pJLS114.

35

An internal deletion mutant removing codons 424 to 476 was constructed by
introducing a Bng site after the codon for aa423 of Hap4 that would exist in addition to
the Bng site present after codon 476. Subsequent digestion of this construct with Bng
followed by ligation resulted in the desired deletion. The additional BglII site was
introduced by PCR using primers ST65 and ST66. The PCR product, spanning codons
330 to 424 (including the desired BglII site) was cloned first into the TA cloning vector
from Invitrogen. The resulting plasmid was then digested with HpaI and BglII to release
the HAP4 fragment encoding amino acids 330-424. This fragment was subcloned into
SmaI-BglII treated pJLSSS, which provided the Gal4 DNA-binding domain and also
HAP4 codons 476-554.

Plasmids expressing lexA-Hap4 fusion proteins were constructed using various
combinations of the PCR oligonucleotides shown in Table 1. Two plasmids were central
to the subcloning strategy. The plasmid pDB20.lexA is a 211 origin, URA3 marked
plasmid that contains the ADH I promoter and terminator flanking lexA. A polylinker
with the sequence CTG-GCG-GCC-GCG-CGC-AAG-CTT is present immediately
downstream of lexA (the CT G in bold type is the final lexA codon that immediately
precedes the polylinker). The Not! site in the polylinker, highlighted in italic type, was
used in all subclones. The plasmid pRS4l4 (Sikorski and Hieter, 1989) is a low copy
TRPI marked plasmid. PCR fragments spanning Hap4 codons 330-424, 330- 476, 424-

476, 424-554, 476-554, or 13424-476 were cloned into the Invitrogen TA vector, excised

with Bag] and ligated into pDB20.lexA. For all other constructs discussed, PCR products
were digested with Bag] and subcloned directly into pDB20.lexA. Once in pDB20.lexA,

the DNA fragments encoding the various lexA-Hap4 fusion proteins flanked by the

36

ADHI promoter and terminator were subcloned into pRS414 either as XmaI-Sall, Sac]-

SalI, or BamHI fragments. All sequences were verified by dideoxy sequencing.

Deletion mutagenesis of Hap4 aal-330

To define a minimal activating region within Hap4 amino acid 1-330, I
constructed deletion mutants of the HAP4 gene fragment encoding amino acids 1-330 and
fused them to lexA. These deletion mutants were constructed using various combinations
of PCR oligonucleotides shown in Table l. The PCR products were digested with EagI
and subcloned directly into pDB20.lexA. Once in pDB20.lexA, the DNA fragments
encoding the various lexA-Hap4 fusion proteins flanked by the ADHI promoter and
terminator were subcloned into pRS414 either as XmaI-SalI, SacI-Sall, or BamHI

fragments. All sequences were verified by dideoxy sequencing.

B-Galactosidase Assays

Plasmids expressing lexA-Hap4 fusions were transformed into yeast strains
JSY03, JSY05, or L40, all of which have an integrated lacZ gene with eight lexA binding
sites upstream. Eight ml cultures were grown to OD595=0.5. B-Gal assays were

performed as described (Guarente, 1983; Miller, 1972). Three independent assays were

performed for each mutant.

Immunoblot Assays

To assess the steady-state level of each lexA-Hap4 fusion protein it was necessary

to use a plasmid with a 2n origin transformed into yeast strain J YSO3. 10 ml cultures

37

were grown to an approximate OD595=0.5, harvested by centrifugation, and resuspended
in 0.25 ml of buffer containing 100 mM Tris-Cl pH 8.0, 20% glycerol, lmM DTT.
Extracts were prepared by vortexing six times at top speed in 15-second bursts, waiting
on ice 15-seconds between bursts, in the presence of glass beads (425-600 microns from
Sigma catalog # G-8772) added to the level just below the meniscus. Extracts were
transferred to clean microfuge tubes and centrifuged at 4° C 14000 rpm for 5 min.
Relative protein concentrations were determined for each extract using a dye-binding
assay (Bradford, 1976). Equal amounts of protein were loaded onto 12.5% SDS-PAGE
gels. Following electrophoresis, proteins were electrophoretically transferred onto
nitrocellulose. Membranes were blocked in 7% powdered milk, 20 mM Tris-Cl pH 7.5,
137 mM NaCl, 0.0038M hydrochloric acid, 0.01 % Tween 20 and then incubated with a
mixture of three different mouse monoclonal antibodies (mABs) raised against lexA
produced by Yuri Nedialkov: 2 jig/ml YN-lexA-2-l2, 4.3 [lg/ml YN-lexA-6-10, and 2
ug/ml YN-lexA-l6-7. Incubation with primary mABs was followed by incubation with a
secondary antibody directed against mouse IgG, conjugated to horseradish peroxidase
(Sigma). Milk (1.5%) in 20 mM Tris-Cl pH 7.5, 137 mM NaCl, 1M hydrochloric acid,
and 0.01% Tween 20 was included as a blocker in all antibody incubations. Blots were
developed using an enhanced chemiluminesence system (ECL, Amersham or

Renaissance, DuPont NEN Life Science Products).

Point Mutant Construction
Clustered point mutations within the HAP4 region from codons 359 to 476 were

constructed using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA). The

38

oligonucleotides used are described in Table 2. The mutations were introduced directly
into a low copy TRPl-marked plasmid from which lexA-Hap4 fusion protein was
expressed from the ADHI promoter. The presence of the correct base-pair changes and

the absence of secondary mutations were confirmed by DNA sequence analysis.

Growth Curve

To compare the ability of Hap4 aal -330 to support growth on a non-fermentable
carbon source with that of full length Hap4(aa1-554) 1 performed a growth curve
experiment. The yeast strain DMYl48 was transformed with plasmids encoding either
wild type or mutant Hap4 protein. DMYl48 transforrnants were initially grown to
saturation (OD595 >15) in selective glucose-containing media. A portion of the saturated

culture, 0.2 ml, was used to inoculate a 100 ml culture of YP-lactate. Growth was

monitored by OD595.

39

 

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Strain Genotype Reference

DMYl48 ura3-52, leuZDl, his3DZOO, trpID63, D. McNabb, personal
hap4::hisG hap5::hisG communication
MAT-a

JSY03 ura3::lexA80p-lacZ, lysZ::lexA8op-URA3, This work
leu2-3,112, trpIA99, adeZ, his34200
MAT—a

JSY05 ura3::lexA8op-lacZ, lysZ::lexA8op-URA3, This work
leu2-3,112, trpl A99, ade2, his3A200,
gcn5::hisG
MAT-a

L40 his3A200, trp1-901, leu2-3,112, ade2, lysZ- (Hollenberg, et al., I 995)

801 am, URA3::(lexA0p)8-lacZ,
LYS2::(lexA0p)4-HIS3
MAT-a

TABLE 3. Description of yeast strains.

44

RESULTS

The observation that HAP4 (aa330-554) is able to function independent of ADA2,
ADA3, and GCN5 while VP16 tested in the same context is dependent on ADA2, ADA3,
and GCN5 indicates that the HAP4 activation domain is intrinsically ADA2, ADA3, and
GCN5 independent. I wanted to know what defined such an ADA independent activation
domain and thus I set out to identify and characterize the HAP4 activation domain. I
constructed an extensive set of deletion mutants of the HAP4 330—554 region, fused to
the lexA DNA binding domain, and tested their ability to activate transcription of a lacZ
reporter gene. The transcriptional activity of these mutants was intially tested using a
lacZ reporter gene on a high copy (211.) plasmid, pLGSDS, in the yeast strain PSY316.
The results obtained using this system were fraught with significant intra- and inter-assay
fluctuations, making analysis difficult. Hoping to eliminate such fluctuations, I
constructed the yeast strain J SY03 (genotype shown in Table 3) that had an integrated
lacZ reporter gene with eight lexA binding sites upstream.

Quantitation of transcriptional potential of the deletion mutants using J SY03 is
shown in Figure 1. Neither N-terminal deletion to residue 359 that resulted in the 359-
476 construct, nor C-terminal deletion to residue 476 that resulted in the 330-476
construct, caused in a significant loss of activation potential. Further N-terminal deletion
to residue 400 or C-terminal deletion to residue 464 resulted in a reduced effect on
activation. The positive contribution made by the 464-476 region is obvious (compare
330-476 with 330—464, 359-476 with 359-464, and 400-476 with 400-464). The 464-476

region is essential, however, only when aa400 is the N-terminal boundary. Otherwise,

45

the 464-476 region is necessary only to achieve maximal activation (compare 330-476

with 330—464 and 359-476 with 359-464).

46

Hap4 Segment Fused to lexA B-Galactosidase

Units
U 390:110
359 464 160:10
803140
533
230140
540:40
3:2
020
17301260

 

FIGURE 1. Specific activities of deletion mutants of the Hap4 C-terminal domain
(aa 330-554). The indicated Hap4 regions were fused to lexA and expressed

from the ADHI promoter on TRPI marked ARS/CEN plasmids. The lexA-Hap4
expression plasmids were transformed into J SY03 which contains an integrated
lacZ reporter gene with eight upstream lexA binding sites. B-galactosidase units
were measured in extracts of cells grown to mid-log phase, and are reported as
nmoles of o-nitrophenol/minute/mg protein. lexA alone has no activation potential.

47

Curiously, the deletion mutant lacking the 424-476 region is active although
neither half has significant activity. It is possible that by joining amino acid 423 with
amino acid 477 an artificial activation domain was created. Alternatively, this may be a
case of potent synergy between two very weak activation domains, as neither the 330-424
nor the 476-554 region possessed any significant independent activation potential.
However, this result does seem to indicate that while the 359-476 region is sufficient to
activate transcription, it is not necessary for such.

Immunoblot analysis shown in Figure 2 indicate that all mutant lexA-Hap4 fusion
proteins are present in detectable, although different, amounts except 330-554. Thus
differences in trans-activation potential between deletion mutants cannot be attributed to
differences in expression or stability of the deletion mutant proteins. It should be noted
that a low copy ARS/CEN plasmid was used to express the lexA-HAP4 fusion proteins in
the B-galactosidase assay whereas a high copy 2}; plasmid was used to express the same
constructs for the immunoblot. This is necessary because the ARS/CEN plasmids did not
produce detectable amounts of protein.

The results shown in Figure] demonstrate that the boundaries of the activation
domain lie between amino acids 359-400 and amino acids 464-476. As shown in the
cases of VP16 (Cress and Triezenberg, 1991; Regier, et al., 1993; Sullivan, et al., 1998),
p53 (Horikoshi, et al., 1995) and GCN4 (Drysdale, et al., 1995) activation domains are
dependent on hydrophobic residues. The 359—476 region of Hap4 possesses seven
different clusters of hydrophobic amino acids: L365/L366, 1370/L371,
1390/W391/Y393/L394, Y427/L428/F429/L430, I4SOIIA52, F456/Y 458, and

L466/M467 (also seen in Figure3). We hypothesized that some of these clusters would

48

FIGURE 2. A. Immunoblot analysis of yeast cells expressing deletion mutants of the
HAP4 gene fused to lexA. B. Immunoblot analysis of yeast cells expressing deletion
mutants and point mutants of the HAP4 gene fused to lexA. The indicated HAP4 deletion
mutants were fused to lexA and expressed from the ADHI promoter on a URA3 marked
21.1 plasmid. Aliquots of whole cell extracts were electrophoresed in SDS polyacrylamide
gels and blotted to nitrocellulose. The filters were incubated with a mixture of three
different polyclonal serums directed against the lexA protein and developed using
enhanced chemiluminescence.

49

359-476

124-329

 

50

365 370
NDDNMSL LNLPI LiEETVSSG

390
I

 

 

 

 

 

 

 

 

WNY LJPSSSSQQ

 

DDKVEEN

 

QDSSRALKKNTNSEKAN' Q

427

 

AKNDETYL F LQDQDESADS

 

 

 

 

 

 

 

 

 

 

 

450 456

HHHDELGSEITLAQDKFSY LI
466

PPTLEEL EEQDCNNGRS

 

 

 

FIGURE 3. The amino acid sequence of the Hap4 359—476 region. Hydrophobic
residues are in larger type. Boxed areas highlight clusters of hydrophobic amino
acids. Each hydrophobic residue within a given cluster was changed to serine.
The resulting mutant was expressed as a lexA fusion from the ADHI promoter
on aTRPI marked ARS/CEN plasmid. The lexA-Hap4 expression plasmids
were transformed into JSY03 and tested for the ability to activate transcription
of a lacZ reporter gene.

51

be essential for this region of Hap4 to active transcription. Therefore mutants were
generated that encoded a change of each hydrophobic amino acid within any given
cluster to serine. The resulting mutants were tested for their ability to activate
transcription of an integrated lacZ reporter gene with eight lexA binding sites upstream.

The results shown in Figure 4 reveal that L36SS/L366S, 137OS/L37IS had no
effect on the activation potential of Hap4 359-476 and I4SOS/L4528 had only a moderate
negative effect. Hap4 359-476 Y427S/L428S/F429S/L43OS resulted in a S-fold
reduction in activation potential. In contrast, 139OS/W39IS/Y393S/L394S,
F456S/Y4588, and L466S/M467S resulted in a greater than lO-fold reduction in
activation potential. These results are consistent with the previous analysis of the
deletion mutants. The region between amino acid 359 and 400 of Hap4 was necessary
only for maximal activation in the absence of GCN5 (see Figure 10) and neither
L36SS/L3668 nor I37OS/L37 18 had any effect on the activation potential of Hap4 359-
476. The region between amino acid 464 and 476 of Hap4 is essential for the activation
potential of the 359-476 region and L466S/M467S resulted in a greater than 10—fold
reduction in activation potential. The immunoblot seen in Figure 5 reveals that all of the
mutations that resulted in a decrease in trans-activation potential had no effect on protein
stability. Thus differences in trans-activation potential between mutants cannot be
attributed to differences in expression or stability of the mutant proteins.

To determine whether or not the 359-476 region was sufficient to carry out all
known transcriptional activation functions of Hap4, a fusion of the 359-476 region to
Hap4 1-330 was planned. The possibility that Hap4 possesses multiple activation

domains was raised when in the course of this pursuit Hap4 1-330 was unexpectedly

52

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53

WT 124-329

L139S/Il40S

F456S/Y 4SSS/L459S/L46GS/M467 S
L466S/M467 S

F456S/Y4588/L459S

I4SOS/L4SZS
Y427S/L4288/F429S/L4308
139OS/W39IS/Y393S/L394S
I37OS/L37 lS

L3658/L3668
WT 359-476

 

FIGURE 5. Immunoblot analysis of yeast cells expressing point mutants of the HAP4
gene fused to lexA. The indicated HAP4 point mutants were fused to lexA and expressed
from the ADHI promoter on URA3 marked Zu plasmid. Aliquots of whole cell extracts
were electrophoresed in SDS polyacrylamide gels and blotted to nitrocellulose. The filters
were incubated with a mixture of three different monoclonal serums directed against the
lexA protein and developed using enhanced chemiluminescence.

54

discovered to be able to support growth on a non-ferrnentable carbon source. Inferred
from this result was that the 1-330 region of Hap4 possesses transcriptional activation
potential, despite a previous report to the contrary (Forsburg and Guarente, 1989). The
original Hap4 work was carried out in the yeast strain BWGl-7a (Forsburg and Guarente,
1989). In this context the 1-330 region of Hap4 was unable to support growth on a non-
ferrnentable carbon source. However, when tested in DMYl48, a more robust yeast
strain, Hap4 1-330 was able to support growth on a non-fermentable carbon source
(Figure 6).

The less potent activation domain resident within 1-330 is unable to support
growth of some yeast strains on a non-fermentable carbon source but does nonetheless
possess transcriptional activation potential. When tested directly, the 1-330 region of
Hap4 when tethered to lexA was able to activate transcription of a lacZ reporter gene,
however with significantly less vigor than 330-554 (see Figure 7). The boundaries of the
activation domain resident within amino acids 1-330 of Hap4 were thus sought. Deletion
mutants of Hap4 (1-330) fused to the lexA DNA binding domain were constructed as
shown in Figure 7 and were tested for their activation potential using the integrated lacZ
reporter strain JSY03. Deletion of the N-terminus up to residue 123 resulted in no loss of
activation potential; however, further deletion to amino acid 160 resulted in an almost
complete loss of activation potential. Thus the N-terminal boundary of the activation
domain present within 1-330 must lie between amino acid 124 and amino acid 160.

Deletion of C-terminal Hap4 sequences to aa224 resulted in a total loss of
activation potential (see result for 1-224 in Figure 7) whereas Hap4 (1-270) maintained

activation potential. The C-terminal boundary of sequences needed for efficient

55

00595

+ CONTROL

+ HAP41-554

 

1 .5
1
0.5
0
0 20 40 60 80 100
TIME (HOURS)

FIGURE 6. Hap4 (aal-330) is capable of supporting growth of the yeast strain
DMYl48 in lactate medium. Growth assesement was carried out in liquid medium
minus tryptophan containing 2% lactate as the sole carbon source. DMYl48 yeast
were transformed with TRPI marked ARS/CEN plasmids expressing either

Hap4 1-330, Hap4 1-554, or the empty parental vector. Single colonies were
picked and grown to saturation in media lacking tryptophan and containing 2%
glucose. These cultures were used ti inoculate media lacking tryptophan and
containing 2% lactate as the sole carbon source. OD595 readings were

recorded at the indicated times.

56

Hap4 Segment Fused to lexA B-Galactosidase

Units
1#0 40° 1 3°
1 103 2 i 1
1_224 2° 1 3
96—224 70 11°
”—270 28° 3 6°
96—329 74° 315°
”#29 928 3- 333
1H0 6 i 2
1H” 2° 1110
217 329 48 .1: 12

FIGURE 7. Specific activities of deletion mutants of Hap4 N-terminal domain

(aa 1-330). The indicated Hap4 regions were fused to lexA and expressed from

the ADH I promoter on TRPI marked ARS/CEN plasmids. The lexA-Hap4
expression plasmids were transformed into JSY03 which contains an integrated
lacZ reporter gene with eight upstream lexA binding sites, B-galactosidase units
were measured in extracts of cells grown to mid-log phase, and are reported as
nmoles of o-nitrophenol/minute/mg protein. lexA alone has no activation potential.

57

transcriptional activation therefore lies between amino acid 224 and amino acid 270.
Immunoblot analysis indicated that the deletion mutant proteins are expressed in
detectable, although not similar, amounts with the exception of 96-329 and 124-329. For
the reasons stated above, high copy plasmids were used for the immunoblot analysis.

We hypothesized that the 124-329 region of Hap4 would also be dependent on
hydrophobic amino acids for the trans-activation function that it possessed. Since the
124-160 region of Hap4 was essential for the trans-activation capability of the 124-329
region we looked in this region of the protein for clusters of hydrophobic amino acids.
Two hydrophobic clusters within the 124-160 region were identified and point mutants
where serine was substituted for most of the residues within the clusters were constructed
(see Figure 8). The mutants constructed were Ll39S/Il4OS and Fl488/L149S/F1518 and
their trans-activation potential was quantified. The results shown in Figure 9 reveal that
Ll39S/Il4OS mutations had little to no effect on the trans-activation potential of the 124-
329 region of Hap4 whereas the Fl488/Ll49S/F lSlS mutation resulted in a complete
loss of activation potential of this region. The determination of the relative abundance of
the Ll39S/Il4OS mutant protein is shown in Figure 5. The determination of the relative
abundance of the F1488/Ll49S/F1515 mutant protein is underway.

The ability of Hap4 to activate transcription is less dependent on Ada2, Ada3, and
Gcn5 than other transcriptional activators (e.g. Gcn4 and VP16) (Berger, et al., 1992;
Pina, et al., 1993). Thus the ability to activate transcription in the absence of any of these
factors should be a reasonable “quality control” test for any activation domain identified.

The ability of 359—476 and 124-329 to function as transcriptional activators in the Agcn5

strain JSY05 was therefore tested. Both 124-329 and 359-476 are dependent on Gcn5 to

58

EKRKIGRHIQTIDE

 

 

KLINDSNYLAFLKFD

 

 

 

 

 

 

DLENEKFR

FIGURE 8. The amino acid sequence of the Hap4 124-160 region. Hydropho-
bic residues are in larger type. Boxed areas highlight clusters of hydrophobic
amino acids. Each hydrophobic residue within a given cluster was changed to
serine. The resulting mutant was expressed as a lexA fusion from the ADHI
promoter on aTRPI marked ARS/CEN plasmid. The lexA-Hap4 expression
plasmids were transformed into JSY03 and tested for the ability to activate
transcription of a lacZ reporter gene.

59

 

1250T

.3

5 1000-

G

0

fl

2 750-

n

a 500-

3?

8 250-
o.

 

 

WT L139$ F1488
I14OS L149$
F1513

FIGURE 9. The contribution of hydrophobic residues to the activation potential of the
Hap4 124-329 region. Each of the Hap4 mutants described in Figure 4 was fused to lexA
and assayed for the ability to activate expression of a lacZ gene with eight lexA binding
sites upstream integrated into the genome of the yeast strain L40. Bars indicate mean
activity (with standard deviation) of B-galactosidase in yeast cell abstracts from three
parallel cultures. B-galactosidase activities in extracts containing only lexA were
negligible. B-galactosidase units were measured as nmoles of o-nitrophenol/minute/mg
protein.

60

attain maximal transcriptional activation potential. However, the absence of Gcn5 more
significantly affects the activation potential of the 124-329 region (Figure 10). The
activation potential of the 124-329 region is reduced 4.2 fold in the absence of Gcn5
whereas the activation potential of the 359-476 region is only reduced 1.6 fold in the
absence of Gcn5. A corallary of this finding is that the 1-330 region of Hap4 might not
be able to support growth on a non-fermentable carbon source in the absence of Gcn5.
When growth of DMY45 harboring a plasmid that expressed either Hap4 1-330 or Hap4
1-554 on a non-fermentable carbon source was monitored, Hap4 1—330 was unable to
support growth in this media whereas Hap4 1-554 was able to support growth (Figure

l 1). Thus it appears that the 124-329 region is a Gcn5 dependent activation domain

whereas the 359—476 region is a Gcn5 independent activation domain.

61

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63

Discussion

The use of deletion mutagenesis as a tool for the study of activation domain
structure and function has yielded a great deal of information. This strategy has resulted
in the demonstration that multiple activation regions can reside within a single protein, as
in the cases of GCN4 (Drysdale, et al., 1995; Jackson, et al., 1996), VP16 (Cress and
Triezenberg, 1991; Regier, et al., 1993; Sullivan, et al., 1998; Walker, et al., 1993), p53
(Lin, et al., 1994), and RelA (Blair, et al., 1994). Detailed study of each motif
independently has revealed a functional dependence of each of these on the hydrophobic
character of specific amino acids. These studies imply that activation domains are
constructed from repeated motifs that depend on hydrophobic residues.

VP16 is an example of a transcriptional activator that contains multiple regions,
each dependent on one or two particular residues, capable of transcriptional activation.
The activity of the VP16N region is dependent on F442 (Cress and Triezenberg, 1991;
Regier, et al., 1993), and the activity of the VP16C region is dependent on F473/F475 for
the ability to activate transcription (Sullivan, et al., 1998). In contrast, GCN4 contains
seven hydrophobic clusters that make redundant contributions to transcriptional
activation (Jackson, et al., 1996). These examples demonstrate the existence of regions
of varying complexity.

Despite a similar dependence on key amino acids with hydrophobic sidechains,
transcriptional activators function via distinct mechanisms. The ADA-dependent versus
ADA-independent phenomenon is an example of how some activators (e. g. VP16 and

Gcn4) show a requirement for certain co-factors whereas others (e. g. Hap4 and Gal4) are

able to function independently of the same co-factors. A similar theme can be seen for
regions within a given protein. For instance, VP16C and VP16N have been shown to
function via distinct mechanisms. The VP16N region efficiently de-represses an
integrated reporter in mammalian cells, whereas the VP16C region is unable to do so
(Horn, 1999). In contrast, VP16C stimulates TFIID recruitment via TFIIA in vitro
whereas VP16N is unable to do so (Kobayashi, et al., 1998). Tethering regions together
that employ different mechanisms of action results in synergy with respect to trans-
activation potential. This synergistic affect can be seen when comparing the ability of
VP16N with that of VP16N+C to activate transcription. A similar result has been
demonstrated for Spl and VP16N+C (He and Weintraub, 1998). It is purported that the
multimerization of activation domains of distinct functions (i.e. Spl and VP16 N+C)
results in synergistic increases in activator potency. However, evidence for “domain
synergy” also exists whereby multimerization of two or more identical subunits also
results in synergistic increases in trans-activation potential (Emami and Carey, 1992).
Thus synergy does not require the presence of two distinct regions.

The yeast activator Hap4 is an example of a prototypical ADA-independent
transcriptional activator. To better understand the essential characteristics of an ADA-
independent activator, we set out to define and characterize the region(s) within Hap4.

Deletion mutant analysis revealed that two activation regions were resident within
Hap4. The N-terminal region resides within the 124-329 region of Hap4 and the C-
temlinal region resides within the 359-476 region of Hap4. Identification of the N-
terminal region represents a novel finding, as previous results indicated that the 1-330

region of Hap4 was devoid of trans-activation potential (Forsburg and Guarente, 1989).

65

Hap4N represents an ADA-dependent region whereas Hap4C represents an ADA-
independent region and thus the implication is that these two regions activate
transcription through unique pathways.

The relative activities of the deletion mutant proteins shown in Figures 1 and 7
may be misleading because all deletion mutant proteins were not present at equivalent
levels. Equal amounts of total protein were loaded into each lane shown in Figure 2,
however, the immunoblot indicates that the amount of lexA-Hap4 present varies.
Therefore, for instance, it may not be true that lexA-Hap4 (aa330-476) is 2-fold more
potent a transcriptional activator than lexA-Hap4 (aa330-554) because, as seen in Figure
2, there is a great deal more lexA-Hap4 (a330-476) present than lexA-Hap4 (aa330-554).
Thus the specific activity of lexA-Hap4 (aa330-554) is actually likely to be much higher
than that of lexA-Hap4 (aa330-476). We used a low copy vector in the activity assay and
a high copy vector in the immunoblot because the amount of protein produced from the
low c0py vector was undetectable by immunoblot. We are unable to use the high copy
vector in the activity assay because over-expression of Hap4 (aa330-554) is toxic.
However, while we cannot confidently assess the specific activities of the various
constructs, we are able to accurately identify minimal Hap4N and Hap4C activation
regions.

Both Hap4N and Hap4C depend on hydrophobic residues for the ability to
activate transcription. The effect of mutation of clusters of hydrophobic amino acids to
serine within Hap4 359-476 and 124-329 can be grouped into three distinct classes:
mutations that have no effect on trans-activation potential of Hap4 359-476

(L139S/Il4OS, L36SS/L366S, and I37OS/L37IS), mutations that had a moderate negative

66

effect on the activation potential (I4508/L4528), and mutations that had a significant
negative effect (greater than S-fold) on the trans-activation potential

(F 1488/Ll49S/F ISIS, 139OSIW39lS/Y393S/L394S, F456S/Y458S, and L4668/M467S).
I will consider possible explanations for these results below.

The C-region of Hap4 contains two clusters, L365/L366 and I370/L371, and the
N-region one cluster, Ll39/Il40, that when all of the hydrophobic residues present within
them are changed to serine, have no effect on trans-activation potential. The simplest
interpretation of this result is that these residues play no role in the activation of
transcription. Of course, it is also possible that these clusters play redundant roles. In
the C-region, L365/L366 and 1370/L37l may be redundant to themselves or to other
unidentified elements within the 359-476 region of Hap4. To determine whether they are
redundant to each other, simultaneous mutation of L365, L366, 1370, and L371 to serine
would be necessary. In the N-region other as yet unidentified hydrophobic residues that
contribute to the activation potential of the 124-329 region may exist. A systematic
mutagenesis scheme would answer this question.

The I4508/L4528 mutation has a moderately negative effect on trans-activation
potential of Hap4 359-476. Thus 1450 and L452 might contribute to the overall trans-
activation potential of Hap 359~476 but are not the sole determinants of this. For
example, these residues may partially contribute to direct interactions with target proteins
but mutation of these two residues to serine does not totally abrogate the interaction.

Alternatively, they may make structural contributions that are essential for target

interaction.

67

The F148S/Ll49S/F1518 mutations within Hap4N and the
I39OS/W39IS/Y393S/L394S, F456S/Y4588, and L466S/M467S mutations within
Hap4C have a significant negative effect on the trans-activation potential of each sub-
domain. These data indicate that these residues make critical contributions to target
interaction, either directly or indirectly.

By nature, hydrophobic amino acid side-chains have a propensity to be buried in
protein tertiary structures. The finding that Hap4 is dependent on hydrophobic amino
acids for the ability to activate transcription may indicate that the role of hydrophobic
amino acids within the activation domain of Hap4 is to facilitate formation of a structure
that is essential for Hap4 to activate transcription. However, this model becomes less
attractive because other activation domains are typically unstructured unless
accompanied by their target molecule. An alternative model proposes that critical
hydrophobic residues remain exposed on the surface of the activation domain and directly
interact with its target. The interaction of activation domain with target thus results in
hydrophobic amino acid side-chains buried in protein quaternary structure.

The central role of hydrophobic residues in directing protein-protein interactions
has been demonstrated in the cases of the p53 transcriptional activation domain
interacting with its repressor MDM2, glucocorticoid steroid receptor interacting with
GRIP-l, and VP16 with TBP. Crystallographic analysis revealed that the essential
hydrophobic amino acid side-chains of GRIP-l, p53, and SRC-l fits into a hydrophobic
groove within the glucocorticoid receptor, MDM2, and the peroxisome proliferator-

activated receptor-y respectively (Feng, et al., 1998; Kussie, et al., 1996; Nolte, et al.,

1998). Fluorescence spectroscopy techniques indicated that regions surrounding both

68

F442 and F473 within VP16 are more ordered in structure in the presence of TBP as
opposed to TFIIB (Shen, et al., 1996). We hypothesize that a similar hydrophobic
surface of Hap4 may interact with target proteins at key steps along the pathway to
transcriptional activation. The high copy suppression strategy described in the following
chapter represents an attempt determine if the hydrophobic residues identified are
involved in target protein interaction.

The amino acid sequence surrounding L466 and M467 of Hap4 is similar to
regions of GCN4 (L123 and F124) and VP16-C (M478 and F479) known to be important
for their ability to function as transcriptional activators GDrysdale, etal., 1995; Sullivan,
et al., 1998). In the sequence alignment displayed in Figure 12, L466 and M467 of Hap4
correspond to the residues M478, F479 of VP16 and L123, F124 of GCN4. However,
both VP16 and GCN4 are Ada2, Ada3, and GCN5 dependent
activators and thus the 466-467 region of Hap4 is not likely to be the sole determinant of
the Ada2, Ada3, and GCN5 independent nature of the 359-476 region of Hap4.

More likely candidates for the determination of Ada2, Ada3, and GCN5
independence are I390/W391/Y393/L394. Simultaneous mutation of each of these
residues to serine results in an ablation of trans-activation potential. Deletion of amino
acids 359-399 results in a significant loss of activation potential and more importantly a
loss of GCN5 independence (see Figure l and Figure 9). Taken together, these results
indicate that the determinants for Ada2, Ada3, and GCN5 independent activation reside
within the 359—399 region of Hap4 and are likely to include I390/W391/Y393/L394

either totally or

69

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70

in part. To test this hypothesis, we could compare the ability of the
I39OS/W 391S/Y 393S/L394S and Y427S/14288/F429S/L43OS Hap4 mutants to activate
transcription of the lacZ reporter gene in a gcn5 yeast strain, expecting that activation by
the Y427S/L4288/F429S/L43OS mutant to be affected and the
I39OS/W39lS/Y39BS/L394S mutant to be unaffected by the deletion of gcn5.

The recent discovery of a Hap4 homologue in K. lactis (Bourgarel and Nguyen,
1999) is very interesting. The existence of a Hap4 homologue allows for cross species
comparisons of the homologues and indeed the process has already begun. Sequence
alignment shows only two well-conserved regions of the protein exist: part of the
Hap2/3/5 interaction region and the region that corresponds to aa462-472 of Hap4 from
S. cerevisiae (see Figure 13). Not only does the 462-472 region have a motif of amino
acids that is reminiscent of GNC4 and VP16 activating regions as mentioned above, but
my work has also shown the importance of the 462-472 region of S. cerevisiae Hap4, as
the L4668/M467S double point mutant has the most severe effect on the activation
potential of the 359-476 region of Hap4. I plan to focus my attention on this region and
determine whether or not it is possible to identify its mechanistic target. If it is possible
to identify the mechanistic target of this part of Hap4, it will be possible to look for

conserved homologues in K. lactis as well.

71

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72

CHAPTER THREE

Identification of Ha Activation Domain Tar et 8

INTRODUCTION

An abundance of data indicates that transcriptional activators are able to
physically interact with numerous factors. The relevance of these interactions is not
always as clear. These factors fall into two different classes: general transcription factors
(GTFs) and coactivators. Coactivators are distinct from GTFs in that they are
dispensable for basal-level transcription in vitro but are required for activated
transcription in vivo or in vitro. Our initial interest in Hap4 stemmed from the fact that
while other activators required the coactivators Ada2, Ada3, and GCN5 for their ability
to activate transcription in viva, Hap4 did not. If Hap4 does not target Ada2, Ada3, and
GCN5, then what piece of the transcription apparatus does it target? It is possible that
Hap4 targets other coactivators, GTFs, or both.

Transcriptional activation requires accessory factors, variously termed co-
activators, adaptors, and mediators, in addition to the GTF proteins needed for basal
transcription. In some cases co-activators appear to bridge the interaction between gene-
specific activator proteins and GTFs, whereas in other cases coactivators facilitate
chromatin remodeling. Several functionally distinct classes of coactivators have been
described in detail in the introductory chapter. These include TAF components of TFIID,
the SRB/mediator complex that associates with RNAPH to form holoenzymes, SAGA
and related complexes that catalyze nucleosomal histone acetylation, and SWIISNF and

related chromatin remodeling complexes. The GTFs TFIIA, TFIIB, TBP, and TFIIH also

73

have been implicated as transcriptional activator targets. A more detailed description of
their role in activated transcription follows.

TFIIA is required for transcriptional activator response in viva (Ma, et al., 1993;
Yokomori, et al., 1994). Transcriptional activators such as Zta, VP16, and T antigen bind
directly to TFIIA, and this binding correlates with the ability to stimulate TFIIA-TFIID-
TATA (DA) complex formation (Damania, et al., 1998; Kobayashi, et al., 1995;
Kobayashi, et al., 1998; Ozer, et al., 1994; Sun, et al., 1994; Yokomori, et al., 1993). A
related hypothesis is that TFIIA affects activator response by inducing an isomerization
of the DA complex. Accumulation of this isomerized complex correlates with
transactivation, although an inability to directly trap this intermediate complex precludes
any direct observation of its activity relative to unisomerized PIC. Isomerization of the
DA complex into a form capable of binding TFIIB is necessary for activation of
transcription (Chi and Carey, 1996). Recently, a mutation in the small subunit of yeast
TFIIA (T 0A2), W76A, that results in a loss of activation potential by Gal4-Hap4 (330-
554) was described (Ozer, et al., 1998). Thus TOA2 may be a target of the Hap4
activation domain resident within the 330—554 region of the protein.

TFIIB has been implicated as the direct target of many gene-specific activators,
leading to the proposal that certain activators stimulate transcription by TFIIB
recruitment (Lin and Green, 1991; Roberts, et al., 1993). However, the model of
activator recruitment of TFIIB to the promoter is controversial. Recruitment of TFIIB to
the promoter was orginally hailed as the “rate limiting step” in PIC formation and thus a
worthy target of transcriptional activators (Lin, etal., 1991). However, a study of VP16

indicated a requirement for more than interaction with TFIIB to obtain full activation

74

because mutations in VP16 that result in a decrease in activation potential do not show a
lower affinity for TFIIB (Walker, et al., 1993). Furthermore, mutations in TFIIB that
disrupt the stability of the TBP-TATA-TFIIB complex do not affect the response of these
complexes to activation by VP16 (Chou and Struhl, 1997). Recent evidence indicates
that TFIIB undergoes a conformational change in the presence of TBP and DNA (Neish,
et al., 1998). It had earlier been noted that TFIIB undergoes a conformational change in
response to VP16 (Roberts, 1994). Thus the role of VP16, and transcriptional activators
in general, has been hypothesized as inducing a conformational change in TFIIB to
facilitate the binding of TFIIB to the TBP-DNA complex. A species-specific region of
yeast TFIIB, which extends from amino acid 144 to amino acid 157, has been identified
(Shaw, et al., 1996). Deletion of this region of yTFIIB severely compromised the ability
of Hap4 to activate transcription while having little affect on the activation potential of
Hapl , GCN4, or Gal4. Thus TFIIB may be a target of the Hap4 activation domain,
although it is formally possible that this TFIIB mutant is unable to maintain a critical
protein-protein contact necessary at the promoter tested with some protein other than
Hap4.

TBP binding to transcriptional activators such as VP16, Spl, and Gal4 has been
demonstrated in vitro (Emili, et al., 1994; Ingles, et al., 1991; Melcher and Johnston,
1995). Moreover, mutational analysis of TBP has identified TBP residues that disrupt
activator function in viva (Amdt, et al., 1995; Kim, et al., 1994; Kim and Roeder, 1997;
Lee and Struhl, 1995; Liljelund, et al., 1993). These findings have lead to several
hypotheses as to the relevance of this interaction. Primary among these hypotheses is

that TBP recruitment to the promoter is assisted by transcriptional activators. The fact

75

that TBP tethered to the promoter via a DNA-binding domain relieves the requirement
for an activation domain seems to support the notion that TBP recruitment could be a
mechanism employed by transcriptional activators (Chatterjee, 1995; Klages, 1995;
Majello, et al., 1998; Xiao, et al., 1995). This result is consistent with the findings that
TBP-TATA recognition may be the rate limiting step in transcription in vivo that can be
enhanced by activators (Klein, 1994), and that overexpression of TBP in viva stimulated
transcription from TATA containing reporter genes (Colgan and Manley, 1992). TBP
can exist as a dimer in soultion but binds DNA as a monomer, raising the possibility that
the function of certain transcriptional activators is to dissociate the TBP dimer, allowing
TBP to interact with the TATA-element (Coleman and Pugh, 1997; Coleman, et al.,
1995; Taggart and Pugh, 1996). A mutation that disrupts the transcriptional activation by
VP16 was found on the DNA binding surface of TBP (Kim, et al., 1994). Four different
clusters of hydrophobic residues within TBP that are essential for activator function were
identified through the systematic mutation of residues predicted by the crystal structure to
be surface exposed in human TBP (Bryant, et al., 1996). Lastly, it has been proposed that
TBP undergoes a conformational change that affects its binding to TATA in response to
transcriptional activators such as VP16 (Liljelund, et al., 1993).

TFIIH interacts with the activation domains of both VP16 and p53 (Xiao, et al.,
1994). Recently, a connection between several activation domains and the kinase activity

of TFIH has been demonstrated. The RARa activation domain AF-l can bind to TFIIH

and be phosphorylated by TFIH-I both in vitro and in viva (Rochette-Egly, et al., 1997).
This phosphorylation is required for in viva activation by RARa. Likewise, p53 can be

phosphorylated by the TFIIH kinase in vitro and this enhances the DNA binding activity

76

of p53 (Lu, et al., 1997). The HIV-1 transactivator Tat binds TFIIH both in vitro and in
viva and stimulates the phosphorylation of the RNAPII CTD by TFIIH (Garcia Martinez,
et al., 1997). Overall, these results indicate that the link between TFIIH and
transcriptional activation domains may involve either protein modifying the action of the
other.

This chapter describes several genetic strategies designed to identify the target(s)

of the Hap4 activation domain present within the 330—554 region of the protein.

EXPERIMENTAL METHODS

Yeast Strains
The yeast strains used for the experiments detailed in this chapter are described in

Table 4.

Ethyl Methane Sulfonate Mutagenesis

Strategies that employed ethyl methane sulfonate (EMS) mutagenesis were
carried out on BCYOS, L40, AMR70, and JSY03 yeast strains. In all cases, yeast
containing a lexA-Hap4 expressing plasmid were grown to a density of 2 x 107 cells/ml
in four 10 ml cultures of appropriate drop-out media. Cells were spun down and washed
twice with about 5 ml of sterile water each time. Cells were resuspended in 3 ml of 100
mM KHPO4 pH7.0 (Phosphate buffer). Phosphate buffer was made fresh by mixing 4.62
ml of 1M KH2P04 and 7.38 ml of 1M K2HP04 in a final volume of 120 ml. EMS was

added to the four tubes in the following amounts: 0, 10, 15, and 20 ul. Cells were

77

incubated at 30°C for one hour with shaking. After one hour, 10 ml of 5% sodium
thiosulfate (Na28203) freshly made and filter sterilized was added to neutralize the EMS.
Cells were spun down and waste was disposed as hazardous waste. Cells were washed
twice with about 5 ml of phosphate buffer and then cells were resuspended in 4 ml of
phosphate buffer. Each culture was diluted by a factor of 105 and 200 pl of each was
plated onto YPD. The amount of EMS that resulted in 50% mortality was determined.
That amount of EMS was then used to treat a fresh 10 ml culture. Upon final
resuspension in phosphate buffer, yeast were split up into as many aliquots of ~2 x 106
cells as possible and incubated in 2 ml of appropriate glucose containing media at 30°C
for 4-5 hours with shaking. Cells were plated onto appropriate media containing FOA at
a concentration of 2 g/liter.

(NOTE: This mutagenesis protocol should this be performed in several replicates
to avoid the “jackpot eflect”, which is the repeated isolation of a particular mutant

farmed early in the process that expanded throughout the population. )

Mutant Isolation Using S-Fluoroorotic Acid
Colonies that survived on S-fluoroorotic acid (FOA) plates (Guthrie and Fink,
1991) were replica plated onto X-gal plates (Rose, et al., 1990) using a nitrocellulose

filter. Blue colonies were indicative of false positives and white colonies were harvested

for further analysis.

78

Yeast Strain Production

To obtain a yeast strain that contained the trpI A99 allele, as well as integrated
URA3 and lacZ reporter genes with lexA binding sites upstream, BCYOS (obtained from
Barak Cohen in Roger Brent’s lab) was mated with TAT7 (obtained from Eric Andrulis
in Rolf Stemglanz’s lab) and sporulated (Rose, et al., 1990). BCYOS was the source of
the integrated URA3 gene with upsdtream lexA binding sites whereas TAT7 was the
source of the integrated lacZ gene with upstream lexA binding sites. J SYO2 resulted
from one of the segregants of that mating. J SYO2 contained integrated URA3 and lacZ
reporter genes with lexA binding sites upstream but not the trpI A99 allele. The trpI A99
allele was introduced using the plasmid pJL228. The plasmid pJL228 was first cut with
XhoI in order to linearize it. The resulting linear piece of DNA that contained a URA3
gene was transformed into J SY02. Yeast that grew in the absence of uracil were assumed
to be the result of integration of the linear DNA into the genome of J SY02. Yeast was
then grown in the presence of FOA to promote the excision of the URA3 gene. The

resulting strains, ahving the trpl A99 allele was named JSY03.

High Copy Suppression of Hap4 Point Mutants

L40 cells that contain mutant lexA-Hap4 expressing plasmids are sensitive to 3-
AT as a result of an inability to promote high levels of expression of the HIS3 gene. The
HIS3 gene product is inhibited by 3-AT, and the level of resistance to 3-AT is an
indicator of the trans-activation potential of a given lexA-Hap4 359-476 mutant. We
hypothesized that suppression of 3-AT sensitivity may result as a consequence of over-

expression of a mechanistic target of Hap4 359—476. To identify yeast genes which,

79

when highly expressed, might suppress 3-AT sensitivity, these yeast strains were
transformed with a LEU2 marked high copy S. cerevisiae genomic library (YEP13 based)
obtained from American Type Culture Collection (ATCC #37323). The library has a titer
of 2.0 x 1010 colony forming units per milliliter with l x 104 clones needed for complete
coverage of the yeast genome. 150,000 colonies were harvested from plates and plasmid
DNA was recovered using the Wizard® Plus Maxiprep DNA Purification System from
Promega. L40 cells harboring mutant versions of lexA-Hap4 359-476 were transformed
with the library DNA (Agatep, et al., 1998). L40 contains a HIS3 gene with lexA binding
sites located upstream. Transformants were plated directly onto plates that contained
amounts of 3-aminotriazole (3-AT) known to be toxic. Upon isolation of suppressors I
will verify the linkage of the suppression phenotype to the high copy yeast library
plasmid. Once this has been accomplished I will sequence the ends of the insert to
identify which fragment of genomic DNA is present. Database searches will allow for
the identification of the genes included on the fragment. The genes will be PCR
amplified and sub-cloned into high copy vectors separately and tested for their ability to
suppress the mutant phenotype. If there is a large number of genes to process, genes with

known roles in gene expression will be tested first.

80

 

 

Strain Genotype Reference

AMR70 his341200, lysZ-801 am trpI-901, leu2- (Hallenberg, et al., 1995)
3,112, URA3::(leanp)8 -lacZ
MAT-at

BC Y05 ura3, trpI, his3, leu2, lysZ B. Cohen, personal
leu2::lexA( 6ap)-LE U2 communication
lys2::lexA(8ap)-URA3
MAT-a

DEY1053 met13, rho', MAT-a D. McNabb, personal

communication

F W294 his4-917}; lysZ-I 73R2, trpI A63, (Gansherafi‘, et al., 1995)
leuZAI, ura3-52, spt3A202
MAT-a

F W463 his4-91 7}; lys2-I 73R2, trpI A63, (Eisenmann, et al., 1994)
leuZAI, ura3-52, spt8A302::LEU2
MAT-a

F Y833 his3A200, lys2A202 trpIA63, leuZAI, (Winston, et al., 1995)
ura3-52, MAT- a

FY1093 his4-917}: lys2-1 73R2, "pl/.163, (Roberts and Winston,
leuZAI, ura3-52, ade8, 1996)
spt7A402::LE U2
MAT- a

F Y I 106 his4-9I 7;; lys2-I 73R2, trpIA63, (Roberts and Winston,
leuZAI, ura3-52, spt20A100::URA3, 1996)
MAT-0t

JSY02 trpI A63, ura3::(leanp)8-lacz, This work
lys2::lexA(8ap)-URA3,
leu2::lexA( 60p )-LE U2, his3delt0200,
MAT-a:

JSY03 ura3::lexA8ap-lacZ, lys2::lexA8ap- This work
URA3, leu2-3,112, trp1A99, ade2,
his3A200, MAT-a

TAT7 his3A200, trp1-901, leu2-3,112, ade2, E. Andrulis, personal
lys2-801 am, ura3::(leanp)8-lacz, communication
LYS2::(leanp)4-HIS3, MAT- a

L40 his3A200, trpI-901, leu2-3,112, ade2, (Hallenberg, et al., 1995)

lys2-801am, URA3:.°(lem0p)8-lacZ,
LYS2::(lexA0pfl-HIS3, MAT- a

TABLE 4. Description of yeast strains.

81

RESULTS

Hap4 Activation Domain Target Identification

The basic strategy employed to identify the target(s) of the Hap4 activation
domain resident within the 330-554 region of the protein was to monitor the activation
potential of a lexA-Hap4 fusion in strains that contained reporter genes with lexA binding
sites upstream. Yeast strains that harbored a mutation in a gene encoding a required
target of the Hap4 activation domain would be unable to support activation by lexA-Hap4
and thus would produce a different phenotype than those yeast that were able to support
activation by lexA-Hap4. Four different strategies were used with the goal of identifying
targets of the Hap4 activation domain. I will describe the logic of each strategy. In the
cases where the strategy failed, I will describe what we learned from the experience and

how the knowledge was applied to the subsequent strategy.

L40/AMR70

A screen was designed that took advantage of the integrated lacZ gene with lexA
binding sites upstream, present in the isogenic yeast strains L40 and AMR70. Yeast
unable to support activation by lexA-Hap4 (330-554) will stay white when grown in the
presence of the chromogenic substrate X-gal. Such yeast were considered as potentially
harboring mutations in some Hap4 specific target, collected, and further characterized.
Carrying out the screen simultaneously in the two isogenic yeast strains allowed for the

expedient arrangement of mutants into complementation groups.

82

Yeast mutants were obtained by treating 2x 108 cells of the yeast strains L40 and
AMR70 with ethyl methane sulfonate (EMS) so that 50% mortality was achieved.
Following mutagenesis and recovery, equal amounts of both mutant strains were
transformed with low copy TRPI marked vectors encoding a fusion of the DNA-binding
domain of the bacterial protein lexA to either Hap4(S), made up of the 330-476 region of
Hap4, or Hap4(L), made up of the 330-554 region of Hap4. (The rationale behind using
Hap4(S) is that it is known to be a more potent trans-activator than Hap4(L). Hap4(L)
may therefore be a more sensitive indicator of mutation.)

Transformants were plated on plates that lacked tryptophan but included the
chromogenic substrate X-gal. Failure of the lexA-Hap4 fusions to drive an integrated
lacZ reporter gene should result in white colonies when the yeast are grown in the
presence of X-gal. Therefore white colonies among blue colonies were considered
mutants. White colonies were picked and plated on plates lacking tryptophan. The
phenotype was confirmed by replating on X-gal plates. Because the L40 yeast strain also
contained an integrated HIS3 with upstream lexA binding sites I was able to test any
putative mutants for their resistance to 3-aminotriazole (3-AT). The putative mutants
were subjected to increasing amounts of 3-AT (OmM, 2.5mM, SmM, 25mM, and 50mM).

Sixty-nine putative mutants were processed, thirty-five of which showed no
sensitivity to 3-AT and were thus discarded. The remaining thirty—four putative mutants
were processed, but were difficult to handle. All of the remaining putative mutants
showed dramatically reduced transformation efficiencies (greater that 100 fold), and
manipulations that involved plasmid transformation were therefore difficult. However,

the biggest problem faced was that the mutant phenotypes would disappear. Yeast taken

83

from frozen stocks of the supposed mutant would not display the mutant phenotype.
Thus, it was the instability of the mutant phenotypes that resulted in abandonment in

favor of a different strategy.

BCYOS

The previous strategy was a screen, I reasoned that a strategy based on selection
would allow for many more yeast to be processed. Thus a new strategy was developed
based on the activity of the URA3 product which converts 5-fluoroorotic acid (FOA) into
fluorouracil. Fluorouracil is a competitive inhibitor of thymidilate synthase and thus
toxic to yeast. The strain used in this strategy was BCY05 (from Barak Cohen) which
has an integrated copy of URA3 with lexA binding sites upstream. Yeast will be able to
grow in the presence of FOA if activation by lexA-Hap4 is disrupted. Yeast capable of
growing in the presence of FOA were considered as potentially harboring mutations in
some Hap4 specific target, collected, and further characterized.

2x 108 cells of BCY05 were treated with EMS so that 50% mortality was

achieved. The surviving yeast were transformed with a low copy TRPI marked plasmid
that carried a lexA-Hap4 (330-554) fusion. Transformants were plated on media that
lacked tryptophan and included 0.1% FOA. Survivors were collected and plated on
lactate media and tested for their ability to grow at 30°C. (I reasoned that if the putative
mutants were indeed defective for Hap4 activation they should not be able to survive on
lactate plates.) Those that did grow at 30°C were tested for their ability to grow at either
16°C or 37°C. Petite mutants, which failed to grow on lactate, were identified by

crossing to DEY1053 and not considered further. (DEY1053 is a mo strain and thus is

84

unable to compliment the inability of other rho' strains to grow on lactate.) BCY05 also
contains a LE U2 gene with upstream lexA binding sites. Putative mutants were also
tested for their ability to grow on media lacking leucine as a further criterion. Of the 167
putative mutants that survived FOA, 16 failed to grow on lactate medium at 30°C. Of the
remaining putative mutants, 6 failed to grow on lactate medium at 16°C and were
considered cold sensitive (cs) while 17 failed to grow on lactate medium at 37°C and
were considered temperature sensitive (ts).

I chose to first concentrate on those putative mutants that were unable to grow on
lactate media at 30°C because they displayed the most severe phenotype and I thought
that these contained mutations that would ultimately be the easiest to characterize. I
attempted to clear these mutants of the TRPI marked plasmid carrying lexA-Hap4 by
passaging them 5-10 times in rich media (YPD). In many cases I was unable to revert to
tryptophan auxatrophy by clearing the plasmid because the plasmid was already cleared.
The explanation for this finding became apparent when I realized that the trpI allele
present in BCY05 is a point mutant and thus prone to reversion. The selection pressure
against activation by lexA-Hap4 in the presence of FOA lead to a loss of the plasmid
expressing it. It would follow that the yeast in which this happened would now be trp’
because the plasmid contained the only functional TRPI gene in the cell. However, some
yeast reverted from up to TRP“. These yeast were able to survive in the presence of
FOA because they did not harbor the plasmid that expressed lexA-Hap4, and they were
able to survive in the absence of tryptophan because they now contained a functional
TRPI gene. In those few cases where it was possible to clear the TRPI marked plasmid

carrying lexA-Hap4 from the putative mutants, upon retransfomation of the TRPI marked

85

plasmid carrying lexA-Hap4 no mutant phenotype existed. I reasoned that plasmid based
mutations were the cause of the previously observed mutant phenotypes, even though the

plasmids in question were not subjected to EMS.

JSY03

The critical flaw in the previous strategy was the fact that BCY05 is a tryptophan
auxatroph as a result of a single point mutation in the TRPI gene, making this an unstable
phenotype. I thus developed a new strategy in the hopes of identifying Hap4 activation
domain targets that could provide an insight into mechanistic properties of the Hap4
activation domain. The new strategy remained based on the activity of the URA3 gene
product, which converts 5-fluoroorotic acid (FOA) into fluorouracil. The strain used in
this strategy, J SY03, has an integrated copy of URA3 with lexA binding sites upstream
and, importantly also containing the trplA99 allele. Yeast will be able to grow in the
presence of FOA if activation of the URA3 gene by lexA-Hap4 is disrupted. Yeast
capable of growing in the presence of FOA will be considered as possibly harboring
mutations in some Hap4 specific target, collected, and further characterized.

There are two significant differences between the previous failed strategy and the
current strategy. The first difference is that J SY03 is a tryptophan auxotroph as a result
of the total deletion of the TRPI gene (i.e. trp1A99) instead of a point mutation in the
TRPI gene as BCY05 is. The number of false positives that were simply TRPI revertants
was too high in the previous strategy. The second change is that instead of transforming
the lex-Hap4 expressing plasmid after EMS treatment, I mutagenized JSY03 cells that

had already had the plasmid present. The resulting increase in the background of false

86

positives due to plasmid based mutations is balanced by the ability to increase the total
number of yeast screened and ultimately selected. A minor difference is that J SY03
contains an integrated lacZ reporter gene with lexA binding sites upstream. This permits
easy verification and quantification of the mutant phenotype.

J SY03 cells were transformed with a low copy TRPI marked plasmid expressing
the lexA-Hap4 (330-554) fusion protein. These cells were treated with EMS so that 50%
mortality was achieved. Cells were split into aliquots of ~2x10° cells and revitalized in
trp'lglucose medium for 5 hours. The aliquots were then plated onto 61 trp’l 0.2% FOA
plates and grown at 30°C. 8000 colonies grew on the 61 plates. When plated on X-gal
plates (to test for activation of the lacZ reporter gene), ~10% turned blue indicating that
the ability to grow in the presence of FOA was likely the result of a mutation within the
URA3 gene. Therefore these putative mutants were no longer considered.

To further screen for mutations affecting activation by Hap4 we reasoned that if
lexA-Hap4 (330-554) is unable to activate transcription then endogenous Hap4 should be
unable to activate transcription. If endogenous HAP4 is unable to function as a
transcriptional activator the yeast will be unable to grow on a non-fermentable carbon
source such as lactate. Thus failure to grow on lactate is an expected mutant phenotype.
I tested 6 colonies from each initial selection plate for their ability to grow on lactate. Of
the 366 colonies tested 42 were unable to grow on lactate. These 42 were then cleared of
the TRPI marked plasmid and retransformed with a freshly prepared version of the same
plasmid to determine if the FOA resistant phenotype and the white on X-gal phenotype
was the result of a defect in the plasmid. Freshly transformed cells were grown on up

plates, colonies replica plated onto X—gal plates, and the blue/white phenotype assayed in

87

a qualitative manner. 13 of the 42 putative mutants seemed to turn blue slowly and 2
remained white. The ability of these 15 putative mutants to activate the transcription of a
lexA-lacZ reporter gene was quantified. This test yielded 4 “mutants” defective for
trancriptional activation by lexA-Hap4 (330—554).

Tetrad analysis revealed that the lactate phenotype and the inability to activate
transcription of the integrated lacZ reporter were not linked. Upon making this discovery
I realized that I may have been tracking mutations in the reporter genes themselves and
not mutations in targets of Hap4. In order to answer this question I transformed with a
plasmid borne lacZ reporter and tested whether the putative mutant strains would support
activation by lexA-Hap4. Indeed, the strains were able to support activation of the
plasmid borne lacZ reporter. Thus I infer that the mutations I followed were in the
reporter genes themselves. This could be determined experimentally by introducing a
new integrated reporter gene and testing for its function. If the new reporter gene is
functional then we can be confident that the original reporter gene was indeed mutant. If
the new reporter gene is not functional, we would presume that the strain in question
possesses a mutatibn that influences integrated reporter gene function in trans while
having no effect on plasmid based reporter gene function.

We reasoned that the cause for the elevated mutation rate may be the use of FOA.
Growth on FOA results in nucleotide starvation, specifically thymidylate, which in turn
would result in an increased mutation rate. I therefore repeated the process in the absence
of EMS treatment and selected for spontaneously occurring FOA-resistant yeast with the
hope that they contained mutations in mechanistic targets of the Hap4 330-554 region.

J SY03 cells were transformed with a low copy TRPI marked plasmid expressing the

88

lexA-Hap4 (330-554) fusion protein. I plated ~2x107 of these cells onto a total of 20 trp’l
0.2% FOA plates and grew them at 30°C. After three days at 30°C 20 FOA resistant
colonies appeared.

I transformed the FOA-resistant cells with a plasmid-bome lacZ reporter and
tested for the putative mutant strains to support activation by lexA-Hap4. All but one of
the putative mutant strains was able to support activation of the plasmid borne lacZ
reporter and therefore were discarded. The ability of the single remaining mutant to
support trans-activation by lexA—Hap4 is shown in Figure 14. The ability of lexA-Hap4
to activate the expression of a lacZ reporter gene was reduced lO-fold in the
spontaneously occurring mutant. (The inflated numbers seen in this assay are likely due
to the high copy reporter plasmid used.)

To determine if the defect in trans-activation was specific to Hap4 or more
general in nature, the ability of the putative mutant to support trans-activation by lexA-
GNC4 was also assessed. Figure 15 shows that neither lexA-Hap4 nor lexA-GCN4 was
able to activate the expression of the integrated lacZ reporter gene. (The ~10 fold
difference between the data seen in Figure 14 and that seen in Figure 15 with regard to
the trans-activation potential of lexA-Hap4 is likely due to the increased number of lacZ
reporter genes present in Figure 14.) While it may be premature to say that the defect is
general in nature, this preliminary evidence indicates that the mutation is not in a factor

specific to Hap4. Further characterization of this mutant yeast strain is underway.

High Copy Suppression

89

High copy suppression is a strategy whereby the mutant phenotypes that are the

result of an enfeebled activator protein are suppressed as a result of an increased amount

90

15000

 

   

.4.
S
8 12000
H
E 9000 -
i
2 6000 -
ti
3 3000 -
o .
STRAIN: WT Mutant
ACTIVATOR: lexA-HAP4 lexA-HAP4

FIGURE 14. The ability of a spontaneously occurring JSY03 mutant to sup-
port trans-activation by lexA-Hap4 (aa330-554). Hap4(aa330—554) was fused
to lexA and assayed for the ability to activate expression of a lacZ gene inte-
grated into the genome of the yeast strain that is the derivative of JSY03 as
well as a lacZ gene present on a high copy LE U2 marked plasmid. Both lacZ
genes had eight lexA binding sites upstream. Bars indicate mean activity
(with standard deviation) of B-galactosidase in yeast cell abstracts from three
parallel cultures. B—galactosidase activities in extracts containing only lexA
were negligible. B-galactosidase units were measured as nmoles of

c " l L ' ’...L....t..’...g protein.

91

2000

1500 -

1000 -

Beta-Galactosidase Units

500 -

 

 

 

o I I
STRAIN: Wl' Mutant WT Mutant

ACTIVATOR: lexA-Hap4 lexA-Hap4 lexA-GCN4 lexA-GCN4

FIGURE 15. The ability of a spontaneously occurring JSY03 mutant to sup-
port trans-activation by lexA-Hap4 or lexA-GCN4. Either Hap4(aa330-554)
or GCN4 (aa9-l72) was fused to lexA and assayed for the ability to activate
expression of a lacZ gene with eight lexA binding sites upstream integrated
into the genome of the yeast strain that is a derivative of JSY03. Bars indi-
cate mean activity (with standard deviation) of B-galactosidase in yeast cell
abstracts from three parallel cultures. [3- -galactosidase activities in extracts
containing only lexA were negligible. B- -galactosidase units were measured
as nmoles of n " I " ....... t-.’...g protein.

 

92

of target(s). The Hap4 point mutations that result in the loss of activation potential may
diminish the ability of Hap4 to maintain the protein-protein contacts needed to activate
transcription. The binding equilibrium may be shifted to the point where the mutant
protein is not engaged with its target long enough to result in trans-activation. The
presence of an increased amount of target protein may shift the equilibrium back to a
range that is sufficient for trans-activation. Thus taking advantage of the point mutants
described in Chapter 2, I have developed, but not completed, a new strategy that is based
on high copy suppression with the goal of identifying Hap4 activation domain target(s)
that could provide an insight into mechanistic properties of the Hap4 activation domain.
One advantage of this strategy is that it allows for quick identification of the gene
responsible for the suppression.

In this strategy I will employ the L40 yeast strain described earlier. Taking
advantage of the integrated HIS3 gene with upstream lexA binding sites, I will titrate the
levels of 3-AT to which the various Hap4 point mutants are resistant. I will then
transform a high copy yeast library and select for suppression of the 3-AT sensitivity
previously demonstrated. Upon isolation of suppressors I will verify the linkage of the
suppression phenotype to the high copy yeast library plasmid. Once this has been
accomplished I will sequence the ends of the insert to identify which fragment of
genomic DNA is present. Database searches will allow for the identification of the genes
included on the fragment. The genes will be PCR amplified and sub-cloned into high
copy vectors separately and tested for their ability to suppress the mutant phenotype. If

there is a large number of genes to process, genes with known roles in gene expression

93

will be tested first. For a discussion of experimental plans for establishing the existence

of specific protein-protein interactions see the Discussion section of Chapter 2.

SPT3I'7/8/20

The genetic screens described to this point represent one strategy for identifying
putative targets of the Hap4 activation domain. An alternative strategy is to test for
genetic interactions between Hap4 and genes already implicated in co-activator
mechanisms from other studies. We therefore tested the ability of Hap4 330-554 to
activate transcription in yeast strains that lacked SPT3, SPT7, SPT8, or SP720. These
SPT genes are part of the SAGA complex and are known transcriptional co-factors.
Hap4 (aa330-554) is able to function independently of many of the SAGA complex
members, most notably Ada2, Ada3, and Gcn5. However, the ability of Hap4 (aa330-
554) to function independently of some of the SAGA complex members that does not
mean that Hap4 (aa330-554) is not dependent on other members of the complex and thus
on the integrity of the complex as a whole.

I tested the ability of lexA-Hap4 330-554 to activate the transcription in spt3,
spt7, spt8, or spt20 deletion mutant strains. The lacZ reporter gene with lexA binding
sites positioned upstream was located on a LE U2 marked plasmid with a 2n origin. The
results of this test can be seen in Figure 16. The deletion of SPT3 or SPT8 had no
negative effect on the trans-activation potential of Hap4 330-554. In contrast, deletion of
SP17 or SP720 resulted in a total ablation of the trans-activation potential of lexA-Hap4

330-554. Irnmunoblots seen in Figure 17 show that deletion of either SPT? or SPT20 did

94

not result in a decreased amount of lexA-Hap4 protein. Therefore, the SPT7 and SPT20

gene products represent putative mechanistic targets of Hap4 (aa330-554).

95

10000 -

Beta-Galactosidase Units

 

 

spt3 spt7 spt8 spt20 WT

FIGURE 16. The effect of deletion of either SPT3, SP77, SPT8, or SPT20 on the
ability lexA-Hap4(aa330-554) to activate transcription. Hap4(aa330-554) was

fused to lexA and assayed for the ability to activate expression of a lacZ gene

present on a high copy LEU2 marked plasmid both with eight lexA binding sites
upstream in yeast strains lacking either .SPT3, SP77, SPT8, or SPT20 Bars indicate
mean activity (with standard deviation) of B—galactosidase in yeast cell abstracts from
three parallel cultures. B-galactosidase activities in extracts containing only lexA were

negligible. B-galactosidase units were measured as nmoles of o-nitrophenol/minute/mg
protein.

96

   
    
    

Wild Type
Aspt7

Aspt20

FIGURE 17. Immunoblot analysis of HAP4 (aa330-476) fused to lexA

in either wild type, Aspt7, or Aspt20 yeast cells. HAP4 (aa330-476)

fused to lexA was expressed from the ADHl promoter on a URA3 marked 21.1
plasmid. Aliquots of whole cell extracts were electrophoresed in SDS polyacrylamide
gels and blotted to nitrocellulose. The filters were incubated with a mixture of three
different monoclonal antibodies directed against the lexA protein and developed
using enhanced chemiluminescence.

97

DISCUSSION

While many lessons were learned along the way, the fatal flaw in all of the
selection strategies, with the possible exception of the spontaneously occurring mutant
strategy, was that I was using a potent activator and an integrated URA3 with 8 binding
sites for that activator upstream. The combination of FOA and 8 lexA-Hap4 molecules
bound upstream of URA3 proved too toxic to the yeast - not likely to be reversed by a
simple point mutation in a Hap4 activation domain target. Perhaps one lexA-binding site
upstream of the URA3 gene would have been better because it would have reduced the
synergistic effect of having 16 activation domains present upstream. In any case, for
such a strategy to be successful one needs a system where a modest change in activity
results in a discemable phenotypic change.

The use of growth on lactate as a secondary screen may not have been
appropriate. The logic held that if the strain in question harbored a mutation in a target of
the Hap4 330-554 activation domain that rendered it non-functional, then this mutation
should cause the endogenous Hap4 to also be non-functional and thus unable to grow on
lactate containing medium. However, the selection was set up using only one of the two
activation domains resident within Hap4. It is possible that the target of the 330-554
region of Hap4 is different from the target of the 1-330 region of Hap4. If this is the case
then it is possible that yeast harboring a mutation in a target of the 330-554 region of
Hap4 would still be able to grow on lactate as a result of a functional activation domain
present within the 1-330 region of Hap4.

Lastly, all of the strategies used, with the exception of the high copy suppression

strategy, were based on trying to break something; namely the link between the Hap4

98

activation domain and its mechanistic target. These strategies thus aimed for going from
a “working” situation to a “broken” situation and selecting for that. I think that going
from “broken” to “working” is a much better way to go and is the aim of the high copy
suppression strategy. There are many ways to break something, but when dealing with a
specific deficiency there are few ways to remedy the situation. (Note: Going from
“working” to “broken” is a problem when using selection strategies, when using
screening strategies the “working” to “broken” method is tried and true.)

Despite these problems, I do have one possible mutant of interest that is a product
of a “working” to “broken” strategy and am currently attempting to characterize it.
Among the things that I need to know are whether the defect is the result of a mutation
within a single gene, whether or not there are any growth phenotypes associated with this
mutation, and whether or not the mutation is recessive or dominant. If the mutation is
recessive, in one gene, and has a growth defect associated with it, I will then be able to
clone it by complementation.

The finding that lexA-Hap4 (aa330-554) is dependent on the SPT7 and SPT20
gene products is very interesting. In the absence of the SPT7 and SPTZO gene products
the SAGA complex is disrupted (Sterner, et al., 1999). Hap4 (aa330-554) is able to
function independently of many of the SAGA complex members, most notably Ada2,
Ada3, and GCN5. However, the ability of Hap4 (aa330-554) to function independently
of some of the SAGA complex members does not mean that Hap4 (aa330-554) is not
dependent on other members of the complex and thus an the integrity of the complex as a
whole. The SAGA complex has functions other than those provided by the ADA gene

family, such as SPT 3 and SPT8 interaction with TBP, and perhaps this is a functional

99

hint at that point. It is possible that SPT7 and SPT 20 are either direct targets of Hap4
(aa330-554) or part of another complex that is part of the pathway used by Hap4 (aa330-
554) to activate transcription. However, it should be noted that the effects of SPT7 and
SPT20 are very general. No activator tested to date is able to work in the absence of
SPT7 and SPT20, a list that includes Gal4, GCN4, and Hap4 tested from a natural
promoter (D. McNabb, personal communication). Perhaps SPT7 and SPT20 are general

factors needed for all trans-activation.

100

W

Future Directions and Persmctives

I have established that Hap4 contains two independent activation regions, each of
which depend on hydrophobic amino acids to function as a transcriptional activator. The
two regions differ in their dependence on GCN5, thus implying that they employ distinct
and different mechanisms in the activation of transcription. My attempts to identify a
mechanistic target of the Hap4C region have not been as successful. However, I will
detail future experiments that may result in a successful identification of a mechanistic
target of the Hap4 359-476 region. I will also attempt to put my work into the larger
context of studies of activation domains and mechanisms.

The results presented in Chapter 2 shows that clusters of hydrophobic amino acids
make contributions to the activation potential of Hap4. These clusters contain anywhere
from two to four residues. Whether or not all of the residues within an identified cluster
make equal contributions to the overall activation potential of Hap4 has not been
established. In order to determine the contribution of each residue identified they would
have to be changed individually to serine or, in the case where more than two residues
exist within a cluster, combinatorial mutants would need to be constructed.

I chose to change the hydrophobic residues within the identified clusters to serine
because serine is both small and non-hydrophobic as opposed to alanine, which, while it
is small, possesses some hydrophobic character. The question of whether or not alanine
substitutions at the identified positions would result in an equivalent loss of trans-

activation potential would be useful information. If changes to alanine result in similar,

10]

yet less severe, phenotypes, then the hydrophobic character of alanine would be useful in
the high copy suppression strategy. If it is true that hydrophobic character is necessary
for activator-target interactions, then alanine would be able to contribute in this way.

In Chapter 3 I described my attempts to identify the target of the Hap4 C-region.
A similar approach can be used with the Hap4 N-region to answer the question of
whether or not these spatially distinct activation domains have different targets.

With the high copy suppression strategy we may identify putative targets of Hap4.
These targets may be known transcription factors or be of have a yet unidentified
function. In the event that we are able to identify previously undiscovered genes that
encode Hap4 activation domain targets, we would construct strains in which the wild-
type gene is disrupted. A large deletion would be constructed in the plasmid-bome gene,
and a selectable marker (such as URA3 gene) would be inserted. This cassette would be
transformed into a diploid strain, selecting for integration of the URA3 marker gene. The
presumably heterozygous diploids would be sporulated and tetrads dissected, where a 2:2
ratio of viability would indicate that the gene is essential. If the gene is non-essential, the
different growth phenotypes that are associated with deletion of the gene would be
catalogued.

If the identified gene is non-essential, the efficiency of activation by Hap4wt,
lexA-Hap4N, and lexA-Hap4C in the deletion strain could be tested in order to determine
whether both activation regions of Hap4 are non-functional in the deletion strain. The
determination of whether or not the identified target is Hap4 specific or is required for the

function of multiple activation domains could be determined by assaying the ability of

102

lexA-Hap4, Gal4, and GCN4 fusion proteins to activate a lacZ gene with upstream lexA
sites.

High copy suppression may indicate a genetic interaction between the activation
domain tested and the target identified. To determine if the two identified players
physically interact, other tests must be carried out. If an epitope-tagged target protein is
able to interact with lexA-Hap4, then the lexA monoclonal antibodies that we possess
could be used to verify the physical interaction by immunoprecipitation experiments.
Quantitative gel shifts or Bia-Core experiments could be used to determine the affinity of
the identified target for Hap4. If no direct physical interaction between Hap4 and the
genetically identified target is demonstrated, then the exact role of this factor will be an
open question, one that will need to be answered.

An epitope-tagged target protein would also be useful in determining if the newly
identified factor is a part of any larger complexes and what these complexes are. It is
possible that it may be part of an already identified complex such as SAGA or SWIISNF
or it may be part of a yet identified complex. Conventional chromatography methods
such as ion-exchange and size-filtration columns could be used to crudely purify the
complex(s) that contain this factor if the factor is part of a previously unidentified
complex. Affinity chromatography using the epitope tag would allow further purification
of the complex(s).

The implication of this work on the field as a whole rests largely on whether or
not we are able to establish a target for either of the activation domains resident within
Hap4. The fact that these activation domains are dependent on hydrophobic amino acids

is a new finding for Hap4, but a well-established feature of many other transcriptional

103

activators. The use of point mutants within Hap4 for the high copy suppression strategy
is a novel approach for establishing activation domain targets and allows for immediate
biochemical verification of the phenomenon through the establishment of physical
interactions if a direct interaction occurs. This links the phenotype to the activation
domain itself through the physical interaction data.

Whether or not new factors are identified in the search described above or a
previously identified factor is implicated in the Hap4 pathway, the result will add another
layer of complexity onto the ever-expanding complex universe that is the field of gene
expression.

Reductionism is giving way to more holistic approaches for attacking the
unsolved questions that remain in the field of gene expression; approches that are carried
out in natural contexts. The work of the reductionist was valuable, for the in vitro work
identified the basal transcription factors necessary for promoter-directed transcription.
However, the discovery of activated transcription quickly introduced a new level of
complexity into the picture and things have been getting more complicated ever since.
For instance, detailed structural analysis of activators has proven very difficult. Simple
patterns of residues do not seem to hold the key to how activators are differentiated. It
will be necessary to study activators in the presence of targets in a natural context to
gain some insight into how specificity is achieved. I hope that my high copy
suppression strategy is a step in the right direction.

An example of how interconnections may be missed if one is to focus only on a

single track can be found in the study of co-activators and co-repressors. Co-activators

and co—repressors provide for the integration of signals from diverse signal transduction

104

pathways and translate them into coordinated transcriptional responses. The role of co-
activators and co-repressors in the response of the steroid receptors to hormones provides
an example of how integration is accomplished in nature. It is also a good example of
how complicated the picture can be. For instance, the co-activator GRIPl modulates the
activity of the progesterone, estrogen, retinoic acid, and retinoid X receptors (Hong, et
al., 1996; Voegel, et al., 1996) while SRC-l modulates the activity of a partially
overlapping group that includes the progesterone, estrogen, retinoic acid, retinoid X,
glucocorticoid, and thyroid receptors (Kamei, et al., 1996; Onate, et al., 1995; Yao, et al.,
1996). Each of these steroid receptors is likely to control the expression of a different
regulon, so the fact that they all interact with two common factors does not simply
represent a point of convergence. The controlled expression of the co-activator targets
provides a means of regulating the action of various receptors. Another interesting
possibility is that the ligand-receptor interaction may influence the affinity of the receptor
for a co-activator, thus providing another level of control.

The challenge that remains for those in the field of gene expression is how to
make sense of all of the data; to integrate it, if possible. It is no longer possible to study
just one thing to the exclusion of all else - be it one activator, one complex, or even one
pathway. One must now fit data into the constantly changing big picture. Genetic
techniques will be invaluable in this pursuit. The in viva validation of the in vitro work
is a complicated, but necessary, step in our understanding of gene expression.

The most recent approaches to comprehensively address these issues are the
related fields of genomics, which attempts to establish circuits of gene expression, and

proteomics, which attempts establish protein families based on structure or sequence

105

similarity. Studies using these techniques may provide the foundation and context for
interpreting mechanistic studies in control of gene expression. However, as with any new
avenue of investigation there are pitfalls. Genome-wide analysis will yield vast amounts
of data, but how to interpret it will be the challenge. For instance, examples of large
fluctuations in specific mRNA levels can occur as a result of a given treatment without a
corresponding alteration in protein levels. Are the fluctuations in mRNA levels
significant in this case? Despite such issues, these techniques will allow for a broader
perspective and will allow for a better understanding of the regulatory circuits that exist
within the genome.

The variety of genomic sequencing initiatives that make genome-wide analysis
possible also allows for cross-species awareness. Homologues of a given protein can be
identified in other species, the extent to which it has been conserved can be determined,
and an understanding and appreciation for what they might be doing in these other
species may be attained. One can find out if they are involved in similar pathways and
what context they are found in. This may then influence the nature of the questions asked
in other organisms. For instance, if I am able to identify a mechanistic target of Hap4 in
S. cerevisiae, does it exist in K. lactis and does the Hap4 homologue in K. lactis target it
as well? This may lead to an identification of conserved transcriptional regulatory
circuits in K. lactis. If the landscape is different in K. lactis, it may lead to the discovery

of new transcriptional regulatory circuits.

106

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