Revealing the Mechanism of HSP104 Transcription Initiation in

the Yeast S.cerevisiae

Thesis submitted for the degree of

“Doctor of Philosophy”

By

Melanie R. Grably

318013778

Submitted to the Senate of the Hebrew University

March 2008

This work was carried out under the supervision of:

Professor David Engelberg

This thesis is dedicated to my family for their support.

To the wonderful people I had the chance to work with throughout the years.

To Irith for her endless (motherly) advice friendship and loving support.

Finally to Dudi, for his endless PATIENCE, understanding, motivation, belief in this

project, and last but definitely not least his friendship.

Thank you.

i

Summary

All living organisms are continuously exposed to sub-optimal growth conditions and

have therefore developed appropriate responses in order to survive. The response to

many of these stressful conditions is mostly regulated at the level of transcription, leading

to up-regulation of the expression of many stress-related genes. Many genes are up-

regulated in response to any given stress (the ‘general stress response’) and some of these

genes are induced most strongly in response to a specific stress (the ‘specific stress

response’). Important aspects of the co-regulation of many genes by the general response

and of specific regulation by the specific stress responses have been revealed. Yet, the

relationship between these two responses is not understood. In this thesis we study the

transcriptional regulation of HSP104 of the yeast S.cerevisiae, a gene highly expressed in

response to heat shock. An early analysis of the HSP104 promoter region, through its

analysis via 5’deletion, mutagenesis and heterologous studies, showed that two different

families of transcriptional activators regulate the expression of this gene. i) Hsf1, which

upon activation by heat shock binds Heat Shock Elements (HSEs). ii) Msn2/4, which

upon activation by a broad range of stresses, bind STress Response Elements (STREs)

and are negatively regulated by the Ras/cAMP/PKA pathway. This analysis also showed

that the promoter displays a highly flexible mode of activation. It is maximally activated

in the presence of both sets of transcriptional activators but can also be activated by either

one alone. We were also able to map the elements in the promoter responsible for basal

activity (the sequence between -334 to -300) and those essential for heat shock-regulated

activity (47). These results were obtained primarily during the course of my M.Sc.

studies.

In this thesis, I describe the continuation of the effort to reveal the mechanism of

transcriptional activation of the HSP104 promoter. I describe four experimental routes.

One, continuing the approach used in the studies described above, we proceeded with

additional 5’ deletions of the HSP104 promoter (particularly the fragment between -334

and -300) attempting to obtain a finely tuned map of the sequence(s) responsible for the

basal activity of HSP104 and for other unexpected properties of the sequence between -

334 to -300. Two, using chromatin immunoprecipitation (ChIP), we monitored some of

the major changes occurring in vivo on the promoter following stress. Three, using a

ii

genetic approach, we identified components of the basal transcription machinery that are

important for HSP104 promoter activity. Four, using a combination of ChIP experiments

and a genetic approach, we sought possible regulators of Hsf1.

Through the deletion analysis, we found that important part of the properties of the

34bp between -334 and -300 could be accounted for by a short HSE-like sequence

residing in -305. Using ChIP assays we show that under optimal growth conditions

nucleosomes on the HSP104 promoter contain mostly acetylated H3 and H4. However,

following heat shock there is a rapid, but transient, decrease in the concentration of

acetylated histones on the promoter which seems to be partly mediated by Msn2/4.

Namely, the Ras/PKA pathway controls H3 and H4 acetylation state via Msn2/4, thereby

governing induction of the promoter. We further show that the decrease in acetylated H3

and H4 on the promoter occurs via two distinct mechanisms. Finally, we show that Hsf1

binding to the promoter is constitutive regardless of stress conditions, but is reduced in

ras2Δ cells. Using the genetic approach, we found that Rpb4, components of the

SRB/MED coactivator complex, or of the SAGA and SWI/SNF complexes are critical for

proper HSP104 transcription. We also identified components of the basal transcription

machinery (primarily of the SAGA complex) that are critical for Hsf1 activity.

These four approaches combined allow the establishment of a model describing

the series of molecular events occurring on the HSP104 promoter before and after heat

shock.

Contents

Summary………………………………………………………………....................i-ii

Introduction

The cellular stress response………………………………………………………… 2-3

General mechanisms leading to transcription initiation……………………………..3-5

Transcription under stress in S.cerevisiae…………………………………………...6-7

The HSE/Hsf1 system……………………………………………………………….7-8

The STRE/Msn2/Msn4 system……………………………………………………..8-9

Hsf1 and Msn2/4 can exclusively or cooperatively activate the yeast HSP104 gene. 10

HSP104 promoter analysis…………………………………..................................10-18

Goals of Study…………………………………………………………………...18-19

Experimental Procedures

Yeast strains, plasmids and media………………………………………..............19-20

Chromatin immunoprecipitation………………………………………………….20-21

RNA preparation and S1 analysis……………………………………………………21

Preparation of cell lysates and western blot analysis……………………...................21

β-Galactosidase assay……………………………………………………..................22

Results

The upstream 34bp fragment of the HSP104 promoter possesses unusual modular

properties………………………………………………………………………….22-28

In response to heat shock, acetylated H3 histones dissociate from the promoter

whereas acetylated H4 histones undergo deacetylation…………………………..29-32

No specific HDAC is responsible for the deacetylation of H4 on the HSP104

promoter……………………………………………………………………………...33

Hsf1 constitutively binds the HSP104 promoter…………………………………34-36

SAGA, SRB/MED and SWI/SNF are important for HSP104 promoter activity....36-43

Regulation of Hsf1…………………………………………………………..........44-47

Discussion………………………………………………………………………...47-52

References………………………………………………………………………..52-61

2

INTRODUCTION

The cellular stress response

Cells are continuously exposed to suboptimal growth conditions, generally

termed cellular stresses. They have developed therefore various strategies in order to

survive and even further proliferate and function under these stresses. At the

molecular level these strategies include the activation of several biochemical

machineries. First, the machinery that imposes growth arrest (3, 54, 76, 101, 106,

111, 130) in order to prevent DNA synthesis and proliferation under stress and

damaging conditions. Second, the induction (primarily at the transcriptional level) of

a small number of genes whose protein products are involved in combating the stress

and in repairing the damage inflicted (16, 20, 44, 51, 69). Cell cycle arrest could be

relieved when repair activity is completed and protective systems are active. Third,

the activation of cell death systems, that occurs if the damage is not repairable (5, 11).

This thesis focuses on the mechanisms responsible for the induction of gene

expression in response to stress. The genes expressed in response to stress could be

categorized into two different groups. One group includes genes whose expression is

required to combat the specific stress inflicted (the “specific stress response”). The

other group includes genes that encode repair and protective proteins, but their

activity is not directly relevant to the stress inflicted. They probably serve as a “just

in case” protective measure (the “general stress response”). For example, upon heat

shock, there is specific expression of heat shock protein genes (HSPs) (14, 16, 51, 81,

85), most of which are chaperones that prevent protein aggregation and maintain

proteins in their soluble and active form. However, in parallel to the induction of

HSPs, the cell also induces expression of genes whose products are responsible for

dealing with oxidative stress and/or DNA damage (20, 44, 51, 85). Similarly, when

cells are exposed to DNA damaging agents, some HSPs are induced in parallel to the

induction of DNA repair systems. The induction of many stress-related genes,

including many that are not relevant to the specific stress inflicted, renders the cell

resistant to other stresses or to more severe stresses, a phenomenon known as cross-

protection and thermotolerance (81, 107, 108).

Although revealed to a certain level in prokaryotes, many aspects of the

molecular basis of the cellular stress response in eukaryotes are still enigmatic. It is

not understood, for example, how cells sense stresses such as heat shock (what

3

cellular receptor is responsible for sensing elevated temperature?), pH, or high

concentrations of free radicals. It is also far from understood how the stress signal is

transmitted to the nucleus and affects the relevant transcriptional activator(s). Finally,

it is not known how the transcriptional machinery functions under conditions in which

many proteins are denatured or inactivated. Does the same basal transcriptional

system function under optimal conditions and under stress?

This study approaches some of these unresolved matters by addressing the

mechanism of transcription initiation under stress of one single gene, HSP104 of the

yeast Saccharomyces cerevisiae. It begins by a comprehensive analysis of the

HSP104 promoter aimed at identifying the major cis-elements involved (most of this

work was carried out in my M.Sc. studies and is therefore presented primarily in the

"Introduction" section). It continues by measuring changes in chromatin organization

that occur on the promoter in response to stress and further continues to the

identification of components of the basal transcription machinery, specifically critical

for HSP104 transcription.

Prior to focusing on what was known about HSP104 transcription when this

thesis was initiated (see page 10), I shall describe our current understanding of

transcription regulation in general, and our current knowledge of stress signaling and

stress-activated transcription factors in yeast.

General mechanisms leading to transcription initiation

Transcription initiation in eukaryotes is a complex reaction involving dozens

of proteins. The complexity of the reaction is further increased by the fact that many

aspects of it could be specific for some groups of genes and even for any given gene.

Yet, there are several major common themes in transcription initiation of all genes

transcribed by RNA PolII. It is clear that transcription initiation of all these genes

requires the presence of the core RNA PolII (12 subunits in yeast) along with proteins

forming the so called preinitiation complex [(PIC), also known as basal transcriptional

complex, reviewed in (28, 78)]. There are still debates whether this complex is

actually preformed or whether the components forming PIC are sequentially recruited

to promoters upon activation. Included in this basal transcriptional complex is the

TATA binding protein (TBP). TBP is found in a multiprotein complex called TFIID

which includes the TBP in addition to 14 proteins known as TBP associated factors

(TAFs). Curiously, although most genes require the presence of one or more TAFs

4

for their transcription, some 16% of the genes of S.cerevisiae do not need TAFs for

their transcription (59, 71, 113). However, the majority of TAFs are essential for

viability [reviewed in (48)], as are 10 of the twelve subunits of RNA PolII (23, 24).

Following binding of TFIID, more protein complexes are recruited to the promoter,

i.e., the TFIIB complex, which assists RNA PolII in selecting the transcription start

site. Then, the RNA PolII holoenzyme along with TFIIF, TFIIE, and TFIIH associate

with the promoter and form the PIC. Following establishment of PIC, promoter

melting and transcription initiation occur and are followed by hyperphoshorylation of

the C-terminal domain (CTD) of the RNA polymerase through the TFIIH kinase

activity. This leads to promoter clearance and elongation of transcription [reviewed

in (28)].

All of the above events do not occur automatically since inactive promoters

(such as promoters of heat shock genes in cells not exposed to stress) are not

accessible to TBP and the subsequent complexes. Such promoters are part of DNA

that is wrapped around histone proteins (two H2A-H2B heterodimers and a H3-H4

tetramer) which form nucleosomal structures. These nucleosomal structures are

further compacted into tightly super-coiled structures called chromatin. Therefore,

the binding of the basal transcription machinery and the formation of PIC are

hindered by these nucleosomes that have to be remodeled to enable transcription

initiation to occur. Thus, many preceding steps should take place in order to allow the

formation of PIC and transcription initiation. There is no general mechanism(s)

leading to chromatin remodeling and transcription initiation of all genes and each

promoter is activated in its own unique and specific way (2, 29, 42). Nevertheless, a

general mechanism leading to gene activation is believed to be the following. Upon

an activating signal there is binding of proteins known as transcriptional activators to

enhancer elements (also called upstream activating sequences) in the relevant

promoter and unbinding (where applicable) of transcriptional repressors. When

bound to enhancers, transcriptional activators recruit chromatin modifying complexes.

The main purpose of these chromatin modifications is to induce “melting” of

nucleosomal structures in order to reduce the histone-DNA interaction which enables

the assembly of PIC. Chromatin modifying complexes could be divided into two

general groups: i) Factors that, through the hydrolysis of ATP molecules, induce

conformational and spatial changes of nucleosomes (1, 43). ii) Factors that covalently

modify histones by either acetylation, phosphorylation, sumoylation, or methylation

5

(70, 73, 79, 122). It is believed that one of the major events leading to the "opening"

of chromatin is the acetylation of histones on conserved lysine residues. This

acetylation probably reduces interactions between histones and DNA. Acetylated

histones (generally H3 and H4) then facilitate access of other chromatin remodeling

factors which impose additional spatial changes on chromatin, followed by the

recruitment of basal transcription factors and RNA PolII [reviewed in (28, 78)].

These modifications are actually critical for proper gene activation. The above

concept is supported by numerous studies (2, 13, 21, 36, 64, 100, 103), but recent

studies reported that on some promoters it is not histone acetylation, but rather histone

deacetylation, that is related to the activation of these promoters. Particularly,

Deckert and Struhl showed that in yeast, histones H3 and H4 undergo deacetylation

on some stress-activated and galactose-induced promoters (31). They further showed

that, depending on the inducer, one single promoter can undergo different chromatin

modifications. For example, an increase in histone acetylation in response to one

stress and a decrease in histone acetylation in response to another stimulus (31).

Following chromatin remodeling and establishment of the preinitiation

complex on the promoter, transcription initiation could be further enhanced by co-

activator complexes such as SRB/MED (8, 53, 63, 125). It should be appreciated that

a plethora of factors could join any of these multi-protein complexes changing their

composition and catalytic properties from one promoter to another. The

transcriptional activators responsible for initiating the cascade of events leading to

transcription initiation are even more specific, usually involved in activation of a

limited number of genes. Many different transcriptional activators are expressed in

the cell, each responding to a narrow subset of signals, and some to a single signal

(i.e., hormones, growth factors). The different modifications occurring on the

chromatin and the enzymes involved in these modifications are also different from

promoter to promoter. Similarly, components composing the co-activator complex

SRB/MED could also vary on promoters [reviewed in (8, 10)] and even components

making up the RNA PolII holoenzyme could differ according to cellular conditions

and the particular promoter. In fact, even one subunit of RNA PolII, Rpb4, is

dispensable for cell proliferation and seems to be required only under stress

conditions (23, 89, 97, 98).

6

Transcription under stress in S.cerevisiae

Although several signal transduction pathways and some stress-induced

transcriptional activators have been identified (37, 38, 41, 87, 92, 96, 102, 133), we

have only partial answers to some of the major questions raised above with respect to

sensing the stress and in turn activating transcription. This issue is nevertheless better

understood in S.cerevisiae than in any other experimental system. A large number of

stress responsive systems have been discovered in this organism including several

transcriptional activators whose activity is induced by specific stresses (e.g. yAP1,

Gcn4, Gln3) (41, 84, 88, 92). Another activator, Hsf1, that is induced mainly by

elevated temperature, but also by other stresses [(52, 83, 90, 133) and see below] and

yet two more activators, Msn2 and Msn4 that are activated in response to any stress

[(17, 87, 110) and see below]. As promoters are usually complex, containing several

enhancer elements, it is most probable that none of these activators is acting alone on

target promoters, but cooperate with one of the other stress-induced activators (4, 29,

47), or with other activators, not necessarily induced by stress (39, 53, 65, 72, 80, 93,

123). It is still unclear how two or more activators co-act on the same promoter.

Recent studies addressed the changes in the organization of chromatin that

occur on stress responsive promoters upon activation (21, 36, 128, 135, 136). It was

found that many promoters undergo extensive chromatin remodeling (i.e.,

nucleosomal disassembly following histone acetylation) upon activation and that the

complexes responsible for this modification are in fact recruited by transcriptional

activators (21, 36, 128, 135, 136). It should be noted that most studies address the

question at the whole genome level and their conclusions are therefore grossly

general. The epistatic relationships between recruitment of transcriptional activators

and changes in chromatin structure are not well established in many cases. Also, it is

also not fully understood how RNA PolII and the factors of the basal transcription

machinery function under stresses such as heat shock, when many proteins are

denatured. One of the RNA PolII subunits, Rpb4, is essential only under stress, and

seems to be involved in the induction of some stress related genes (23, 89, 97, 98). It

may also function as a stabilizer of RNA PolII under stress (23, 104). It is not clear

whether other components of the PIC are specifically important for transcription

under stress.

In an attempt to understand these aspects of the mechanisms of transcriptional

activation under stress, we have been focusing on the HSP104 promoter. This

7

promoter manifests some activity under non-stressed conditions that is dramatically

increased under stress [see Fig. 2 and (4, 16, 47, 121)]. In the first stage of the study,

we cloned and mapped the cis-elements responsible for basal promoter activity and

those for stress induced activity [see details below and in (47)]. We found that stress

responsive activity resides between -300 and -120, a fragment that contains binding

sites for the transcriptional activators Hsf1 and Msn2/4 [see details of promoter

analysis in (47) and below in page 10 under “Hsf1 and Msn2/4 can exclusively or

cooperatively activate the yeast HSP104 gene”]. The mechanisms by which Hsf1 and

Msn2/4 modulate the promoter and render it active are not known.

The HSE/Hsf1 system

Hsf1 binds the Heat Shock Element (HSE: a repeat of the pentanucleotide 5'-

nGAAnnTTCn-3') present in the enhancer region of promoters of many genes

encoding heat shock proteins as well as a few other promoters (81, 82, 85, 109).

There is a cluster of four HSEs in the HSP104 promoter (Fig. 1). The HSE/Hsf1

system was reported in all eukaryotes studied. In S.cerevisiae HSF1 is essential for

viability (119).

It is not known how Hsf1 is regulated in lower or in higher organisms, but

phosphorylation (25, 26, 55, 58, 66, 67, 83, 90, 119, 133), oxidation (77, 94, 137)

and/or sumoylation (6, 56, 57, 60) may be involved. Yet, none of these modifications

play a crucial role in Hsf1 activation (99). They are probably involved in just fine

tuning Hsf1’s activity. Thus, signal transduction cascades controlling Hsf1 in yeast or

mammals are not well defined (25, 26, 30, 40, 52, 66, 94, 105, 120). Recently,

however, HSR1, an RNA molecule, has been shown to be essential for the activity of

Hsf1 in mammalian cells, raising a novel and attractive way for regulating

transcriptional activators (112). In yeast, regulation of Hsf1 may also involve

phosphorylation (119), but as in mammalian cells, the kinase(s) involved and the

effect of phosphorylation on Hsf1 activity are not known. Also, it was recently

reported that in S.cerevisiae, trehalose, a disaccharide known to function as a

chemical chaperone in yeast cells, also regulates Hsf1’s activity in response to heat

shock (27). In addition, it was suggested that PKA is responsible for Hsf1 regulation

(40), but many other studies showed that PKA has just a minor effect on Hsf1 [see

more details below under “Hsf1 and Msn2/4 can exclusively or cooperatively activate

the yeast HSP104 gene” and in (35, 47)].

8

In mammalian cells, Hsf1 is a monomeric cytoplasmic protein, that in

response to stress is recruited to the nucleus, trimerized and binds DNA (45, 90, 118,

133, 134). By contrast, in yeast, Hsf1 was shown to be constitutively homotrimerized

and to constitutively bind HSEs (61, 117). However, a more detailed study, that

analyzed Hsf1 binding in vivo using ChIP asays, suggested that some promoters bind

Hsf1 only following stress, similar to the case in mammalian cells (51, 135). It was

found that in Drosophila, upon activation, Hsf1 binds HSEs and recruits mediator

complexes to heat shock loci as part of a cascade of events leading to transcription

activation. In fact, this recruitment seems to involve direct interaction between Hsf1

and components of the mediator complex (95). In addition, Hsf1 of mammalian cells

has been shown to interact in vitro and in vivo with the chromatin remodeling

complex SWI/SNF (123). Chromatin remodeling activities on purified nucleosome

templates were also shown to be dependent upon their recruitment via Hsf1 (123).

These reports indeed lead to the finding that in yeast, interactions between Hsf1 and

components of the mediator do exist and that mediator complex can be recruited to

promoters via Hsf1 (39).

The STRE/Msn2/Msn4 system

As is shown in Figure 1, the promoter of HSP104 contains several repeats of

the sequence 5’ AGGGG 3’ or 5’ CCCCT 3’. These sequences are known as STress

Response Elements (STREs). STREs were originally identified in the promoters of

CTT1 and DDR2 genes [encoding the cytoplasmic catalase and DNA damage

response proteins respectively (68, 132)] whose transcription are highly induced

under oxidative stress and exposure to DNA damaging agents respectively.

Unexpectedly, it was found that CTT1 transcription was also elevated in response to

heat shock, although it does not contain any HSE (132). Furthermore, it has been

shown that DDR2 can be transcriptionally activated not only by DNA damaging

agents, but also by thirteen other stresses including osmotic shock, nitrogen starvation

oxidative stress and stationary phase (126). Promoter analysis of CTT1 and DDR2

revealed that transcription activation in response to all these stresses is dependent on

short sequences that were termed STREs (68, 86). STREs were then identified in the

promoters of hundreds of stress related genes (17, 91). The promoter of HSP104

contains three classical STREs positioned at -172, -220 and -252bp from the ATG

[Fig. 1 and ref. (47)].

9

Given that STREs are activated by many stresses and in turn induce the

transcription of hundreds of genes, many of them not to a full extent, they clearly

belong to the general, non-specific stress response (81, 107), part of the “just in case”

expression of genes, not directly relevant to the stress inflicted.

STREs serve as binding sites for two transcriptional activators, containing

Cys2His2 zinc fingers, known as Msn2 and Msn4 (87, 110). Since Msn2/4 are able to

bind STREs, they are activators of the many STRE containing genes (e.g., CTT1,

DDR2, HSP12). Their transcriptional activity is stimulated by a broad range of

stresses. Indeed, the msn2∆msn4∆ double mutant shows up to ten fold reduction in

the basal and induced expression of many stress related genes and similar reduction in

the activity of STRE-dependent reporter genes (16, 44, 102, 115, 127). While the

means by which Msn2/4 are activated remain elusive, it is well established that the

Ras/cAMP/PKA pathway directly inhibits Msn2/4 translocation to the nucleus (46).

Therefore, in yeast cells deleted for RAS2 or in mutants with low cAMP levels [in

yeast, Ras proteins induce cAMP production and consequently PKA activity (18, 19)],

Msn2/4 are constantly localized to the nucleus and the cells exhibit high and relatively

constitutive expression of many stress related genes (9, 86, 116, 121, 129).

Conversely, cells expressing the constitutively active mutant of Ras2 (RAS2val19) are

hypersensitive to stress and are defective in proper expression of stress related genes,

because Msn2/4 are constantly cytoplasmic (9, 46, 86, 116, 121, 129). Another level

of Msn2/4 regulation is probably protein stability. In response to numerous stresses

such as heat shock or ethanol, Msn2 has been shown to be highly unstable (33). The

instability of Msn2 is related to its nuclear localization as it is highly degraded in an

msn5 mutant [MSN5 encodes a nuclear exportin involved in the nuclear export of

many proteins (12, 32, 62)], demonstrating that constitutive nuclear localization is

detrimental to Msn2 stability (15, 33, 75). Interestingly, studies have also shown that

in response to heat shock, Msn2 is phosphorylated directly by Srb10 (Srb10 is a

component of the mediator co-activator complex SRB/MED with intrinsic kinase

activity) thereby downregulating its activity. Additional evidence demonstrating the

negative effects of Srb10 on the activity of Msn2 is that srb10 mutant cells have high

basal transcript levels of many Msn2 target genes (15, 22, 74).

10

Hsf1 and Msn2/4 can exclusively or cooperatively activate the yeast HSP104 gene

The results described in the following paragraph describe the first phase of my

analysis of HSP104 transcription initiation. As most of the data obtained in this phase

were achieved during the course of my M.Sc. studies, they are described in this

section of the “Introduction”. It should be appreciated however, that some

experiments were completed during the course of my Ph.D. studies and formed the

basis for the continuation of the work described in “Results”. These latter

experiments are also described here in order to maintain the coherence of the

“Introduction”.

HSP104 promoter analysis

As the first step towards analyzing the HSP104 promoter, we investigated the

sequence of the upstream region of HSP104 as it appears in the Saccharomyces

Genome Database (SGD website). We identified several putative cis-elements

reaching up to 700bp upstream of the first AUG codon. These putative elements

included various HSEs and STREs (Fig. 1).

Figure 1. Sequence corresponding to 750bp of the HSP104 promoter region. Letters in green correspond to the most 5’nucleotide in each of the deletion constructs used in our study. +1 is the first nucleotide of the coding sequence. Putative STREs are shown in red italics, STRE-like elements in pink and HSEs in blue; the putative TATA box is marked in green. The transcription initiation region is underlined in black.

-713-750 AAGGGCACTG CTAGCTCAGC CGGAACCTAA ATTGATTAGA GTTAGCGCTA -700 GAAACCGTGG ATGTTCAGGA CTAACGTACG ATCTACAATA TATCACCGAG

-641-650 CCGGGGAAAT TCGATGAGGT AGTAGAACAA GATGGCGTTA AAATTGTCAT-600 CGATTCAAAG GCGTTATTCA GCATCATTGG AAGTGAAATG GACTGGATCG

-531-550 ACGACAAGTT GGCCTCTAAG TTTGTCTTCA AGAATCCAAA CTCCAAGGGC

-500-500 ACATGCGGTT GTGGCGAGAG TTTCATGGTT TAAAAACCTT CTGCACCATT-450 TTTAGAAAAA AAGAATCTAC CTATTCACTT ATTTATTCAT TTACTTATTT -400 ATTTACATAT TTATCATACA TATTAACATT GAACCCTCCA TCGTGGTAGT

-334 -305-350 GTTTGCTGTT CCTAACTTTT CTTTCGTTGT TCTTGTAGAT ATATATTTTT-300 CCAGAATTTT CTAGAAGGGT TATTAATTAC AATCTTAAAC GTTCCATAAG-250 GGGCCGCGAT TTTTTTGTTC AATTTTCAAC AGGGGGCCCA TCTCAAAGAA-200 CTGCAAATTA TATCACAGTA AAAGGCAAAG GGGCGCAAAC TTATGCAACC -150 TGCCAGATTA TTATATAAGG CATTGTAATC TTGCCTCAAT TCCTTCATAA -100 TTCGTTCCTT TGTCACTTGT TCCTTTTTAC CCTTGAATCG AATCAGCAAT

-50 AACAAAGAAA AAAGAAATCA ACTACACGTA CCATAAAATA TACAGAATAT +1 ATGAAC

STRELegend

HSE

STRE-like (PDS)

TATA box

Transcription initiation region

-713-750 AAGGGCACTG CTAGCTCAGC CGGAACCTAA ATTGATTAGA GTTAGCGCTA -700 GAAACCGTGG ATGTTCAGGA CTAACGTACG ATCTACAATA TATCACCGAG

-641-650 CCGGGGAAAT TCGATGAGGT AGTAGAACAA GATGGCGTTA AAATTGTCAT-600 CGATTCAAAG GCGTTATTCA GCATCATTGG AAGTGAAATG GACTGGATCG

-531-550 ACGACAAGTT GGCCTCTAAG TTTGTCTTCA AGAATCCAAA CTCCAAGGGC

-500-500 ACATGCGGTT GTGGCGAGAG TTTCATGGTT TAAAAACCTT CTGCACCATT-450 TTTAGAAAAA AAGAATCTAC CTATTCACTT ATTTATTCAT TTACTTATTT -400 ATTTACATAT TTATCATACA TATTAACATT GAACCCTCCA TCGTGGTAGT

-334 -305-350 GTTTGCTGTT CCTAACTTTT CTTTCGTTGT TCTTGTAGAT ATATATTTTT-300 CCAGAATTTT CTAGAAGGGT TATTAATTAC AATCTTAAAC GTTCCATAAG-250 GGGCCGCGAT TTTTTTGTTC AATTTTCAAC AGGGGGCCCA TCTCAAAGAA-200 CTGCAAATTA TATCACAGTA AAAGGCAAAG GGGCGCAAAC TTATGCAACC -150 TGCCAGATTA TTATATAAGG CATTGTAATC TTGCCTCAAT TCCTTCATAA -100 TTCGTTCCTT TGTCACTTGT TCCTTTTTAC CCTTGAATCG AATCAGCAAT

-50 AACAAAGAAA AAAGAAATCA ACTACACGTA CCATAAAATA TACAGAATAT +1 ATGAAC

STRELegend

HSE

STRE-like (PDS)

TATA box

Transcription initiation region

11

Using PCR on genomic DNA we cloned a fragment of 713bp of the promoter and

ligated it upstream to a β-galactosidase reporter gene (Fig. 2A). When introduced to

yeast cells, the -713LacZ reporter gene manifested basal activity under non heat shock

conditions which was induced 5.5 fold in response to heat shock (Fig. 2B). This

reporter activity reflected the levels of endogenous HSP104 mRNA that accumulated

thirty minutes after heat shock and dropped after one hour (Fig. 2C). We next

proceeded with 5’-deletions in order to determine the minimal promoter sequence

conferring basal and induced activities. This deletion analysis (Fig. 2A) showed that

a fragment of 334bp upstream from the first AUG gave rise to reporter activities

similar to the full length -713LacZ. A drastic decrease in basal (but not induced)

reporter activity was observed upon further removal of 34bp [-300LacZ (Fig. 2B)].

These results strongly suggested that 334bp of the promoter are essential and

sufficient for the basal activity of HSP104 promoter. 300bp of the promoter are

essential and sufficient for heat shock-induced activity. Namely, the 34bp between -

334 and -300 are dispensable for induced activity, but indispensable for HSP104

transcription under optimal growth conditions. Further analysis of those 34bp is

described below.

12

A)

B) C)

Figure 2. A 334bp fragment of the HSP104 promoter is sufficient and essential for both basal and induced activities in wild-type cells (the SP1 strain). A) Schematic view of various constructs fused to LacZ coding sequence, ranging from -713bp to -300bp of the promoter. B) β-galactosidase activity of the various constructs under optimal growth conditions (30oC) and following heat shock (39oC for one hour). C) S1 analysis of endogenous HSP104 mRNA at various time points during heat shock treatment or under optimal growth conditions.

In order to search for the cis-elements required for the heat shock induced

transcription of HSP104, the sequences downstream to -300 were further analyzed

through 5’deletions. The results are described in detail in (47). Briefly, we found that

upon deletion of the HSE cluster (Fig. 3) between -300 and -286, the reporter gene

remained responsive to heat shock due to the presence of the STREs of the promoter

(reflected by the -284LacZ construct). Removal of the first distal STRE positioned at

-252 (in the -248LacZ construct) almost abolished the responsiveness of the reporter

gene. Only residual activity remained that was just slightly induced in response to

heat shock. This induced activity of -248LacZ is due to the presence of the remaining

STREs positioned at -220 and -172 because their deletion completely abolished

reporter activity (Fig. 3B and 3C). As mentioned above, the general stress response

via the STRE/Msn2/4 system is negatively regulated by the Ras/cAMP/PKA pathway

and many stress related genes are upregulated in ras2∆ cells (9, 86, 116, 121, 129).

-713

-300 ATG

-641 ATG

-531 ATG

-334 ATG

-500 ATG

HSE

HSE cluster

STRE-like

STRE

Legend

BamHI-3’

ATG

5’-XhoI

LacZ coding seq

100 bp

SP1

Time in 390C 0’ 15’ 30’ 60’5hrs300C

HSP104

ACTIN

0

100

200

300

400

500

600

-713 -641 -530 -500 -334 -300

390C

300CSP1

-713

-300 ATG

-641 ATG

-531 ATG

-334 ATG

-500 ATG

HSE

HSE cluster

STRE-like

STRE

Legend

BamHI-3’

ATG

5’-XhoI

LacZ coding seq

100 bp

-713

-300 ATG

-641 ATG

-531 ATG

-334 ATG

-500 ATG

HSE

HSE cluster

STRE-like

STRE

Legend

BamHI-3’

ATG

5’-XhoI

LacZ coding seq

100 bp

-300 ATG-300 ATG-300 ATG

-641 ATG-641 ATG

-531 ATG

-334 ATG-334 ATG-334 ATG

-500 ATG-500 ATG-500 ATG-500 ATG

HSE

HSE cluster

STRE-like

STRE

Legend

BamHI-3’

ATGATGATG

5’-XhoI

LacZ coding seq

100 bp100 bp

SP1

Time in 390C 0’ 15’ 30’ 60’5hrs300C

HSP104

ACTIN

0

100

200

300

400

500

600

-713 -641 -530 -500 -334 -300

390C

300CSP1

0

100

200

300

400

500

600

-713 -641 -530 -500 -334 -3000

100

200

300

400

500

600

-713 -641 -530 -500 -334 -300

390C

300C

390C

300CSP1

13

Indeed, as can be seen in Figure 4A, activity of -334LacZ under non-heat shock

conditions is derepressed and highly active in ras2∆ cells. Deletion of the HSE

cluster (-284LacZ reporter) had no effect on the activity of the promoter in ras2∆

cells. A decrease in promoter activity was in fact measured only following deletion of

sequences corresponding to STREs. These data indicated that in ras2∆ cells HSEs

play no role in HSP104 activation and that all STREs present are spontaneously

functional. These conclusions were further reinforced by mutating the various STREs

(singly and in combination) in the HSP104 promoter [data not shown; described in

(47)]. To further study the functionality of the STREs on the HSP104 promoter, we

measured the activity of the various reporter genes in msn2∆msn4∆ strain (Fig. 5A).

A)

B) C) Figure 3. A 260bp region of the HSP104 promoter is responsible for the induced activity of the promoter in SP1 cells. Deletion of STRE at -252 reduces overall activity and almost entirely abolishes response of the reporter gene to heat shock. A) Schematic view of constructs whose activities are shown in B). B) β-galactosidase activity of the various constructs at 30oC and following heat shock at 39oC. The inset graph corresponds to the activity of the shorter constructs. Note the different scale used. C) Fold induction of the activities of each construct in response to heat shock.

100 bp

-230 ATG

ATG-215

ATG-200

ATG-180

ATG-160

-248 ATG

-284 ATG

-280 ATG

-260 ATG

-300 ATG

5’-XhoI BamHI-3’

LacZ coding seq

-222 ATG

-334 ATG

HSE

HSE cluster

STRE-like

STRE

Legend

100 bp100 bp

-230 ATG-230 ATG-230 ATG

ATG-215 ATG-215 ATG-215

ATG-200 ATG-200

ATG-180 ATG-180

ATG-160 ATG-160

-248 ATG-248 ATG

-284 ATG-284 ATG-284 ATG

-280 ATG-280 ATG-280 ATG

-260 ATG-260 ATG-260 ATG

-300 ATG-300 ATG-300 ATG

5’-XhoI BamHI-3’

LacZ coding seqLacZ coding seq

-222 ATG-222 ATG

-334 ATG-334 ATG-334 ATG

HSE

HSE cluster

STRE-like

STRE

Legend

Fold Activation

-334 3.6

-300 9.5

-284 9.1

-280 12.3

-260 10.5

-248 2.2

-230 1

0

50

100

150

200

250

300

350

400

450

1

-334 -284 -260-300 -280

0

11

2

23

3

4

45

5

1

390C

300C

-248 -222 -200 -160-230 -215 -180

SP1 Fold Activation

-334 3.6

-300 9.5

-284 9.1

-280 12.3

-260 10.5

-248 2.2

-230 1

0

50

100

150

200

250

300

350

400

450

1

-334 -284 -260-300 -280

0

11

2

23

3

4

45

5

1

390C

300C

-248 -222 -200 -160-230 -215 -180

Fold Activation

-334 3.6

-300 9.5

-284 9.1

-280 12.3

-260 10.5

-248 2.2

-230 1

0

50

100

150

200

250

300

350

400

450

1

-334 -284 -260-300 -280

-334 -284 -260-300 -280

0

11

2

23

3

4

45

5

1

390C

300C

-248 -222 -200 -160-230 -215 -180

-248 -222 -200 -160-230 -215 -180

SP1

14

We first noticed that the activity of the full length -334LacZ was decreased

when compared to wild type, indicating some possible role for Msn2/4 in the basal

activity of HSP104 (compare Fig. 5 with Fig. 3). However, unexpectedly, the reporter

gene was induced to maximal levels even in the absence of these two transcriptional

activators suggesting that the induced activity could be solely provided by the

HSE/Hsf1 system. Indeed, when we deleted the HSE cluster (reflected by the -

284LacZ construct), and subjected msn2∆msn4∆ cells to heat shock, the reporter gene

was no longer induced in response to heat shock, confirming that in msn2∆msn4∆

cells, heat shock responsiveness of the reporter gene is due to the HSEs alone (Fig. 5).

This conclusion is further strengthened by measurements of the mRNA levels of

HSP104 in msn2∆msn4∆ cells which are normally induced in response to heat shock.

A) B)

Figure 4. HSP104 expression is derepressed in ras2∆ cells and is regulated exclusively through STREs. A) Deletion of each STRE causes a decrease in basal (30oC) β-galactosidase activity of the promoter (compare 260 vs. 248; 222 vs. 215; and 180 vs. 160). The graphs shown are different in scale. The numbers above some bars describe the fold reduction in activity as compared with the previous bar. B) S1 analysis of HSP104 mRNA in ras2∆ and RAS2val19 cells.

Our deletion analysis, in combination with the effects of the point mutations,

strongly suggests that the derepression of the HSP104 promoter in ras2∆ cells is

mediated exclusively via STREs. These STREs must be recognized by Msn2 and

Msn4, as they are not functional in msn2∆msn4∆ cells (see Fig. 5).

Time in 390C 0’ 15’ 30’ 60’5hrs300C

SP1ras2∆HSP104

ACTIN

SP1RAS2val19 HSP104

ACTIN

Time in 390C 0’ 15’ 30’ 60’5hrs300C

SP1ras2∆HSP104

ACTIN

HSP104

ACTIN

SP1RAS2val19 HSP104

ACTINSP1RAS2val19 HSP104

ACTIN

HSP104

ACTIN

0.00

100.00

200.00

300.00

400.00

500.00

600.00

700.00

800.00

900.00

1 0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

1-334 -284 -260 -230 -215 -180-300 -280 -248 -222 -200 -160

3.3

6.5

63

-HS

ras2∆

0.00

100.00

200.00

300.00

400.00

500.00

600.00

700.00

800.00

900.00

1 0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

1-334 -284 -260 -230 -215 -180-300 -280 -248 -222 -200 -160

3.3

6.5

63

-HS

ras2∆

15

A) B)

Figure 5. Msn2/4 contribute to the basal and induced activities of the HSP104 promoter. A) The β-galactosidase activity of each reporter was measured in msn2∆msn4∆ cells under optimal growth conditions of 30oC and following heat shock at 39oC. Note that the scale used in the inset graph is different. B) S1 analysis of HSP104 mRNA in msn2∆msn4∆ cells.

Hence, we expected that knocking out MSN2 and MSN4 genes in a ras2∆

background would eliminate the derepression observed in this strain. Unexpectedly,

the activity of the -334LacZ construct in ras2∆msn2∆msn4∆ was similar to its activity

in ras2∆ (compare Fig. 6A with Fig. 4A). Further intriguing was the severe decrease

in activity observed for the -300LacZ construct. Thus, in ras2∆msn2∆msn4∆ cells,

the region between -334bp and -300bp seems to have acquired some increased

activity although this sequence was absolutely dispensable for promoter activity in

ras2∆. This observation underscores the importance of the upstream 34bp. All

constructs, downstream of -300LacZ, also displayed very low activity in

ras2∆msn2∆msn4∆ cells (Fig. 6).

Sp1msn2∆msn4∆

Time in 390C 0’ 15’ 30’ 60’ 5hrs300C

HSP104ACTINSp1msn2∆msn4∆

Time in 390C 0’ 15’ 30’ 60’ 5hrs300C

HSP104ACTINHSP104ACTIN

0

50

100

150

200

250

300

350

1

-334 -300

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1

-284 -260 -230 -215 -180-280 -248 -222 -200 160

300C

390Cmsn2∆msn4∆

0

50

100

150

200

250

300

350

1

-334 -300

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1

-284 -260 -230 -215 -180-280 -248 -222 -200 160-284 -260 -230 -215 -180-280 -248 -222 -200 160

300C

390Cmsn2∆msn4∆

16

A) B)

Figure 6. HSP104 expression in ras2∆msn2∆msn4∆ cells. A) Sequences between 334 and 300bp of HSP104 are important for the β-galactosidase activity of the promoter in ras2∆msn2∆msn4∆ cells at 30oC. A change in scale used in the right hand graph. The 25.5 fold decrease in activity was obtained by dividing the activity of -334LacZ by that of -300LacZ. B) S1 analysis of HSP104.

In order to unambiguously assess that the sequences identified in our study are

indeed independently responsible for the heat shock responsiveness of the HSP104

promoter, we fused the sequences from -334 to -160, or -305 to -160 (these sequences

contain all elements responsible for the induced activity, but lack the basal promoter

region) to the CYC1 minimal promoter and checked whether the sequences derived

from the HSP104 promoter could now render the CYC1 promoter heat shock

responsive [the native CYC1 promoter is normally not induced by heat shock (data not

shown)]. Briefly, we found that the HSP104 enhancer region is indeed sufficient for

rendering the heterologous promoter responsive to heat shock (Fig. 7A). Also, the

heterologous reporters were constitutively elevated in ras2∆ cells. Namely, the

sequence we defined as an enhancer is indeed, independently, necessary and sufficient

for promoter activation and regulation in response to heat shock and in response to the

Ras pathway. Notably however, we also observed that the heterologous promoters

displayed lower basal activity compared to their homologous counterparts (compare

Fig. 7A and 2B and data not shown). These results suggest that the element we

defined as essential for the basal transcription activity (i.e., the 34bp between -334

and -300) are specific for the HSP104 promoter and functions together with its own

basal promoter and cannot function with another.

0

100

200

300

400

500

600

700

1

-334 -300 -284 -280 -2600

2

4

6

8

10

12

14

16

1

-248 -230 -222 -215 -200 -180

300C

25.5

ras2∆msn2∆msn4∆

0

100

200

300

400

500

600

700

1

-334 -300 -284 -280 -2600

2

4

6

8

10

12

14

16

1

-248 -230 -222 -215 -200 -180

300C

25.5

ras2∆msn2∆msn4∆

Sp1ras2∆msn2∆msn4∆HSP104

ACTIN

Time in 390C 0’ 15’ 30’ 60’5hrs300C

Sp1ras2∆msn2∆msn4∆HSP104

ACTIN

HSP104

ACTIN

Time in 390C 0’ 15’ 30’ 60’5hrs300C

17

A) B)

Figure 7. Sequences between -334 and -160bp of the HSP104 promoter contain all elements responsible for the heat shock response and the Ras response. A) β-galactosidase activity of the chimeric -334HSP104-CYC1-LacZ and -305HSP104-CYC1-LacZ constructs were assayed in the wild type strain SP1 at 30oC and at 39oC. ‘-‘ is the inverted and ‘+’ is the native orientation of the HSP104 insert with respect to the CYC1 promoter. B) Same constructs as in A) assayed in ras2∆ cells under the same experimental conditions.

To summarize the results obtained (see Table 1), we showed, through a

combination of a genetic approach and a molecular approach (5’deletions of the

HSP104 promoter, point mutations, fusion to a heterologous promoter and the use of

several yeast mutants) that the HSE/Hsf1 and the STRE/Msn2/4 systems cooperate to

achieve maximal inducible expression. However, in the absence of one set of factors

(e.g., in msn2∆msn4∆ cells or in constructs lacking HSEs) proper induction of

HSP104 promoter is achieved exclusively through the other. We also showed that

HSP104 is constitutively derepressed in ras2 cells. This derepression is evoked

exclusively via STREs with no role for HSEs. Strikingly, in ras2∆msn2∆msn4∆ cells,

we observed that the HSP104 promoter is also derepressed via the upstream 34bp.

Thus, appropriate transcription of HSP104 is usually obtained through cooperation

between the STRE/Msn2/4 and HSE/Hsf1 systems, but each factor could activate the

promoter on its own, backing up the other. Transcription control of HSP104 is

therefore adaptive and robust, ensuring proper expression under extreme conditions

and in various mutants. Finally, we identified a 34bp fragment residing upstream to

the HSP104 enhancer that is critical for the promoter basal activity. This fragment is

highly specific to this promoter and can acquire, under particular conditions, Ras

responsive properties (i.e., in ras2∆msn2∆msn4∆ cells).

020406080

100120140160180200

1

units

+334-CYC1min -334-CYC1min +305-CYC1min

300C

390C

SP1

020406080

100120140160180200

1

units

+334-CYC1min -334-CYC1min +305-CYC1min

300C

390C

SP1

0

100

200

300

400

500

600

700

1

units

+334-CYC1min +305-CYC1min

ras2∆300C

390C

0

100

200

300

400

500

600

700

1

units

+334-CYC1min +305-CYC1min

ras2∆300C

390C

18

Table 1. Summary of the role of various fragments in HSP104 promoter

Goals of Study

In this thesis, I describe the continuation of the effort to reveal the mechanism of

transcriptional activation of the HSP104 promoter. The overall goal is to obtain

sufficient data that will allow the establishment of a global model of the molecular

events leading to the activation of the HSP104 promoter. To achieve this goal, I

undertook four experimental routes. One, continuing the approach used in the studies

described above, we proceeded with additional 5’ deletions of the HSP104 promoter

(particularly the fragment between -334 and -300) attempting to identify the

sequence(s) responsible for the basal reporter activity of HSP104 and the unexpected

activity observed in the ras2∆msn2∆msn4∆ strain. Two, using chromatin

immunoprecipitation (ChIP), we monitored some of the major changes occurring in

vivo on the promoter following stress. Three, using a genetic approach, we identified

components of the basal transcription machinery that are important for HSP104

promoter activity. Four, using a combination of ChIP experiments and a genetic

approach, we sought possible regulators of Hsf1.

Through the deletion analysis, we found that important properties of the 34bp

between -334 and -300 could be accounted to a short HSE-like sequence residing in -

305. Using ChIP assays we show that under optimal growth conditions nucleosomes

on the HSP104 promoter contain mostly acetylated H3 and H4. However, following

heat shock there is a rapid, but transient, decrease in the concentration of acetylated

histones on the promoter which seems to be partly mediated by Msn2/4. It seems that

the Ras/PKA pathway controls H3 and H4 acetylation state via Msn2/4, thereby

governing induction of the promoter. We further show that the decrease in acetylated

H3 and H4 on the promoter occurs via two distinct mechanisms. Finally, we show

that Hsf1 binding to the promoter is constitutive regardless of stress conditions, but is

reduced in ras2∆ cells. Using the genetic approach, we found that Rpb4, components

--300 to 300 to --285285--300 to 300 to --285285--300 to 300 to --285285

19

of the SRB/MED coactivator complex, or of the SAGA and SWI/SNF complexes are

critical for proper HSP104 transcription. We also identified components of the basal

transcription machinery (primarily of the SAGA complex that are critical for Hsf1

activity.

These approaches combined allow the establishment of a model describing the

series of molecular events occurring on the HSP104 promoter before and after heat

shock.

EXPERIMENTAL PROCEDURES

Yeast strains, plasmids and media

Yeast strains used were from either the SP1 or the BY4741 genetic backgrounds

(Table 2). Yeast cultures were usually grown in YPD medium (1% yeast extract, 2%

peptone, 2% glucose). HA-Hsf1 containing the native HSF1 promoter was cloned in

pRS306 (114) expressed from its own promoter and was integrated in the URA3

locus. -334LacZ and -260LacZ were described in (47). The HSELacZ construct is

composed of the HSE cluster from HSP104 subcloned in 4 repeats upstream to the

CYC1 minimal promoter. Medium used for selecting transformants and for growth of

plasmid harboring cultures was the synthetic media, SD (0.17% yeast nitrogen base

without amino acids and NH4(SO4)2, 0.5% ammonium sulphate, 2% glucose and 40

mg/ml of the required nutrients). If plasmid inserted was integrative, cells were

grown (following selection) in YPD medium. Single base pair deletions of the

HSP104 promoter from -305 to -286 were done using PCR and fused to β-

galactosidase reporter gene in -178trp using 5’XhoI and 3’BamHI restriction sites

(thereby removing the CYC1 minimal promoter). Constructs in which an internal

fragment of 78bp was deleted (∆78 promoter family) were obtained by subcloning the

various promoters into a modified pBluescript (SK+) in which the XbaI and ApaI

restriction sites were eliminated. Following subcloning, the plasmids were digested

using XbaI and ApaI enzymes thereby creating the ∆78 promoter family. Modified

promoters were then transferred back to the -178trp plasmid in XhoI and BamHI sites.

The HSFp-HA-HSF vector was constructed the following way. The promoter region

of HSF was obtained via PCR with 5’KpnI and 3’ClaI flanking restriction sites. The

HSF promoter was subcloned upstream of a BS-HA-HSF vector digested with the

20

same enzymes. BS-HA-HSF was obtained using a PCR fragment of HSF coding

region with 5’NcoI and 3’EcoRI flanking restriction sites. HSF coding region was

then cloned in frame to HA epitope in the pBluescript plasmid. The resulting HSFp-

HA-HSF was transferred to the pRS306 shuttling vector using KpnI-EcoRI restriction

sites. In order to integrate pRS306-HSFp-HA-HSF in the yeast genome, the vector

was digested with BsmI (which cuts uniquely in the URA3 marker).

Table 2. Strains used in this study

a represents knocked out gene

Chromatin immunoprecipitation

Cells were grown in YPD to A600 of 0.8. Samples of 200ml were heat shocked at

39oC and were collected at time points indicated in each experiment. Cultures were

then treated for cross linking with 40ml of 11% formaldehyde in 0.1M NaCl, 1mM

EDTA, 50mM Hepes-KOH pH7.5 (final concentration of 1% formaldehyde). Samples

were then incubated at room temperature for 20min with occasional swirling. 60ml of

3M glycine were added with an additional incubation of 5min. Cells were pelleted

and washed twice with cold TBS and once with 10ml cold FA lysis buffer/0.1%SDS

(50mM Hepes-KOH pH7.5, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1%

sodium deoxycholate, 1mM PMSF). Cells were then broken with glass beads

(vortexing 30" x 10) in 1ml cold FA lysis buffer/0.5%SDS. 6.5ml of cold FA lysis

buffer/0.1%SDS was added and the supernatant was centrifuged at 45 000 rpm in a 50

Ti Beckman rotor for 20min at 4oC. Pellets were then "resuspended" in 1.2ml cold

FA lysis buffer/0.1%SDS and transferred to 1.5ml eppendorf tube. Samples were

then sonicated to get, in average, 400bp length DNA fragments (between 100-

1000bp). Samples were then centrifuged at 15000 xg for 10min at 4oC. 3.25ml of

cold FA lysis buffer/0.1%SDS was added and samples were aliquoted and frozen in

liquid nitrogen. For IPs, 0.5ml of chromatin solution was incubated with Ab pre-

Strains Genotype Reference SP1 MATa his3, leu2, ura3, trp1, ade8 Can. Toda et al.(1985) TK161R2V Isogenic to SP1 but RAS2val19 Toda et al.(1985)

SP1ras2∆ Isogenic to SP1 but ras2::LEU2 Engelberg et al. (1994)

SP1msn2∆msn4∆ Isogenic to SP1 but msn2::HIS3 msn4::URA3 Stanhill et al.(1999) SP1ras2mmsn2∆msn4∆ Isogenic to SP1 but ras2::LEU2 msn2::HIS3 msn4::URA3 Stanhill et al.(1999) BY4741 MATa his3∆1, leu2∆0, ura3∆ Euroscarf collection BY4741xª∆ Isogenic to BY4741 but x::KanMx Euroscarf collection

21

bound to protein G sepharose beads (Amersham) for 2hrs at 4oC (Ab. used: 1µg anti-

HA 3F10 (Roche), 2µl anti-acetyl-Histone H4 (Upstate), 1µl anti-acetyl-Histone H3

(Upstate), 2µg anti-total H3 (abcam) and 8µl anti-histone H4 non-acetylated

(Serotec)). Beads were then washed, each time for 5min at room temperature in the

following order: twice in 1.4ml of FA lysis buffer/0.1%SDS, twice in 1.4ml FA lysis

buffer/0.1%SDS/500mM NaCl, once in 1.4ml 10mM Tris-HCl pH8, 250mM LiCl,

1mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, and once in 1.4ml TE (10mM

Tris-HCl pH8, 1mM EDTA). Immunoprecipitated material was then eluted at 65oC in

0.25ml of 50mM Tris-HCl pH7.5, 10mM EDTA, 1% SDS. The sup. was then

transferred to a fresh tube containing 0.250ml TE (in parallel, 50µl of chromatin

solution which had not undergone immunoprecipitation was used (and termed whole

cell extract,WCE) and followed same treatment as IP'ed material). 20µl of 20mg/ml

pronase (Boehringer Mannheim) was added and samples were incubated at 42oC for

two hours and then transferred to 65oC overnight for decrosslinking. 50µl of 4M LiCl

was then added and DNA was extracted with phenol-chloroform in LETS (0.1M LiCl,

0.5M EDTA, 0.01M Tris-HCl pH7.4, 0.2% SDS) and then chloroform. DNA was

then precipitated. IP's were resuspended in 200µl TE and a 1:500 stock dilutions were

prepared for input DNA. PCR reaction was done in 50µl.

RNA preparation and S1 analysis

Overnight cultures grown in YPD were diluted to A600 of 0.25 and further grown to

A600 of 0.8 at 30oC. 20 ml samples were heat shocked at 39oC for the specified time.

Protocols for RNA extraction and S1 analysis are described in (47).

Preparation of cell lysates and western blot analysis

100ml cell cultures were grown to an A600 of 0.8. Cultures were split to 20ml samples

and each sample was treated for 5, 10, or 15 minutes at 39oC and one sample was

maintained under control conditions of 30oC. Cells were pelleted and the protocol

proceeded as described in (7). SDS-polyacrylamide gel electrophoresis, Western blot,

and ECL reaction were performed as described in (7). For HA-HSF, detection

antibody used was 12CA5 mouse anti-HA at a 1:1000 concentration. Secondary

antibody used was diluted to 1:10000 concentration.

22

β-Galactosidase assay

Overnight cultures were diluted to A600 of 0.15 and further incubated at 30oC until

cultures reached A600 0.3-0.4. Following procedures were performed as described in

(47).

RESULTS

The upstream 34bp fragment of the HSP104 promoter possesses unusual

modular properties

Our previous analysis of the HSP104 promoter revealed that 334bp upstream

of the coding sequence are required for both basal and induced activities whereas

300bp are essential and sufficient for heat shock induced activity only. That is, the

34bp between -334 and -300 are indispensable for the basal activity of the promoter.

Yet, although supporting basal transcription, this 34bp are highly specific to the

HSP104 promoter because they played no role when the upstream fragment of the

HSP104 was fused to the minimal CYC1 promoter. Namely, when cloned upstream

to the CYC1, the fragment between -334 to -160 and the fragment between -305 to -

160 manifested the same, very low basal activity [Fig. 7 and (47)]. These results

suggest that, the activity of the 34bp fragment is not only specific to HSP104, but is

somehow cooperating with the HSP104 minimal promoter (-160-+1) to impose a

relatively high basal activity. Finally, under particular conditions (i.e., in the

ras2∆msn2∆msn4∆ strain) the 34bp acquire new properties and become Ras2

responsive (Fig. 6). Given the importance and the specificity of the 34bp, we

considered that perhaps, there is a shorter cis-element within this sequence. To

address this matter we designed a series of deletion constructs that were planned to

eliminate some potential HSEs present in the 34bp region, as well as a possibly

functional, although non-canonical, TATA box which is also present in this fragment

(Fig. 8A). We also planned a series of constructs that enabled us to monitor the

possible interplay between the 34bp and the minimal promoter. The latter was

achieved by deleting an internal 78bp fragment within the HSP104 promoter which

deletes the two most distal STREs (i.e., at -252 and -220), while leaving intact the

majority of the HSE cluster and the most proximal STRE, residing at -172 of the

23

promoter. This deletion created a promoter containing essentially only the elements

responsible for the promoter basal activity, i.e., the 34bp, separated by a short DNA

sequence, are fused to the basal promoter region (-160 to +1).

The various deletion constructs were tested first in wild type cells. As shown

in Figure 8B, sequential deletion of the putative HSEs in the 34bp (-328LacZ, -

317LacZ, -311LacZ) or further deletion of the putative TATA box (-305LacZ) did not

result in a significant decrease in the activity of the reporter genes (Fig. 8B see also

Fig. 9). Namely, we could not account the decrease in reporter activity upon removal

of the entire 34bp to the particular deletions of the sporadic HSEs or of the non-

canonical TATA box. We did observe a significant decrease in basal promoter

activity when we further removed the 5bp between -305 to -300 (Fig. 8B). All

deletion constructs lost their basal activity when the internal 78bp were removed

(compare the six bars at the left to the six bars at the right in Fig. 8B), but remained

nevertheless responsive to heat shock. These results show that the presence of the

34bp upstream fragment and the minimal -160-+1 fragment is not sufficient to impose

basal promoter activity. Perhaps these two fragments must reside in a very particular

spatial and distal orientation towards each other. An alternative explanation may be

that the missing downstream STREs are required not only for induced activity, but

also for the basal activity and are cooperating with the upstream 34bp. In addition,

when we mutated the STRE positioned at -172 we observed no further effect in the

activity of the reporter, suggesting that this STRE could be less functional or perhaps

may require the presence of the more distal STREs in order to be functional [compare

in Figure 8B 300∆78 and 300stre3m∆78; see also in (47)]. We next introduced the

various reporters to ras2∆ cells (Fig. 8C). As expected, we measured high and

spontaneous activity of the reporter gene under optimal growth conditions, which

could not be further induced in response to heat shock (upper panel). Notably, the

activity of the -300LacZ construct was lower, suggesting that in order to achieve full

response to the Ras system, the HSP104 promoter requires the 34bp upstream

fragment in addition to STREs (47). Upon deletion of the internal 78bp fragment

from all constructs, we observed two important changes in the activity of the reporters

(Fig. 8C lower panel). First, the spontaneous activity of the reporters was now

dramatically reduced; further reinforcing the fact that the HSP104 promoter is indeed

Ras responsive via two distal STREs. Second, deletion of the Ras responsive

sequences converted the reporter to being heat shock responsive, most probably due

24

to the fact that the HSEs are present and are not Ras-regulated (Fig. 8C lower panel;

compare with upper and note the difference in scale between the two graphs). We

next introduced these reporters to ras2∆msn2∆msn4∆ cells (Fig. 8D) and also

observed the high and spontaneous activity, but to levels that were about half of those

measured in ras2∆ (but still 4-6 fold higher compared to the activity in wild type). In

addition, there is some decrease in activity of most reporters as compared to -

334LacZ. Also, the decrease of the activity of the -300LacZ reporter (compared to its

activity in ras2∆ cells) is very dramatic in this strain (~30 units versus 700 units in

ras2∆). Namely, there are elements in the 34bp that are Ras responsive and can

compensate for the lack of STREs activity (due to the fact that Msn2/4 are missing).

Similar to the case in ras2∆ cells, the equivalent reporters lacking the 78bp

manifested lower basal activity, but were heat shock responsive (Fig. 8D, lower

panel). We also introduced these reporters to msn2∆msn4∆ cells (Fig. 8E) and like

we previously observed, the reporters in which we deleted the sporadic HSEs and

TATA elements do not behave any differently from their full length counterpart -

334LacZ, but the activity was significantly lower compared to that of wild type cells

(compare Figs. 8E to 8B). Also, in msn2∆msn4∆ cells the internal 78bp are not

significant clearly showing that the contribution of the 78bp fragment to basal

promoter activity requires intact Msn2/4.

25

A) B) C) D) E)

Figure 8. Deletion analysis of the 34bp fragment. A) Sequence of the 34bp fragment of HSP104 required for proper basal activity. B) Activity of the various deletions of the 34bp and their ∆78 counterparts (missing enhancer elements) in wild type SP1. C) Same as in B but measured in ras2∆ cells. Note the difference in scale used in upper and lower graphs. D) Same as above but in ras2∆msn2∆msn4∆. Note the difference in scale used in upper and lower graphs. E) Same as above but measured in msn2∆msn4∆ cells. These are the results of duplicates in a single assay; hence no STD could be calculated.

34bp 5’ ttttctttcgttgttcttgtagatatatatttttcc 3’putative HSEputative TATA box

34bp 5’ ttttctttcgttgttcttgtagatatatatttttcc 3’putative HSEputative TATA boxputative HSEputative TATA box

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26

The systematic 5’ deletion analysis of the upstream 34bp pointed at the

sequence between -305 and -300 as important. This 5bp contains a single HSE site.

Although HSEs are normally active in response to heat shock, this site seems to

partially contribute to the basal transcription activities of the promoter and even to its

Ras2-mediated response. It may also be part of the HSE cluster residing (according

to our original mapping) between -300 and -285. To test the role of these 5bp and the

downstream cluster of HSEs in a most fine tuned manner, we continued and

proceeded with additional 5’deletions in which we systematically deleted a single

nucleotide at a time from -305 to -286 (Fig. 9A). In wild type cells, deletions between

-305 and -302 had no effect. The -302LacZ construct still contains the full putative

HSE sequence. The -301LacZ construct, which lacks only one nucleotide of the

perfect HSE cis-element, demonstrated reduced activity that was actually similar to

that of -300LacZ. We further verified the activity of these deletions in msn2∆msn4∆

cells (Fig 9B). Thus, we were able to map most properties of the 34bp to the single

HSE element at -302. In other words, the HSE positioned at -302 is essential for the

high basal activity of the HSP104 promoter. This result could also be interpreted by

suggesting that the HSE cluster (-302 to -285) should be intact to support basal

transcription. Since Hsf1 is known to be the sole regulator in the absence of Msn2/4

[Fig. 5 and (47)], we also measured the ability of the various constructs to respond to

heat shock in the absence of Msn2/4. Our previous, somewhat crude, mapping

showed that this heat shock response requires the HSE cluster residing between -300

to -285. Our current, fine tuned mapping showed, quite strikingly, that HSEs

upstream to -294 are essential for the heat shock response of HSP104. Additional

constructs downstream to -294bp displayed no activity even though still containing

some HSEs (Fig. 9B). As mentioned in the “Introduction”, the activity of the -

334LacZ reporter in ras2∆msn2∆msn4∆ cells was similar if not identical to that in

ras2∆ cells, but -300LacZ showed very low activity in ras2∆msn2∆msn4∆ and high in

ras2∆ (47). We tested therefore the activity of -302LacZ in ras2∆msn2∆msn4∆ cells.

As shown in Fig 9C, the pattern of activity of the -301LacZ and -302LacZ reporters is

quite similar to that in wild type cells but levels are slightly higher. Importantly, in

ras2∆msn2∆msn4∆ cells, the activity of the -302LacZ reporter is about 10 fold higher

than the activity of -300LacZ. In fact, results obtained in ras2∆msn2∆msn4∆

certainly suggest a role for the HSE at -302 in explaining the high spontaneous

activity of the promoter in ras2∆ cells. Namely, in cells lacking Ras2, and in the

27

absence of Msn2/4, the HSE takes over and allows high spontaneous promoter

activity.

28

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A)

B) C)

Figure 9. Fine tune (single base deletion) analysis of the -305 to -284 fragment. The activity of single base deletions of HSP104 reporters were measured in SP1 (A), in msn2∆msn4∆ (B) and in ras2∆msn2∆msn4∆ (C).

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29

In response to heat shock, acetylated H3 histones dissociate from the promoter

whereas acetylated H4 histones undergo deacetylation

The genetic and molecular evidence obtained so far show that Hsf1 and

Msn2/4 are responsible for both basal and inducible activities of the HSP104

promoter [Figs. 2, 3, 5 and further details in (47)]. We do not know, however, how

these transcriptional activators directly affect the promoter to render it active. As the

major effect of transcriptional activators is remodeling of the chromatin, particularly

imposing histone acetylation or deacetylation, we monitored the acetylation state of

histones H3 and H4 residing on the HSP104 promoter. We performed ChIP assays

that allow in vivo monitoring of the promoter-bound histones. We first tested the

status of H3 histones (Fig. 10). Using anti-acetylated H3 antibodies in the ChIP

assay, we observed that under optimal growth conditions acetylated H3 molecules

occupy the promoter in all strains tested (Fig. 10A). Importantly, however, the

HSP104 promoter in ras2∆ cells is less occupied with acetylated H3 under non-heat

shock conditions (Fig. 10A).

Next, we measured the status of H3 histones following stress. ChIP analysis

shows that in all strains tested, acetylated H3 histones are scarcely detectable on the

promoter, five to seven minutes following heat shock (Fig. 10B). The decrease

observed in acetylated histone H3 on the promoter was very short lived in wild type

and in ras2∆ cells (Fig. 10B). Fifteen minutes after the beginning of heat shock,

acetylated histone H3 was again occupying the promoter at the same level as before

heat shock. In strains with deleted MSN2 and MSN4, acetylated histones did not

reoccupy the promoter indicating that Msn2/4 may have a role in the process (Fig.

10B).

The loss of acetylated H3 from the HSP104 promoter shortly after heat shock

could be achieved by either of the following mechanisms: 1) Histone deacetylation.

2) Specific removal of acetylated histones from nucleosomes. 3) Total disassembly of

nucleosomes. To distinguish between these possibilities, we performed a ChIP assay

using anti H3 antibodies. As shown in Figure 10C, we observed partial reduction in

H3 promoter occupancy following heat shock. At the same time, acetylated H3

molecules are not detected (Figs. 10B and 10C). The results strongly suggest that

acetylated H3 is specifically removed from the promoter in response to heat shock

whereas non-acetylated H3 remains bound. Namely, there seems to be partial

30

nucleosome disassembly involving specifically the removal of only acetylated H3

molecules from the HSP104 promoter in response to heat shock.

A)

B) C) Figure 10. Under non-heat shock conditions, some histone H3 molecules are acetylated on the HSP104 promoter and in response to heat shock, acetylated H3 histones dissociate from nucleosomes. A) ChIP analysis using anti-acetylated H3 antibodies, to monitor the presence of the protein on the HSP104 promoter of the indicated strains, grown under optimal growth conditions. B) Acetylated state of histones H3 was monitored via ChIP assays on the HSP104 promoter of wild type, ras2∆, msn2∆msn4∆ and ras2∆msn2∆msn4∆ cells in response to heat shock. Cross-linking and further processes of the ChIP analysis were performed at the indicated time points. C) Histone H3 undergoes transient remodeling in response to heat shock. Antibody recognizing total histone H3 was used in ChIP assays as well as antibody recognizing acetylated histone H3. WCE= whole cell extract.

IP:totH3

IP:aceH3

WCE

SP1

- 15’5’ 10’Heat Shock

IP:totH3

IP:aceH3

WCE

SP1

- 15’5’ 10’

SP1

- 15’5’ 10’Heat Shock

W.T.

- 15’7’ 10’

IP: ace H3

WCE

Heat Shock

Strain

IP: ace H3

Heat Shock

Strainras2∆

- 5’ 10’ 15’

WCE

msn2∆msn4∆

- 15’5’ 10’

IP: ace H3

WCE

ras2∆msn2∆msn4∆

- 15’5’ 10’

IP: ace H3

WCE

W.T.

- 15’7’ 10’

W.T.

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IP: ace H3

WCE

Heat Shock

Strain

IP: ace H3

WCE

Heat Shock

Strain

IP: ace H3

Heat Shock

Strainras2∆

- 5’ 10’ 15’

ras2∆

- 5’ 10’ 15’

WCE

msn2∆msn4∆

- 15’5’ 10’

msn2∆msn4∆

- 15’5’ 10’

IP: ace H3

WCE

ras2∆msn2∆msn4∆

- 15’5’ 10’

ras2∆msn2∆msn4∆

- 15’5’ 10’

IP: ace H3

WCE

W.T.msn2∆

msn4∆

ras2∆msn2∆

msn4∆

ras2∆

WCE

Strain

IP:ace H3

RAS2val19

W.T.msn2∆

msn4∆

ras2∆msn2∆

msn4∆

ras2∆

WCE

Strain

IP:ace H3

RAS2val19

31

Next we tested the status of H4 histones on the HSP104 promoter (Fig. 11).

Just like we observed in the case of histone H3, acetylated H4 molecules occupy the

HSP104 promoter under optimal growth conditions (Fig. 11A). Also, we detected

that less acetylated H4 histones occupy the HSP104 promoter in ras2∆ cells. These

results strongly indicate a role for Ras2 in promoting acetylated histone occupancy on

the HSP104 promoter. We next monitored the changes occurring to acetylated H4

molecules on the promoter of HSP104 following heat shock (Fig. 11B). Similarly to

acetylated H3 histones, we also observed a transient decrease in acetylated H4

molecules on the promoter in response to heat shock. In wild type cells, following a

fifteen minutes heat shock, acetylated H4 molecules reoccupied the promoter, but not

to the same level as before heat shock. Very little acetylated H4 reoccupied HSP104

promoter in strains with deleted MSN2 and MSN4, just like the case for H3 histones

(Fig. 10B). In an attempt to identify the mechanism leading to the decrease in

acetylated H4 histones in response to heat shock, we measured the presence of non-

acetylated histone H4 on the promoter in response to heat shock. As shown in Figure

11C, we could not measure non-acetylated H4 molecules in wild type cells not

exposed to heat shock (Fig. 11C, left lane). However, five minutes following heat

shock, this promoter becomes occupied with non-acetylated H4. This result is a clear

mirror image to that in Figure 11B, showing the disappearance of acetylated H4 from

the promoter. We therefore conclude that in response to heat shock, histones H4

remain associated with the promoter but undergo extensive deacetylation. A similar

mirror image is observed for the ras2∆ strain. The HSP104 promoter in this strain is

occupied with low levels of acetylated H4 under optimal conditions (Fig. 11A) and in

contrast contains increased levels of non-acetylated H4 (Fig. 11C, lane 5). As in this

strain the promoter is spontaneously active (47), it seems that reduced levels of

acetylated H4 and increased levels of non-acetylated H4 is important for promoter

activity. In summary, H3 and H4 population on the promoter is modified via two

different mechanisms. Acetylated H4 molecules undergo deacetylation whereas

acetylated H3 are removed altogether. These changes that occur in response to heat

shock and in ras2∆ cells correlate with promoter activity.

32

A)

B)

C)

Figure 11. Under non-heat shock conditions, all histone H4 molecules are acetylated on the HSP104 promoter and in response to heat shock, acetylated H4 histones are deacetylated. A) ChIP analysis using anti-acetylated H4 antibodies, to monitor the presence of the protein on the HSP104 promoter of the indicated strains, grown under optimal growth conditions. B) Acetylated state of histones H4 was monitored via ChIP assays on the HSP104 promoter of wild type, ras2∆, msn2∆msn4∆ and ras2∆msn2∆msn4∆ cells in response to heat shock. Cross-linking and further processes of the ChIP analysis were performe at the indicated time points. C) Histone H4 undergoes extensive deacetylation in response to heat shock. Antibody recognizing non-acetylated histone H4 was used for ChIP assays as well as antibody recognizing acetylated histone H4.

IP: ace H4

WCE

- 15’5’ 10’

msn2∆msn4∆

IP: ace H4

WCE

- 15’5’ 10’

ras2∆msn2∆msn4∆

IP: ace H4

WCE

W.T.

- 15’7’ 10’

ras2∆

- 5’ 10’ 15’

IP: ace H4

WCE

Heat Shock

Strain

Heat Shock

Strain

IP: ace H4

WCE

- 15’5’ 10’

msn2∆msn4∆

IP: ace H4

WCE

- 15’5’ 10’- 15’5’ 10’

ras2∆msn2∆msn4∆

IP: ace H4

WCE

W.T.

- 15’7’ 10’

W.T.

- 15’7’ 10’

ras2∆

- 5’ 10’ 15’

ras2∆

- 5’ 10’ 15’

IP: ace H4

WCE

Heat Shock

Strain

Heat Shock

Strain

- 5’ - 5’ - 5’ 10’ 15’ - 5’ 10’ 15’

WCE

Heat Shock

Strain

IP:nonace H4

wt msn2∆msn4∆ ras2∆ ras2∆msn2∆msn4∆

- 5’ - 5’ - 5’ 10’ 15’ - 5’ 10’ 15’

WCE

Heat Shock

Strain

IP:nonace H4

wt msn2∆msn4∆ ras2∆ ras2∆msn2∆msn4∆

IP:ace H4

W.T.msn2∆

msn4∆

ras2∆msn2∆

msn4∆

ras2∆

WCE

Strain RAS2val19

IP:ace H4

W.T.msn2∆

msn4∆

ras2∆msn2∆

msn4∆

ras2∆

WCE

Strain RAS2val19

33

No specific HDAC is responsible for the deacetylation of H4 on the HSP104

promoter

In an attempt to identify the histone deacetylase(s) responsible for the initial

dramatic decrease in histone H4 acetylation, we used a battery of mutants, each

deleted in a gene encoding a known histone deacetylase (taken from the S.cerevisiae

knock-out library). We systematically determined the acetylation state of histone H4

following a five minute heat shock in each of the mutants. As is shown in Figure 12,

in all strains tested, we observed disappearance of acetylated H4 from the HSP104

promoter, as was also observed in the isogenic wild type strain BY4741. Thus there

seems to be no single HDAC responsible for H4 deacetylation on the HSP104

promoter. Some histone deacetylases are probably redundant (34, 73) for

deacetylating H4 on the HSP104 promoter. It may also be that yet another HDAC,

not yet identified and therefore not tested by us, is responsible for H4 deacetylation of

the HSP104 promoter.

Figure 12. None of the deletion mutants of histone deacetylases compromises the deacetylation effect on histone H4. Mutants in which known histone deacetylases are deleted were assayed for the level of acetylated histones H4 in response to heat shock.

WT rpd3∆

hst1∆

hst2∆

hst3∆

hst4∆

- 5’ - 5’ - 5’ - 5’ - 5’ - 5’Heat Shock

Strain

IP:ace H4

WCE

WT rpd3∆

hst1∆

hst2∆

hst3∆

hst4∆

- 5’ - 5’ - 5’ - 5’ - 5’ - 5’- 5’ - 5’ - 5’ - 5’ - 5’ - 5’Heat Shock

Strain

IP:ace H4

WCE

Heat Shock - 5’ - 5’ - 5’ - 5’ - 5’ - 5’

Strain WT hda1∆

hos1∆

hos2∆

hos3∆

sir2∆

IP:ace H4

WCE

Heat Shock - 5’ - 5’ - 5’ - 5’ - 5’ - 5’

Strain WT hda1∆

hos1∆

hos2∆

hos3∆

sir2∆

IP:ace H4

WCE

34

Hsf1 constitutively binds the HSP104 promoter

Our previous promoter analysis and genetic studies revealed that HSP104

transcriptional induction is mediated by some cooperation between Hsf1 and Msn2/4

[(47); see also Figs. 2 and 3]. The dynamics of promoter occupancy by each of these

activators and the mutual relationship between them are not known. To reveal the

dynamics of promoter occupancy by Hsf1, we employed ChIP assays in wild type and

various mutant strains grown under optimal growth conditions and in response to heat

shock. We found that Hsf1 binding is constitutive under all conditions tested in wild

type cells and in the msn2∆msn4∆, ras2∆ and ras2∆msn2∆msn4∆ strains (Fig. 13A).

Notably, basal binding of Hsf1 to the HSP104 promoter in ras2∆ cells is reduced

compared to its binding in other strains (Figs. 13A and B). In order to rule out the

possibility that lower binding of Hsf1 in ras2∆ cells reflects lower steady state levels

of Hsf1 in these cells, we performed western blot analysis and observed that steady

state levels of Hsf1 are in fact identical in all strains and are not affected by heat

shock (Fig. 13C). The lower Hsf1 promoter occupancy in ras2∆ cells may be

interpreted as if the Ras cascade positively regulates Hsf1's DNA binding ability. We

believe, however, that the weaker Hsf1 binding in ras2∆ cells is a result of

constitutive binding of Msn2/4 to the promoter in this strain [(47); and Fig. 4) that

partially disturbs Hsf1’s binding. Indeed, removal of Msn2/4 from ras2∆ cells

(ras2∆msn2∆msn4∆) results in resumption of efficient binding of Hsf1 to the

promoter (Fig. 13A and B). Also, as shown above in ras2∆ cells, Hsf1 is dispensable

for HSP104 promoter activity as in these cells promoter activity was constitutively

high even after all HSEs were deleted [(47); and Fig. 4].

35

A)

B) C) Figure 13. HSF constitutively binds to the HSP104 promoter. A) Wild type, ras2∆, msn2∆msn4∆ and ras2∆msn2∆msn4∆ strains were crosslinked at the indicated time points following heat shock and ChIP was performed on HA tagged Hsf1. B) Samples of ChIP experiments shown in A (from non-heat shocked cells) were loaded on the same gel to compare HSF binding under basal conditions in different strains. C) Western analysis on HA-tagged Hsf1 showing similar protein level in all strains and under all conditions.

HSFp-HA-HSF

- 15’5’ 10’

W.T.

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

empty HSFp-HA-HSF

- 15’ - 5’ 10’ 15’

ras2∆

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

HSFp-HA-HSF

- 15’5’ 10’

W.T.

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

HSFp-HA-HSF

- 15’5’ 10’

W.T.

HSFp-HA-HSF

- 15’5’ 10’

W.T.

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

empty HSFp-HA-HSF

- 15’ - 5’ 10’ 15’

ras2∆

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

emptyempty HSFp-HA-HSF

- 15’- 15’ - 5’ 10’ 15’

ras2∆

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

empty HSFp-HA-HSF

- 15’ - 15’5’ 10’

msn2∆msn4∆

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

empty HSFp-HA-HSF

- 15’ - 15’5’ 10’

ras2∆msn2∆msn4∆

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

emptyempty HSFp-HA-HSFHSFp-HA-HSF

- 15’ - 15’5’ 10’

msn2∆msn4∆

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

emptyempty HSFp-HA-HSFHSFp-HA-HSF

- 15’ - 15’5’ 10’

ras2∆msn2∆msn4∆

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

Integrated vector

IB: α-HA

Heat Shock

Strain

empty HSFp-HA-HSF

- - 5’ 10’ 15’

empty HSFp-HA-HSF

- - 5’ 10’ 15’

W.T. msn2∆msn4∆

empty HSFp-HA-HSF

- - 5’ 10’ 15’

empty HSFp-HA-HSF

- - 5’ 10’ 15’

Integrated vector

IB: α-HA

Heat Shock

Strain ras2∆ ras2∆ msn2∆msn4∆

Integrated vector

IB: α-HA

Heat Shock

Strain

Integrated vector

IB: α-HA

Heat Shock

Strain

empty HSFp-HA-HSF

- - 5’ 10’ 15’

empty HSFp-HA-HSFempty HSFp-HA-HSF

- - 5’ 10’ 15’

empty HSFp-HA-HSF

- - 5’ 10’ 15’

empty HSFp-HA-HSFempty HSFp-HA-HSF

- - 5’ 10’ 15’

W.T. msn2∆msn4∆

empty HSFp-HA-HSF

- - 5’ 10’ 15’

empty HSFp-HA-HSFempty HSFp-HA-HSF

- - 5’ 10’ 15’

empty HSFp-HA-HSF

- - 5’ 10’ 15’

empty HSFp-HA-HSFempty HSFp-HA-HSF

- - 5’ 10’ 15’

Integrated vector

IB: α-HA

Heat Shock

Strain

Integrated vector

IB: α-HA

Heat Shock

Strain ras2∆ ras2∆ msn2∆msn4∆

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

- - - -

HSFp-HA-HSF

W.T.msn2∆

msn4∆

ras2∆msn2∆

msn4∆

ras2∆

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

- - - -

HSFp-HA-HSF

W.T.msn2∆

msn4∆

ras2∆msn2∆

msn4∆

ras2∆

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

- - - -

HSFp-HA-HSF

W.T.msn2∆

msn4∆

ras2∆msn2∆

msn4∆

ras2∆

Integrated vector

IP: HA-HSF

WCE

Heat Shock

Strain

- - - -

HSFp-HA-HSF

W.T.msn2∆

msn4∆

ras2∆msn2∆

msn4∆

ras2∆

36

Next, we attempted to directly measure the occupancy of Msn2 and Msn4 on

the promoter. Similarly to Hsf1, we constructed Ha-tagged proteins and inserted them

in the yeast genome at the URA3 locus. We were unfortunately unsuccessful in

detecting either Msn2 or Msn4 on the HSP104 promoter under normal or heat shock

conditions. Furthermore, we could not measure via ChIP assays Msn2/4 binding in

ras2∆ cells in which these two transcriptional activators are believed to constitutively

bind STREs (data not shown). We tried different variations of tagging Msn2 (either

at the N-terminus or C-terminus) and we also tried to over-express Msn2 under the

Alcohol Dehydrogenase 1 (ADH1) promoter known to be a strong constitutive

promoter. We also tried growing cells in minimal media (YNB) as opposed to rich

media (YPD). In all cases, DNA binding of the proteins was not detected although

the Msn2/4 proteins were confirmed to be expressed and active. It seems that the

inability to measure Msn2/4 binding via ChIP is not specific to our laboratory. There

is no report in the literature on successful ChIP on Msn2 or Msn4. Some reports (part

of all genome binding assay) do report on Msn2/4 binding to some promoter, (but

with very low affinity) (135). We still do not know whether the reason is technical or

conceptual (reflecting no direct association of Msn2/4 with DNA).

SAGA, SRB/MED and SWI/SNF are important for HSP104 promoter activity

The experiments described above analyzed the HSP104 promoter and

provided an insight into the events occurring on nucleosomes located on the HSP104

promoter. The experiments further showed that these events are controlled, at least in

part, by the activators Msn2/4. Having identified the major activators of the promoter

and some of their effects on chromatin organization we sought to identify the basal

transcription factors required for heat shock induced HSP104 transcription. To

uncover these factors, we tested HSP104 promoter activity in various mutants from

the Saccharomyces Genome Deletion Project. Mutants included those lacking a gene

encoding a basal transcription factor. Into each of these mutants we introduced

(separately) three reporter genes: i) -334LacZ, ii) STRE-LacZ and iii) HSE-LacZ.

Activity of the -334LacZ manifests the activity of the full length promoter. Activity

of STRE-LacZ [-260LacZ in (47)] manifests the function of the promoter activity

dependent on Msn2/4. The HSE-LacZ reporter contains the HSE cluster of HSP104

in four repeats fused to the CYC1 minimal promoter. This reporter reflects the

activity of Hsf1. 69 strains (Table 3) were tested with the three different plasmids.

37

Of those, 17 strains demonstrated lowered -334LacZ reporter activity, either under

physiological conditions or under stress conditions (Table 4), while 10 mutant strains

displayed higher activity of -334LacZ (Table 5). Nine mutant strains had no influence

on the activities of neither of the reporters and 34 strains showed reduced HSE-LacZ

activity (see details below in the next section).

38

Table 3. List of mutants in which reporter activity was measured.

Strain Cofatctor complex )of WT(% Gal activity -β

Uninduced Induced HSP104 HSP104

)WTof (% Gal activity -β Uninduced Induced

STRE-LacZ STRE-LacZ

)of WT(% Gal activity -β Uninduced Induced

HSE-LacZ HSE-LacZ ahc1∆ ADA 45 66 94 70 84 80 ada2∆ ADA,SAGA 56 117 57 17 27 65 ada3∆ ADA,SAGA 42 74 8 7 2 1 gcn5∆ ADA,SAGA 23 69 99 17 13 20 ada1∆ SAGA 60 88 72 81 56 84 spt3∆ SAGA 41 81 213 52 22 34 spt7∆ SAGA 58 16 325 37 6 8 spt8∆ SAGA 59 101 77 31 26 37

yer049∆ NuA3 68 89 70 55 71 64 yer1101∆ NuA3 75 115 74 206 79 75

sas3∆ NuA3 3 7 0 2 7 6 eaf3∆ NuA4 49 93 215 173 86 74 bdf1∆ TFIID 225 135 742 122 46 67 bdf2∆ TFIID 48 72 91 127 90 101 elp3∆ HAT 116 185 227 140 156 111 ayt1∆ HAT 52 62 172 96 67 53 hpa2∆ HAT 43 55 124 83 52 53 hpa3∆ HAT 62 88 88 157 29 32 sas2∆ HAT 71 90 0 35 59 60 rpd3∆ HDAC 56 70 142 93 69 65 hda1∆ HDAC 40 58 162 224 44 44 hos1∆ HDAC 56 61 97 132 111 92 hos2∆ HDAC 36 64 33 55 35 26 hos3∆ HDAC 64 65 77 47 23 30 sir2∆ HDAC 38 71 29 77 38 46 hst1∆ HDAC 36 55 80 105 72 56 hst2∆ HDAC 59 85 109 116 54 41 hst3∆ HDAC 59 76 92 82 60 45 hst4∆ HDAC 74 118 87 92 66 62 tfg3∆ Multiple 62 69 259 91 4 23 swi3∆ SWI/SNF 46 96 143 95 2 14 snf5∆ SWI/SNF 60 75 150 133 187 148 snf6∆ SWI/SNF 13 50 0 0 10 23 snf11∆ SWI/SNF 47 75 40 43 121 99 rsc1∆ RSC 70 71 337 212 107 121 rsc2∆ RSC 76 50 300 129 55 72 isw1∆ ISW1 105 125 169 257 99 89 isw2∆ ISW2 40 41 65 53 133 63 chd1∆ Homodimer 74 156 143 152 149 84 not3∆ CCR4-NOT 111 125 111 209 71 56 not4∆ CCR4-NOT 111 109 1161 118 35 39 caf1∆ CCR4-NOT 227 60 237 87 15 13

39

Table 3. Con’t.

Strain Cofatctor complex )of WT(% Gal activity -β

Uninduced Induced HSP104 HSP104

)of WT(% Gal activity -β Uninduced Induced

STRE-LacZ STRE-LacZ

)of WT(% Gal activity -β Uninduced Induced

HSE-LacZ HSE-LacZ caf4∆ CCR4-NOT 51 71 47 60 35 31 caf16∆ CCR4-NOT 61 65 56 83 39 38 caf40∆ CCR4-NOT 103 109 88 142 44 45 caf130∆ CCR4-NOT 65 100 86 82 102 69 dbf2∆ CCR4-NOT 62 51 342 415 32 23 dhh1∆ CCR4-NOT 319 67 873 132 17 7

srb9∆ SRB/MED,CCR4-NOT 150 59 723 104 52 38

srb10∆ SRB/MED,CCR4-NOT 72 15 469 41 13 6

srb2∆ SRB/MED 33 110 154 69 32 94 srb5∆ SRB/MED 10 23 78 23 12 29 srb8∆ SRB/MED 60 28 614 90 69 28 med1∆ SRB/MED 154 107 715 229 42 38 med9∆ SRB/MED 179 93 1550 484 29 32 nut1∆ SRB/MED 135 68 580 249 101 78 rox3∆ SRB/MED 86 41 1605 251 4 16

cdc73∆ Paf1 complex 11 20 11 17 4 12 rtf1∆ Paf1 complex 49 52 100 51 34 28 leo1∆ Paf1 complex 21 29 37 51 34 28 ccr4∆ CCR4-NOT,Paf1 157 43 399 107 11 5 hpr1∆ Paf1,THO/TREX 0.1 1 0 1.5 0 0.1 mft1∆ THO/TREX 4 3 5 0.1 0.4 0.5 tex1∆ THO/TREX 34 38 44 61 32 33 thp2∆ THO/TREX 48 74 192 148 68 57 dst1∆ TFIIS 25 36 182 111 58 40 spt4∆ SPT 10 2 62 8 0.5 0.3 rpb9∆ RNA PolII core 48 62 169 41 34 30 rpb4∆ RNA PolII core 2 0.4 17 0.6 1 0.4

40

Table 4. Strains required for positive regulation of HSP104.

Table 5. Strains which induced an up-regulation of HSP104 (i.e. downregulate HSP104).

Strain Cofatctor complex )of WT(% Gal activity -β

Uninduced Induced HSP104 HSP104

)of WT(% activity Gal -β Uninduced Induced

STRE-LacZ STRE-LacZ

)of WT(% Gal activity -β Uninduced Induced

HSE-LacZ HSE-LacZ bdf1∆ TFIID 225 135 742 122 46 67 elp3∆ HAT 116 185 227 140 156 111 chd1∆ Homodimer 74 156 143 152 149 84 caf1∆ CCR4-NOT 227 60 237 87 15 13 dhh1∆ CCR4-NOT 319 67 873 132 17 7

srb9∆ SRB/MED,CCR4-NOT 150 59 723 104 52 38

med1∆ SRB/MED 154 107 715 229 42 38 med9∆ SRB/MED 179 93 1550 484 29 32 nut1∆ SRB/MED 135 68 580 249 101 78

In order to confirm that the decreased or increased reporter activities measured

in the mutants reflect indeed defects in expression of endogenous HSP104, we

performed S1 analysis to detect endogenous mRNA levels of HSP104. We first

measured RNA levels in strains which demonstrated a decrease in the β-galactosidase

activity of -334LacZ. Most strains listed in Table 4 indeed had lower RNA levels

Strain Cofatctor complex )of WT(% Gal activity -β

Uninduced Induced HSP104 HSP104

)of WT(% Gal activity -β Uninduced Induced

STRE-LacZ STRE-LacZ

)of WT(% Gal activity -β Uninduced Induced

HSE-LacZ HSE-LacZ gcn5∆ ADA,SAGA 23 69 99 17 13 20 spt7∆ SAGA 58 16 325 37 6 8 sas3∆ NuA3 3 7 0 2 7 6 hos2∆ HDAC 36 64 33 55 35 26 sir2∆ HDAC 38 71 29 77 38 46 hst1∆ HDAC 36 55 80 105 72 56 snf6∆ SWI/SNF 13 50 0 0 10 23

srb10∆ SRB/MED,CCR4-NOT 72 15 469 41 13 6

srb2∆ SRB/MED 33 110 154 69 32 94 srb5∆ SRB/MED 10 23 78 23 12 29 srb8∆ SRB/MED 60 28 614 90 69 28 dst1∆ TFIIS 25 36 182 111 58 40 rpb4∆ RNA PolII core 2 0.4 17 0.6 1 0.4 cdc73∆ Paf1 complex 11 20 11 17 4 12 leo1∆ Paf1 complex 21 29 37 51 34 28 hpr1∆ Paf1,THO/TREX 0.1 1 0 1.5 0 0.1 mft1∆ THO/TREX 4 3 5 0.1 0.4 0.5

41

either under physiological conditions or under heat shock conditions when compared

to wild type (Fig. 14). In some strains, however, such correlation was not observed.

The sas3∆ strain is an extreme case of such discrepancy. When we measured β-

galactosidase activity of the various constructs in sas3∆ cells very little or no activity

was detected. We were therefore surprised to see that an impressive amount of

HSP104 mRNA accumulated in these cells in response to heat shock. Other strains in

which RNA levels were not fully correlated to levels of reporter assays are snf6∆ and

srb2∆ that exhibited very low levels of HSP104 mRNA, but normal or close to

normal levels of β-galactosidase induced activity. In addition, in the hos2∆ and sir2∆

strains that showed some decrease in promoter activity (Table 4) endogenous HSP104

mRNA levels were not affected (data not shown). Finally, hpr1∆ cells which showed

very low or non-inducible reporter activity, expressed HSP104 mRNA at basal levels

(not induced) significantly higher than those in wild type cells (Fig. 16). We therefore

considered Hpr1 as a negative regulator of HSP104 rather than a positive regulator.

A B Figure 14. Components from the SRB/MED, SAGA and SWI/SNF complexes and RNA PolII subunit, Rpb4, are important for proper transcription of HSP104. A) S1 RNA analysis of HSP104 mRNA was performed on the indicated yeast strains following heat shock at the marked time points. Table indicates β-galactosidase activity units obtained with the -334LacZ reporter in the same strains before and after heat shock. B) Same RNAs as in A were used to measure ACTIN mRNA levels via S1 analysis as control.

srb2∆

srb5∆

srb8∆

srb9∆

srb10∆

wt

Heat shock

- 5’ 10’

15’

60’

5hrs

300

snf6∆

med1∆

HSP104-LacZ(units)

-HS +HS

51.2 273

15.7 271.7

4.7 56.3

24.2 56.0

59.9 179.6

37.4 56.1

60.6 288.4

7.4 175.6

hst1∆

gcn5∆

spt7∆

cdc73∆

dst1∆

mft1∆

rpb4∆

sas3∆

- 5’ 10’

15’

60’

5hrs

300

HSP104-LacZ(units)

-HS +HS

28.1 161.2

12.8 185.4

31.5 43.6

15.4 103.1

2.0 5.8

6.1 55.0

1.01 1.35

1.82 12.6

time time

HSP104 HSP104

srb2∆

srb5∆

srb8∆

srb9∆

srb10∆

wt

Heat shock

- 5’ 10’

15’

60’

5hrs

300

Heat shock

- 5’ 10’

15’

60’

5hrs

300

snf6∆

med1∆

HSP104-LacZ(units)

-HS +HS

51.2 273

15.7 271.7

4.7 56.3

24.2 56.0

59.9 179.6

37.4 56.1

60.6 288.4

7.4 175.6

hst1∆

gcn5∆

spt7∆

cdc73∆

dst1∆

mft1∆

rpb4∆

sas3∆

- 5’ 10’

15’

60’

5hrs

300

- 5’ 10’

15’

60’

5hrs

300

HSP104-LacZ(units)

-HS +HS

28.1 161.2

12.8 185.4

31.5 43.6

15.4 103.1

2.0 5.8

6.1 55.0

1.01 1.35

1.82 12.6

time time

HSP104 HSP104

Heat shock

- 5’ 10’

15’

60’

5hrs

300

srb2∆

srb5∆

srb8∆

srb9∆

srb10∆

wt

snf6∆

med1∆

hst1∆

gcn5∆

spt7∆

cdc73∆

dst1∆

mft1∆

rpb4∆

sas3∆

Heat shock

- 5’ 10’

15’

60’

5hrs

300

timetime

ACTIN ACTIN

Heat shock

- 5’ 10’

15’

60’

5hrs

300

Heat shock

- 5’ 10’

15’

60’

5hrs

300

- 5’ 10’

15’

60’

5hrs

300

srb2∆

srb5∆

srb8∆

srb9∆

srb10∆

wt

snf6∆

med1∆

hst1∆

gcn5∆

spt7∆

cdc73∆

dst1∆

mft1∆

rpb4∆

sas3∆

Heat shock

- 5’ 10’

15’

60’

5hrs

300

Heat shock

- 5’ 10’

15’

60’

5hrs

300

timetime

ACTIN ACTIN

42

Overall, the results nevertheless show a general correlation between reporter

activity and mRNA levels. Namely, the genes deleted in 15 strains out of the 17

tested, encode proteins that are required for proper induction of the HSP104 promoter.

Our results strongly suggest the involvement of the SAGA complex (represented by

the spt7∆, gcn5∆ mutants), the SRB/MED coactivator complex (represented by the

srb2∆, srb5∆, srb8∆, srb9∆, srb10∆, and med1∆ mutants) as well as the SWI/SNF

chromatin remodeling complex (snf6∆ mutant) in mediating HSP104 transcription

initiation (Table 4). In addition, we also measured promoter activity and mRNA

levels of HSP104 in rpb4∆ cells and observed that HSP104 is barely, if at all, induced

in this strain in response to heat shock. Rpb4 is a RNA PolII subunit which is not

essential for growth under optimal growth conditions, but essential under stress and is

important for the activation of PolII under heat shock conditions (23, 24, 89, 97, 98).

As the components identified are part of the basal machinery we wondered whether

the defective induction of HSP104 in these mutants is specific to HSP104, or whether

the mutants demonstrate a general abnormality in inducing stress genes in response to

heat shock. To this end we measured the mRNAs of HSP26 and SSA3, two HSP

encoding genes known to be induced in response to heat shock. As shown in Figure

15, most of the mutants tested induced HSP26 and SSA3 normally, suggesting that the

failure of the mutants to properly induce HSP104 is specific. We did notice however,

that some mutants, like rpb4∆ and snf6∆, seem to have more general defects in

inducing stress genes. Curiously, SSA3 mRNA levels were spontaneously elevated in

most mutants.

Figure 15. Most factors required for proper induction of HSP104 are specific for this gene. RNA from mutants that demonstrated impaired HSP104 induction in response to heat shock, were used to measure mRNA levels of HSP26 (sample of no heat shock and ten minutes heat shock were used) and SSA3 (sample of no heat shock and fifteen minutes heat shock were used).

HS - + - + - + - + - + - + - + - +

WT dst1∆ hst1∆ srb8∆srb10∆ spt7∆ gcn5∆ srb2∆

HSP26

SSA3

HS - + - + - + - + - + - + - + - +

WT dst1∆ hst1∆ srb8∆srb10∆ spt7∆ gcn5∆ srb2∆

HSP26

HS - + - + - + - + - + - + - + - +- + - + - + - + - + - + - + - +- + - + - + - + - + - + - + - +

WT dst1∆ hst1∆ srb8∆srb10∆ spt7∆ gcn5∆ srb2∆

HSP26

SSA3

- + - + - + - + - + - + - + - +

srb5∆snf6∆ rpb4∆ sas3∆

mft1∆ cdc73∆med1∆

srb9∆

HS

HSP26

SSA3

- + - + - + - + - + - + - + - +- + - + - + - + - + - + - + - +- + - + - + - + - + - + - + - +

srb5∆snf6∆ rpb4∆ sas3∆

mft1∆ cdc73∆med1∆

srb9∆

HS

HSP26

SSA3

43

Next, we monitored HSP104 mRNA levels in strains which, in the reporter

assay, demonstrated an increase in activity (Table 5) and are therefore mutated in

putative suppressors of HSP104 transcription. Most of these strains did demonstrate

spontaneous elevation of HSP104 mRNA levels (Fig. 16) and also manifested higher

induced levels when compared to wild type. bdf1∆, caf1∆, and srb9∆ strains,

however, demonstrated induced levels of HSP104 mRNA close to those of wild type,

and therefore were considered as non-regulators of HSP104 (data not shown).

Combining the results obtained we note that a total of 6 strains are considered to be

mutated in negative regulators of HSP104 transcription. Two of them are mutated in

components of the SRB/MED complex (med9∆ and nut1∆) the same complex that is

also involved in positively regulating HSP104. The fact that components of the same

complex both positively and negatively regulate HSP104 transcription, strongly

suggests that the SRB/MED complex may play a role in the fine tuning of the

transcription initiation of HSP104. The other four mutated strains cannot be grouped.

Figure 16. Some components are involved in down-regulating the transcription of HSP104. A) S1 RNA analysis of HSP104 mRNA was performed on the indicated yeast strains following heat shock at the marked time points (left panel). The right panel shows same RNAs as in the left panel was used to measure ACTIN mRNA levels via S1 analysis as control.

wt

ccr4∆

chd1∆

elp3∆

med9∆

hpr1∆

nut1∆

HSP104

ACTIN

Heat shock

- 5’ 10’

15’

60’

5hrs

300

time

wt

ccr4∆

chd1∆

elp3∆

med9∆

hpr1∆

nut1∆

HSP104

ACTIN

Heat shock

- 5’ 10’

15’

60’

5hrs

300

Heat shock

- 5’ 10’

15’

60’

5hrs

300

time

44

Regulation of Hsf1

As explained in “Introduction”, mechanisms underlying Hsf1

activation/regulation are not revealed. Many regulators of Hsf1 have been identified,

that impose phosphorylation, sumoylation, and oxidation, but none of these

modifications seems critical. Furthermore, elements of the basal transcription

machinery that are required for Hsf1 activity were hitherto not identified. As

described in the previous section, we monitored the activity of the HSE-LacZ reporter

gene in various mutants. This reporter reflects the activity of Hsf1 only, because it

contains only HSEs upstream to the minimal promoter. Therefore, in a strain

demonstrating poor activity of this reporter, Hsf1 activity is compromised (Table 6, of

which 20 mutant strains exclusively affect activity of the HSE-LacZ reporter). Thirty

nine such strains were identified. We did not find even one strain with spontaneous

increased HSE-LacZ activity. Since strains identified are mutated in components of

the basal transcription machinery, they may be compromised in activation of many

stress-induced activators and not specifically Hsf1. In order to assess whether the

effects observed are specific to Hsf1, we tested in some of the strains the activity of

the SV40-LacZ reporter which is activated solely by the yAP-1 transcriptional

activator (Table 7). Except for one strain (sas3∆), all other 11 strains with reduced

HSE-LacZ activity that were tested, showed activity levels of the SV40-LacZ gene

similar to that of wild type (Table 7).

45

Table 6. Strains which down-regulated HSE-LacZ reporter activity (not exclusive).

Strain Cofatctor complex )of WT(% Gal activity -β Uninduced Induced HSP104 HSP104

)of WT(% Gal activity -β Uninduced Induced

STRE-LacZ STRE-LacZ

)of WT(% Gal activity -β Uninduced Induced

HSE-LacZ HSE-LacZ

ada2∆ ADA,SAGA 56 117 57 17 27 65 ada3∆ ADA,SAGA 42 74 8 7 2 1 gcn5∆ ADA,SAGA 23 69 99 17 13 20 spt3∆ SAGA 41 81 213 52 22 34 spt7∆ SAGA 58 16 325 37 6 8 spt8∆ SAGA 59 101 77 31 26 37 sas3∆ NuA3 3 7 0 2 7 6 hpa3∆ HAT 62 88 88 157 29 32 hos2∆ HDAC 36 64 33 55 35 26 hos3∆ HDAC 64 65 77 47 23 30 sir2∆ HDAC 38 71 29 77 38 46 hst2∆ HDAC 59 85 109 116 54 41 tfg3∆ Multiple 62 69 259 91 4 23 swi3∆ SWI/SNF 46 96 143 95 2 14 snf6∆ SWI/SNF 13 50 0 0 10 23 not4∆ CCR4-NOT 111 109 1161 118 35 39 caf1∆ CCR4-NOT 227 60 237 87 15 13 caf4∆ CCR4-NOT 51 71 47 60 35 31 caf16∆ CCR4-NOT 61 65 56 83 39 38 dbf2∆ CCR4-NOT 62 51 342 415 32 23 dhh1∆ CCR4-NOT 319 67 873 132 17 7

srb9∆ SRB/MED,CCR4-NOT 150 59 723 104 52 38

srb10∆ SRB/MED,CCR4-NOT 72 15 469 41 13 6

srb2∆ SRB/MED 33 110 154 69 32 94 srb5∆ SRB/MED 10 23 78 23 12 29 srb8∆ SRB/MED 60 28 614 90 69 28 med1∆ SRB/MED 154 107 715 229 42 38 med9∆ SRB/MED 179 93 1550 484 29 32 rox3∆ SRB/MED 86 41 1605 251 4 16

cdc73∆ Paf1 complex 11 20 11 17 4 12 rtf1∆ Paf1 complex 49 52 100 51 34 28 leo1∆ Paf1 complex 21 29 37 51 34 28 ccr4∆ CCR4-NOT,Paf1 157 43 399 107 11 5 hpr1∆ Paf1,THO/TREX 0.1 1 0 1.5 0 0.1 mft1∆ THO/TREX 4 3 5 0.1 0.4 0.5 tex1∆ THO/TREX 34 38 44 61 32 33 spt4∆ SPT 10 2 62 8 0.5 0.3 rpb9∆ RNA PolII core 48 62 169 41 34 30 rpb4∆ RNA PolII core 2 0.4 17 0.6 1 0.4

46

Table 7. Comparison between HSE-LacZ and SV40-LacZ in various mutant strains.

Strains Cofactor complex )of WT(% Gal activity -β

Uninduced Induced HSE-LacZ HSE-LacZ

)of WT(% activity Gal -β Uninduced Induced

SV40-LacZ SV40-LacZ ada2∆ ADA,SAGA 32 89 125 149 ada3∆ ADA,SAGA 0.5 0.4 111 111 gcn5∆ ADA,SAGA 25 40 137 126 spt3∆ SAGA 32 57 350 194 spt7∆ SAGA 6 10 169 92 spt8∆ SAGA 32 54 313 168 sas3∆ NuA3 8 6 10 9 hpa3∆ HAT 40 45 101 103 hos2∆ HDAC 35 28 67 72 hos3∆ HDAC 40 43 132 101 sir2∆ HDAC 43 47 165 102 hst2∆ HDAC 64 50 102 90

To begin and address the mechanism of action of the mutations responsible for

reduced Hsf1 activity, we tested in some of the strains the ability of Hsf1 to bind in

vivo to the HSP104 promoter. Since we showed that Hsf1 constitutively binds HSEs

on the HSP104 promoter regardless of heat shock conditions, we predicted reduced

DNA binding activity of Hsf1 in strains in which HSE-LacZ activity was low. Quite

surprisingly, we actually observed an inverse correlation between the transcriptional

activity of Hsf1 and its DNA binding activity. As is shown in Figure 17, gcn5∆,

spt7∆, caf1∆, and ccr4∆ cells which demonstrated very poor reporter activity of HSE-

LacZ (Table 6), displayed stronger binding of Hsf1 on the HSP104 promoter

compared to wild type. Perhaps in these strains stronger Hsf1 DNA binding activity

is inhibitory. Transcription repression activity of Hsf1 was previously suggested

(131). It should be noted that the studies on the mechanism of action of the mutants is

only at its beginning and a large scale study is required for revealing the role of each

factor on Hsf1 activity. Nevertheless, the finding of elements of the basal

transcription machinery that are highly specific to Hsf1 is novel and unexpected.

47

Figure17. Hsf1 shows stronger binding in some HSE-LacZ inefficient strains. Indicated strains were crosslinked at the indicated time points following heat shock and ChIP was performed on HA tagged Hsf1.

DISCUSSION

In response to stress, or to fluctuations in optimal growth conditions, cells halt

transcription of most genes. Yet, genes involved in combating stresses are

upregulated. The molecular mechanisms responsible for transcription initiation of

this group of genes are not fully revealed. This work described a systematic study

aimed at revealing these mechanisms in the S.cerevisiae HSP104 gene. This

experimental approach (elaborating on just one promoter) is somewhat different than

current prevailing strategies in the field. Common studies reported in recent years on

transcription initiation in general and in response to stress in particular, addressed co-

regulation of many genes searching for common themes in their regulation (21, 36,

59, 103, 128, 135, 136). These approaches lead on one hand to important discoveries,

but on the other hand major matters are left unresolved. Most important, data

accumulated so far suggest that although common regulatory themes do exist, each

promoter is induced via specific processes. Our long-term intention is therefore to

dissect the processes of transcription initiation under stress of one gene in order to

understand its specific and unique regulation at high resolution and in a highly

detailed manner.

This thesis presents the data obtained so far in the long run toward this

ultimate goal. This data was obtained via three approaches I undertook: I) Promoter

analysis, i.e., identification of various cis-elements which are essential for regulating

promoter activity of HSP104. In order to identify the important elements and the

relationships between them for HSP104 transcription we proceeded with 5’deletions

of the promoter, point mutations and the establishment of heterologous reporter genes.

II) Monitoring modifications of histones occurring on the promoter of HSP104 in

response to heat shock. We also monitored the binding activity of the transcriptional

activator Hsf1 and attempted to measure the binding of Msn2/4 on the HSP104

Heat Shock - - 5’

Integrated Vector empty HSFp-HA-HSF

IP: HA-HSF

WCE

Strain BY4741

- - 5’

empty HSFp-HA-HSF

caf1∆

- - 5’

empty HSFp-HA-HSF

ccr4∆

- - 5’

empty HSFp-HA-HSF

- - 5’

empty HSFp-HA-HSF

gcn5∆ spt7∆

Heat Shock - - 5’

Integrated Vector empty HSFp-HA-HSF

IP: HA-HSF

WCE

Strain BY4741

- - 5’

empty HSFp-HA-HSF

caf1∆

- - 5’

empty HSFp-HA-HSF

ccr4∆

- - 5’

empty HSFp-HA-HSF

- - 5’

empty HSFp-HA-HSF

gcn5∆ spt7∆

48

promoter. III) The identification of components of the basal transcription machinery

involved specifically in transcriptionally activating HSP104.

The results obtained allow us to draw a detailed working model for the

molecular events occurring on the HSP104 promoter in response to heat shock (see

details in the last section of the Discussion). The first aspect of our promoter analysis

dealt with, as mentioned above, analyzing the promoter through 5’deletion [most

aspects of promoter analysis are thoroughly described in (47)]. We sought at this

stage of our study, to identify sequences which are required for the high basal activity

of -334LacZ reporter. We found the putative single HSE site between (-304 and -300)

to have an important role in this promoter activity. In fact, our fine-tuned mapping

effort did not point at an additional particular sequence within the 34bp as responsible

for the activity. We further conclude that the 34bp (particularly the HSE between -

304 and -300) may possibly be required to interact with the specific basal

transcription machinery of HSP104 (this conclusion is also based on the results with

the heterologous promoter, Fig. 7). We therefore attempted to analyze the interaction

between the 34bp with the basal transcription machinery by deleting an internal

fragment of 78bp, thereby bringing the 34bp close to the minimal promoter. The

various ∆78 constructs manifested low basal activity, suggesting that perhaps the

spatial organization between the 34bp and the basal transcription machinery is critical

for the basal activity of the HSP104 promoter. It could also be that STREs, removed

with the internal 78bp, are required for basal activity. The ∆78 constructs provided

important information about the flexibility and modularity of the promoter (Fig. 8).

Normally, there is complete cooperation between STRE/Msn2/4 and HSE/Hsf1

systems for optimal promoter activity. Yet, under specific conditions, such as in

msn2∆msn4∆ cells, HSP104 is solely activated by the HSE/Hsf1 system. Conversely,

in ras2∆ cells, the HSE/Hsf1 system is not required for the activation of HSP104

promoter activity. High activity of HSP104 in this strain is due to the hypothesized

constitutive binding of Msn2/4 to the STREs present on the promoter. This

flexibility and back-up capabilities of the HSE/Hsf1 and STRE/Msn2/4 systems are

now further expanded and include the upstream 34bp that become Ras2 responsive in

cells lacking Msn2/4 and the internal 78bp, that under various conditions, affect not

only induced, but also basal promoter activity. Namely, promoter flexibility is much

stronger than we previously suggested and many cis and trans-elements are backing

up each other to allow promoter activity under various circumstances. Measuring

49

HSP104 mRNA in different mutants show, indeed, the robustness of HSP104

transcription that is rarely affected by the absence of activators and other transcription

factors.

Through monitoring the acetylation states of histones, we suggest that

induction of HSP104 transcription requires that acetylated H3 and H4 molecules be

absent. We observed that under optimal growth conditions all H4 histones on the

HSP104 promoter are acetylated as well as major fractions of H3 histones. Under

these conditions, only in ras2∆ cells, concentrations of acetylated H3 and H4 are

lower and the promoter is even occupied with non-acetylated H4. As in this strain the

HSP104 promoter is constitutively active, there is clear correlation between promoter

activity and non-acetylated histones. Current dogma of gene activation claims that

acetylation of histones followed by the dissociation of nucleosomes from promoters is

required for promoter activation (28, 49, 50). The case for HSP104 is different.

There is no global dissociation of histones from the promoter, but rather a specific

dissociation of acetylated H3 molecules from nucleosomal structures and

deacetylation of H4 molecules. Non-acetylated H3 proteins remain associated with

nucleosomes even in response to heat shock and so do histone H4 molecules that are

now not acetylated. Although not very common, the disassembly and deacetylation

patterns we observe for HSP104 in response to heat shock should not be that

surprising. As described in Deckert et al (31) histone deacetylation can also lead to

transcription initiation of several stress-responsive genes. Also, a recent study also

demonstrated that some promoters, namely GAL induced promoters, are activated by

histone deacetylation (31).

However, our conclusion differs somewhat from that of Deckert and Struhl

(31). These investigators also monitored a dramatic decrease in acetylated histones

on the HSP104 promoter in response to heat shock [18 fold decrease of acetylated H4

and 10 fold decrease in acetylated H3 (31)]. Yet, as there was some decrease in the

level of unacetylated H4 occupancy (30%) they concluded that there is altogether

chromatin remodeling or disassembly of the nucleosomes. We suggest that the

changes on the chromatin should not be taken as all-or-non activity (disassembly, or

deacetylation). There seems to be a series of different events which we partly

revealed; i.e., histones H4 are deacetylated, and acetylated H3 molecules are

disassembled from nucleosomes. Also, these changes are transient and reflect the

dynamic nature of the changes occurring on the promoter during continuous exposure

50

to stress. This change in nucleosome composition may also be part of the shift of the

cell’s response from acute stage to adaptive stage. Perhaps fifteen minutes after heat

shock, nucleosomes are reassembled in a different combination of modified histones

and transcription continues through a different mechanism than that of the first fifteen

minutes following heat shock.

In spite of mass research it is still not possible to describe in fine details events

leading to transcription activation. To a certain extent, it is possible for some

promoters. For instance, the transcription of the HO gene, transcribed during the G1

phase of the cell cycle includes the recruitment (by a transcriptional activator) of the

chromatin remodeling complex SWI/SNF which is followed by the recruitment of

Gcn5 that acetylates histones on the nucleosomes present on the promoter.

Acetylation via Gcn5 then induces the recruitment of another transcriptional activator

to the HO promoter (29). Another study showed that within a group of HSP genes,

thought to be co-regulated, there are differences in the steps leading to their

transcription initiation (36). For instance, HSP12 which showed the highest level of

nucleosome displacement also showed highest level of histone H3 acetylation (36).

SSA4 which showed the lowest nucleosome displacement also demonstrated the

lowest acetylation state. Finally HSP82 also showed an increase in nucleosome

displacement which correlated with an increase with histone acetylation (36) and yet

all three genes are activated in response to heat shock in an Hsf1 dependent manner.

For the first time, we show that transcriptional activation of a single promoter

involves a combination of mechanisms for remodeling nucleosomes; histone

displacement (partial nucleosome disassembly) and histone deacetylation.

What factor(s) is responsible for inducing the changes observed on the HSP104

promoter? Results from our small genetic screen indicate a role for SWI/SNF in

regulating promoter activity and transcription of HSP104. The main role for

SWI/SNF is to promote nucleosome disassembly raising the question whether

SWI/SNF is responsible for disassembling acetylated histone H3 from the HSP104

promoter in response to heat shock. ChIP assays may answer this question by

monitoring acetylation state of histone H3 in response to heat shock in mutant

SWI/SNF strains (snf6∆, for example). Another open question is the identity of the

histone deacetylases (HDACs) involved in deacetylating histone H4 on the promoter

of HSP104 in response to heat shock. It is clear that these enzymes display some

redundancy in the cell (34, 73). In order to target which family of histone deacetylase

51

is responsible for deacetylating histone H4 it will be necessary to construct many

strains with combined deletions of various HDACs. Many transcriptional activators,

such as Hsf1 (in mammals), Gcn4 and Swi5 have been shown to recruit chromatin

remodeling complexes to promoters in vitro and in vivo (29, 124). Does Hsf1 in yeast

play such a role on heat shock induced promoters? As Hsf1 constitutively binds

HSP104 regardless of stress conditions, perhaps some post-translational modification

of the protein could target chromatin modifying complexes to the HSP104 promoter.

An alternative explanation is that Msn2/4 recruit the nucleosome remodeling

enzymes. The fact that the lower levels of acetylated H3 or H4 are found in ras2∆, in

which Msn2/4 are constitutively active and probably constitutively bound, supports

the notion that Msn2/4 are responsible for these changes.

Studies show that in Drosophila, Hsf1 is responsible for recruiting the

mediator complex to heat shock promoters (95). Hsf1 in S.cerevisiae was also shown

to have a role in recruiting the SRB/MED co-activator complex to Hsf1 dependent

promoters (39). Hsf1 may recruit the SRB/MED complex to the HSP104 since we

find that SRB/MED is involved in transcription initiation of HSP104. The

confirmation of such a hypothesis could be achieved by performing a successive

immunoprecipitation followed by PCR (i.e., double ChIP assays, one

immunoprecipitation against Hsf1 followed by another one against a component of

the SRB/MED complex) as components of the mediator complex have been shown to

be associated with the HSP104 promoter in response to heat shock and to other Hsf1

target genes (39). The Srb10 component of the SRB/MED complex seems to be

involved in transcription of HSP104 (Table 4). We observed that at the level of the

reporter gene activity and, more importantly, at the level of RNA production.

However, our reporter studies demonstrated that the activity of a STRE-LacZ reporter

in srb10∆ was significantly up-regulated even under non-heat shock conditions. This

result is in agreement with studies showing that Msn2 is degraded in a Srb10-

dependent manner (39). Regarding HSP104 transcription, this suggests that other

mechanisms seem to override the loss of Srb10 and that probably Srb10 in the context

of the SRB/MED complex has another function.

Through the genetic screen, we also found that Rpb4, a RNA PolII subunit, is

required for HSP104 as well as for HSP26 and SSA3 transcription [Figs. 14 and 15

and (24)]. Indeed, DNA micro array studies show that 98% of genes are

downregulated in response to heat shock in RPB4 null cells (89). Interestingly and

52

perhaps not surprisingly, overexpression of Msn2 could suppress the growth

phenotype observed for Rpb4, but only at 34oC. It would be interesting to test

whether in rpb4∆ cells, Hsf1 binds the HSP104 promoter and whether the

modifications of histones H3 and H4 still take place.

The study presented in this thesis allows establishment of a model describing

the molecular events leading to the transcriptional activation of the HSP104 promoter:

under optimal growth conditions, the promoter is occupied by Hsf1 and acetylated

histones. Under these conditions, the promoter is only partially active via the 34bp

(most probably via the HSE at -304 to -300), but also partially via downstream STREs

(because lower basal levels are measured in msn2∆msn4∆ cells or in constructs

lacking internal STREs, the ∆78 constructs). Upon lowering cAMP levels,

permanently, such as in ras∆2 cells or under stress conditions, cells remove the

negative regulation imposed by the Ras/cAMP/PKA pathway on Msn2/4. This

enables the nuclear localization of the STRE-binding transcriptional activators which

subsequently leads to the transcriptional activation of STRE-containing genes. In

parallel, upon heat shock (which also activates Msn2/4), HSE containing promoters

are transcriptionally activated through the binding of Hsf1. In the case of HSP104,

the binding of both transcriptional activators leads to proper transcriptional activation

of this gene via the recruitment of chromatin modifying complexes (in an Msn2/4

dependent manner) which promote histone H4 deacetylation and disassembly of

acetylated H3 histones from nucleosomes. In addition to these chromatin

modifications, proper transcriptional induction could be enhanced through the

recruitment of the SRB/MED complex [perhaps via Hsf1 (39)]. Finally, the RNA

PolII is recruited, but it must be in its holoenzyme form (i.e., containing all subunits

including Rpb4)

Thus, we now have a comprehensive working model that describes the major

molecular steps leading to the transcriptional activation of the HSP104 gene.

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