Global Repression of Non-Heat Shock Gene Transcription by Activation of Eeat Shock Factor in DrosophiIa

Meredith Leigh Stevens

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Zoology

University of Toronto

O Copyright by Meredith Leigh Stevens (1999)

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Global Repression of Non-Beat Shock Cene Transcription by Activatioa of Heat Shock Factor in Drosophifa

Meredith Leigh Stevens M.Sc. Thesis 1999 Graduate Department of Zoology University of Toronto

Abstract

Cells respond to stress by activation of the heat shock (hs) raponse, regulated by the hs

transcription factor, HSF. In Drosophila hs results in HSF binding at over 200 loci on polytene

chromosomes, concomitant with transcriptional repression at non-hs gene sites. We suggest that

HSF binding causes the preferential recruitment of RNA polymerase 11 (Pol II) to the hs genes,

resulting in the repressed transcription of non-hs genes. To exclude the possibility that non-hs

gene repression was due to efiects of heat, the proline amino acid analogue azetidine-2-

carboxylic acid (AzC) was used to induce the Drosophi[a hs response. At normal temperatures

AzC elicits HSF binding and hs gene transcription in a manner similar to heat but on a longer

time scale. AzC also results in repression of non-hs gene transcription, indicating that it is the

activation of HSF DNA-binding which leads to Pol II recruitment and subsequent global

transcriptional repression.

Acknowledgements

1 would like to thank the following people who have made this work possible:

Dr. J. Timothy Westwood for his insight, mentorship, and support as my graduate supervisor,

Dr. Angela Lange for many hours spent using the fluorescence microscope;

Dr. Barb Funnell for use of her scanner,

Members of the Westwood lab, both past and present, who have shared many laughs and made my experience an enjoyable one: Wing Chang, Jenny Ho, Martin Hyrcza, Kasia Kociuba, Dan Mao, Phi1 Mercier, Jean Paul Paraiso, Tony So. Neil Winegarden, and Tim Westwood;

My whole fmily, especially my parents and Tim Gibson, who have been a constant source of encouragement, love, and understanding.

Table of Contents

Abstract

Acknowledgements

Table of Contents

List of Figures

List of Abbreviations

Chapter 1- General Introduction: The Heat Shock Response A. The Heat Shock Response: Discovery and Early Work B. The Heat Shock Proteins C. Heat Shock Factor D. Regulation of HSF Activation E. Transcriptional Activation of the Heat Shock Genes F. Negative Regulation of HSF Activity

G. Inducer Effects on HSF Activation H. Thesis Objectives

Chapter 2- Azetidine induces Heat Shock Factor DNA-binding and transcriptional activity in both saiivary gland cells and SL2 cells

A. Abstract B. Introduction C. Materials and Methods D. Results E. Discussion

Chapter 3- Heat shock and azetidine cause the relocaiization of RNA polymerase 11 to the heat shock genes resulting in the global repression of al1 other genes

A. Abstract B. Introduction C. Materials and Methods D. Results E. Discussion

Chapter 4- General Discussion A. Possible function(s) of HSF binding at sites other than the major

heat shock gene loci B. HSF binding plays a role in transcriptional repression C. HSF binding at ecdysone-inducible loci D. 1s there a universal inducing signal of the stress response? E- Future Directions

References

List of Figures

Figure 2-1 Five mM azetidine induces HSF binding and heat shock gene puffing in the 42 salivary gland polytene chromosomes of Drosophih in a tirnedependent manner-

Figure 2-2 Fi* mM azetidine induces heat shock gene puffing and HSF DNA-binding 46 in polytene chromosomes at earlier time points than 5 mM azetidine.

Figure 2-3 Azetidine activates HSF DNA-binding to HSEs in Drosophila SL2 cells. 50

Figure 2-4 Azetidine induces hsp70 gene transcription in Drcosophila SL2 ceiis, 53

Figure 2-5 Azetidine results in an apparent increase in HSF hyperphosphorylation. 56

Figure 2-6 Azetidine does not result in the accumufation of hsc70 on the chromatin. 60

Figure 3- 1 Azetidine induces the activation of heat shock gene transcription and the global repression of transcription tiom other genes.

Figure 3-2 Azetidine treatment causes hyperphosphorylated RNA Polperase II to redistribute to the heat shock gene loci.

Figure 3-3 Azetidine induces the redistribution of RNA Polymerase II to heat shock 85 gene loci in hsf mutants.

Figure 3-4 Azetidine induces the activation of heat shock gene transcription and the 87 repression of non-heat shock gene transcription in hsf mutants.

Figure 3-5 HSF binds to major developmental loci following in vivo heat shock. 90

Figure 3-6 Ecdysone puff regression and repression of transcription occur at 74EF and 93 75B in response to heat.

Figure 3-7 Ecdysone puff regression and decreased RNA Polymerase II staining are observeci at 74EF and 75B in response to heat,

Figure 3-8 Azetidine pretreatment shows only a modest effect on transcriptional repression at 74EF and 7SB.

Figure 3-9 Azetidine treatment results in transcriptional repression at 74EF and 75B following ecdysone induction.

List of Abbreviations

ATP AZC bp BrdU BrUTP BSA OC cDNA CN- CTD C- terminal ddHzO DEPC DNP D'IT EDTA EGTA EMSA FITC g grp h H2B HEPES HR HS or hs HSBP-1 hscs HSE HSF hsps M MAPK min ml mm mM mRNA N-terminal PAGE PBS PMSF Pol II Pro rPm

-adenosine triphosphate -azetidine-2-carboxylic acid (azetidine) -base pair -bromodeoxyuridine -brominated uridine triphosphate -bovine senun albumin -degrees Celcius -complimentary DNA -cyanide -carboxy terminal domain -carboxy tennind -double distilleci water -diethylpyrocarbonate -2,4-dinitrophenol -dithiothreitol -ethy lenediamine tetraacetic acid -ethyleneglycol-bis@-arninoethyl ether) N,N,N',N' tetraacetic acid -electrophoretic mobility shift assay -fluorescein isothiocyanate -gram(@ -glucose regulated protein -hour(s) -histone 2B -N-2-hydroxyethylpeperazine-N'-2-ethane sulfonic acid -hydrophobie heptad repeat -heat shock -heat shock factor binding protein -heat shock cognate proteins -heat shock element -heat shock factor -heat shock proteins -molar -mitogen activated protein kinase -minutes -millilitre -millimetre -rnillimolar -rnessenger RNA -amino terminal -polyacrylarnide gel electrophoresis -phosphate buffered saline -phenylmethylsulfonyl fluoride -RNA Polymerase II -praline -revolutions per minute

SDS SL2 TB 1 TBE TE fJ1 Crg

-sodium dodecylsuIfate -Schneider Line 2 -transcription buffer 1 -tris- borate-EDTA -tris-EDTA -microlitre -microgram

vii

CHAPTER 1

General Introduction

The Heat Shock Response

Al1 organisms are capable of responding to adverse fonns of stress in their environment

by evoking a highly conserved and regulated response at the molecular level. The heat shock or

'stress' response involves the rapid activation of heat shock transcription factor (HSF) and the

elevated expression o f heat shock proteins (hsps) which act to repair protein darnage resulting

fiom proteotoxic stress (reviewed in Welch, 1993; Parsell and Lindquist, 1994).

In addition to heat and a diversity of chexnical stresses in the environment (reviewed in

Nover, 1991), the synthesis of heat shock proteins are increased under conditions of fever,

inflamrnatory disease (Kautmann and Schoel, 1994), or ischemia (i3enjamin and Williams,

1994). The hsps also play a role in the immune response (Kaufiann, 1990; Young, 1990);

other pathophysiologicd States such as hypertrophy, neural injury, and aging (Morimoto et al.,

1 994); and in protection fiom stress-induced apoptosis (Kabakov and Gabai, 1997; Mosser et a[. ,

1 997; Buzzard et al., 1 998; Schett et al., 1999)-

The ubiquitous nature of heat shock proteins in biology, and the wide scope of inducers

which elicit a response, highlight the need for a complete understanding of how the heat shock

response is induced and regulated. More irnportantly, the fact that the response is inducible yet

widespread and conserved in virtually al1 organisms makes it an ideal system by which to study

the regulation of eukaryotic gene expression.

A. The Heat Shock Resportse: Discovery and Early Work

The heat shock response was &st described by Ritossa (1962, 1964a) who observed a

new pattern of puf'6ng in the salivary gland chromosomes of Drosophila busckii larvae in

response to heat shock, 2,4dinitrophenol, and sodium salicylate treatments. The stress-induced

puffs were shown to be sites of uridine incorporation, indicative of active transcription (Ritossa,

1964b). Almost ten years passed unhl Tissieres et al. (1974) discovered that heat shock induces

the rapid synthesis of a specific set of proteins, temeci the heat shock proteins, while the

synthesis of normal cellular proteins is greatly inhibited. Al1 prokaryotic and eukaryotic

organisms studied so far have dernonstrated this same response to elevated temperatures and also

a host of other inducers including amino acid analogues, heavy metals (e-g. cadmium), ethanol,

and sulfhydryl reagents (e-g. arsenite) (Nover, 199 1).

In addition to the inçreased synthesis of hsps, several morphological changes can be

observed in ceIIs following heat shock. Within the nucleus heat stress results in a change in

nucleolar morphology, a blockage in the export and assernbly of ribosomes, and the appearance

of actin containing filaments distributed throughout the nucleus (Welch and Suhan, 1985). An

increase in the number of perichromatin granules can also be observed (Welch and Suhan, 1985).

These are thought to be aggregates of unprocesseci RNA which form due to a breakdown in the

splicing process.

Within the cytoplasm heat shock results in the dismption of the cytoskeleton and the

collapse of the intermediate filament network to form large aggregates surrounding the nucleus

(Coss et al., 1982; Glass et al., 1985; Welch and Suhan, 1986). Concomitant with this, the

mitochondna swell and migrate to the perinuclear region (Welch and Suhan, 1986). Heat shock

also results in an increase in the number of lysosomes and the disruption and dispersal of the

Golgi complex throughout the cytoplasm (Welch and Suhan, 1985).

Several biochernical changes occur as a consequence of heat stress including: a rapid

decrease in intracellutar pH, a drop in intracellular ATP levels, and an increase in cytosolic

calcium levels (Stevenson et al., 198 1; Findly et ai., 1983; Weitzel et ai., 1985; Dnunmond et

al., 1986; Winegarden et al., 1996; Zhong et al., 1999).

B. The Heat Shock hoteins

Anfinsen (1973) originally proposecl that the folding of nascent polypeptides in vitro is a

spontaneous process, based solely on the primary structure of the protein. This hypothesis does

not reflect the in vivo situation where the intracellular protein concentration is hi& favouring

the aggregation of unfolded proteins (Georgopoulos and Welch, 1993). It is now known that heat

shock proteins act as rnolecular chaperones to mediate protein folding in the ce11 under nomal

growth conditions as well as under stress.

Heat shock proteins constitutively expressed within the cell at normal growth

temperatures are known as heat shock cognate proteins (hscs). The hscs are homologous to the

heat shock proteins with regard to both amino acid sequence and biochemical properties, and

account for 5-10 % of the total protein mass of the cell (Morirnoto et al., 1994; Craig, 1985).

Hscs play a role in protein synthesis, protein folding, protein degradation, and protein

translocation across intracellular membranes (reviewed in Craig et al., 1994; Frydrnan and Hartl,

1 994; Morimoto et al., 1994).

The hscs may be abundant within the unstressed ce11 but the level of constitutive protein

is not sufficient to deal with the effects of ce11 stress. The heat shock response is induced to

increase the concentration of heat shock proteins within the ce11 and is absolutely essential for

ce11 s u ~ v a l . Work by Craig and Jacobsen (1984) has shown that deletion of the hsp70 gene in

yeast renders cells heat-sensitive. Additionally, Riabowol er al. (1 988) demonstratecl that

injection of hsp-specific antibodies into mammalian cells resulted in the inability of the cells to

survive a heat shock. The phenornenon of "acquired therrnotolerance" fùrther demonstrates the

necessity of the heat shock response as a cellular defense mechanism. If cells are subjected to a

mild heat shock and are then allowed to recover, they are able to survive a subsequent heat shock

that would otherwise be lethal (Gerner and Scheider, 1975). Evidence shows that

therrnotolerance depends on the expression and h c t i o n of hsp70 (Li and Werb, 1982; Riabowol

et al., 1988).

The hsps are among the most conserved proteins in nature and show a high degree of

sequence homology between prokaryotic and eukaryotic forms (Craig, 1985; Lindquist, 1986).

Both constitutive and inducible hsps can be categorized into one of the following major groups,

dependent on the molecular weight of the protein: hsp100, hsp90, hsp70, hsp60, hsp40, srnail

hsps and ubiquitin.

hsp7O

The best characterized, and most prominently induced group of stress proteins is the

hsp70 family (Craig and Gross, 199 I ; ParseIl and Lindquist, 1994). Containing proteins that

range in size fiom 66 kDa to 78 kDa (Tavaria et al., 1996), the hsp70 proteins have been highly

conserved, exhibiting 60-78% identity among eukaryotic proteins and approximately 50%

identity between DnaK, the E. coli hsp70, and the eukaryotic forms (Lindquist, 1986).

Al1 eukaryotic cells contain multiple hsp70 family members. For exarnple, the human

hsp7O family consists of at l e s t 11 genes coding for both inducible and cognate proteins

(Tavaria et al., 1996). Human heat shock cognate proteins have various iniracellular locations

and include: nuclear and cytoplasrnic hsp73 (commonly refmed to as hsc70), grp78 (glucose-

regulated protein; also referred to as BiP) located inside the endoplasmic reticulum, and grp75

found within the matrix of the mitochondria (Welch, 1993). The inducible hsp70, designated

hsp72, is expressed at high levels under conditions of stress and localizes mainly within the

nucleolus (Welch and Feramisco, 1984).

The Drosophifa hsp70 family consists of 5 different genes, also coding for both inducible

and cognate proteins (Lindquist, 1986). Three different heat shock mgnate proteins have been

identified in Drosophifa: a major and a rninor 70 kDa species (hsc70), and also a 72 kDa species

(hsc72) (Palter et al,, 1986). Whereas the major hsc70 and hsc72 are expressed throughout

development, and are localized to the meshwork of cytoplasmic fibres swrounding the nucleus,

the minor hsc70 is only expressed in adult flies (Palter et al., 1986). The inducible members of

the hsp70 family in Drosophila include hsp70 and hsp68 (Lindquist, 1986). Hsp7O is virtually

undetectable at normal growth temperatures and can be induced more than 200-fold upon heat

shock (Velazquez et al,, 1983 ; Mason and Lis, 1 997). This is likely explaineci by the fact that the

Drosophila genorne contains multiple copies of the hsp70 gene, with two copies at the 87A locus

and three copies at the 87C locus (Ingolia et al., 1980). During heat shock, Drosophila hsp70

localizes within the nucleus and shows a specific association with the granulai- region and dense

fibrillar component of the nucleolus; areas involved in the assernbly of small ribonucleoproteins

and pre-ribosomes (Arrigo et al., 1980; Pelham, 1984; Velazquez and Lindquist, 1984; Morcillo

et al,, 1997). Hsp70 also associates specifically with the chromosomes (Amgo et a[., 1980;

Velazquez et al., 1980). Work frorn our laboratory has s h o w that both hsc70 and hsp70 bind at

the sarne 250 sites on the polytene chromosomes following heat shock, indicating that they have

the sarne targets on the chromatin (Kociuba, 1999). Approximately 64% of the hsp70 binding

sites CO-localize with binding sites of the heat shock transcription factor (Kociuba, 1999).

A11 hsp70 proteins consist of a conserved amino-terminal nucleotide-binding domain

(Flaherty et al., 1990) and a l a s conserved carboxy-terminal domain for binding substrates

(Hightower et al., 1994). Hsp70 proteins utilize the energy of ATP to transiently participate in

the process of protein maturation. Both hsp70 and hsc70 have been shown to interact with

proteins undergoing synthesis on the ribosome (Beckmann et al., 1990). Hsp70 is thought to

interact with the nascent polypeptide preventing folding until synthesis is complete, whereupon

hsp70 releases the protein (Welch, 1993).

Hsphsc70 interact with protein substrates via cycles of ATP binding and hydrolysis. In

the ATP-bound state, hsp70 will bind and release polypeptide substrates rapidly ( H d , 1996).

This process has been well characterized for the E. cdi hsp70 homologue DnaK (reviewed in

Frydman and Hohfeld, 1997). The cofactor D n d stimulates the ATPase activity of DnaK. In

the ADP-bound state the interaction of DnaK with the substrate is stable. The cofactor GrpE

binds to the DnaK ATPase domain, stimulating the release of ADP. Subsequent binding of ATP

results in the release of the substrate fiom DnaK. In eukaryotes, hsp40 (Hdjl) has been

characterized as the Dnal homologue (Minami et al., 1996).

Another important fùnction of cognate hsp7O is the transport of proteins between

compartments. BiP and grp75 have both been shown to interact with nascent polypeptides while

they are translocating into the lumen of the endoplasmic reticulurn and the mitochondrial matrix

(Gething et al., 1986; Kang et ai., 1990; Mizzen et al., 199 1 ; Zimmerman, 1998). BiP and grp75

bind to the peptide until translocation is over, preventing folding until the protein is completely

inside the organelle (Welch, 1993). Hsc7O is dso involved in the recyciing of clathnn coated

vesicles by actually releasing or 'bcoating" clathrin fiom the vesicles (Chappe11 et al., 1986).

In the stressed ce11 evidence suggests that hsp70 participates in the processes of acquired

themotolerance and repair of stress-induced protein damage (Tavaria et al., 1996). Work has

shown that hsp70, hsp90, and hsp40 fùnction cooperatively to renature damaged proteins in the

cytoplasm (Schumacher et al., 1996). Hsp7O also appears to fùnction in the repair of damage to

nuclear functions such as splicing (Vogel et al., 1995).

hsplOO

The highly conserved hsp 100 family is compnsed of proteins with molecular weights in

the range 104-1 10 kDa (Kabakov and Gabai, 1997) and c m be found in bacteria, yeast, plants,

and mammals (reviewed in Parsell and Lindquist, 1994). HsplOO proteins are heat-inducible

ATPases that usually contain two essential ATP-binding domains (Parsell et al., 1991; Parsell

and Lindquist, 1994).

Particularly wekl characterized is the yeast hsp lO4 protein which is required for the

induction of thermotolerance (Sanchez and Lindquist, 1990) and the resolubilization of protein

aggregates in yeast (Parsell et ai., 1994). There also seerns to be a fùnctionai relationship

between hsp 104 and hsp70, as hspl04 is also necessary for the restoration of splicing processes

following heat shock (Vogei et a[., 1995). Similarly when hsp70 levels are reduced, hspl04

becomes necessary for survival at hi& temperatures. When hspl04 levels are reduced, hsp70

becomes necessary for the acquisition of thermotolerance (Parsell and Lindquist, 1994).

hsp90

Heat shock proteins compnsing the hsp90 family fall in the range of 82-94 kDa and are

constitutively expressed in abundant proportions (Kabakov and Gabai, 1997). Stress results in a

3 to 5-fold increase in the levels of hsp90 (Welch, 1987). Members of the hsp90 family have

been observed in the cytosol and the endoplasmic reticulurn (grp94) (Caplan, 1999).

Similar to the hsp7O and hsplOO families, hsp90 is an ATPase with an amino-terminal

nucleotide-binding domain. However, binding of ATP to hsp9O is much weaker than for hsp70

(reviewed in Caplan, 1999). Hsp90 is not required for protein fotding, however it is capable of

stimulating folding by other molecular chaperones such as hsp7O and hsp40 (Schumacher et ai.,

1996).

Hsp90 plays a regdatory role for certain protein kinases and steroid hormone receptors

(reviewed in Csermely et ai., 1998). Binding of hsp90 to pp60src results in repression of the

protein tyrosine kinase (Brugge, 1986; Xu and Lindquist, i 990). Hsp90 also binds to steroid

hormone receptors such as the glucocorticoid receptor, thus maintainhg the receptor in an

inactive state. Binding of ligand to the receptor leads to disruption of the complex and thus an

active transcription factor (Beato, 1989; Pratt, 1993). Recent studies also suggest that hsp90 may

negatively regulate the activity of the heat shock transcription factor (Ali et al., 1998; Zou et al.,

1998). This will be discussed in the section entitled, "Negative Regulation of HSF Activity".

hsp60

Hsp6O proteins (and their bacterial counterpart, groEL) are the main components of the

protein folding chaperone machine, promoting the folding of newly translateci proteins in

bacteria and in the mitochondna and chloroplasts of eukaryotic cells. ln both prokaryotes and

eukaryotes, expression of hsp6O increases during heat shock (Parsell and Lindquist, 1994;

Kabakov and Gabai, 1997).

In prokaryotes, groEL utilizes the energy of ATP hydrolysis to promote protein folding in

cooperation with the CO-chaperone g o E S and also DnaK (Georgopoulos and Welch, 1993;

Frydman and Hartl, 1994). In eukaryotes, the ATPase activity of hsp6O is regulated by hsplO

(homologous to groES), and its chaperoning fùnctions are carried out in tandem with hsp7O

(Kabakov and Gabai, 1997).

hsp40

As has been described above, hsp40 is the DnaJ homologue in eukaryotic cells and plays

an important role in stimulating the ATPase activity of hsp70 (DnaK) (Minami et ni., 1996).

After heat shock hsp40 translocates into the nucleus and accumulates in the nucleoli,

colocalizing with hsp70 (Ohtsuka et al., 1993). Hsp40 likely assists hsp70 in the recovery of

nucleolar fûnction following heat shock (Ohtsuka et al., 1993). Recently, D d has been

implicated in the negative regdation of heat shock transcription f a o r activity (Shi et ai., 1998).

This will be discussed fùrther in the section entitled, 'Wegative Regdation of HSF Activity".

Smaii hsps

The small hsp family are the least conserved of al1 the hsp groups and are çompnsed of

proteins 15-28 kDa in sue. F o d in mycobacteria and aii eukaryotes, these proteins are M e r

subdivided into two groups: hsp27 (25-28 kDa hsps) and the crystallins (15-22 kDa hsps). The

small hsps are inducibly and constitutively expressed, and their level of expression changes

throughout development and the ce11 cycle (reviewed in Arrigo and Landry, 1994).

In unstressed marnmalian cells, hsp27 plays an important role in signal transduction to

actin microfilaments by inhibiting actin polymerization and depolymerizing F-actin (Miron et

al., 1991). Though hsp27 is not an ATPase it is still able to act as a chaperone, preventing

aggregation and accelerating protein folding processes (Amgo and Landry, 1994). In heat

stressed cells hsp27 may act to stabilize the actin cytoskeleton (Lavoie et al., 1993) and to speed

the repair of protein aggregates (Kampinga et al., 1994).

Ubiquitin

The ubiquitin systern provides a means, other than lysosorna1 degradation, for protein

degradation in eukaryotes. Protein substrates are targeted for degradation in the proteasorne by

conjugation with multiubiquitin (reviewed in Clechanover, 1994). First identified as a heat

shock protein in chicken ernbryo fibroblasts, ubiquitin synthesis increases after heat shock to

ensure that damaged proteins are degraded rapidly (Bond and Schlesinger, 1985; Parsell and

Lindquist, 1 994).

Other Stress Proteins

Heat shock and other forms of stress will sometimes induce oher proteins which receive

the designation of an hsp. These include: hemeoxygenase (hsp32) which is involved in an

antioxidant defense mechanism (Vile et al., 19941, a collagen-bindîng 47 kDa protein (hsp47)

which participates in procollagen processing and the assernbly of collagen (Nakai et ai., 1992),

and immunophilins or cyciop hilins w hich are peptidy 1 pro1 y l cis-tram i somerases (Gething and

Sambrook, I992)-

C. Heat Shock Factor

Transcription of the heat shock genes is activated by the inducible transcription factor,

heat shock factor (HSF). HSF is synthesized constitutively and is stored in an inactive,

monomeric form (Kingston et al., 1987; Zimarino and Wu, 1987). During penods of stress HSF

trimerizes to fonn the active transcription factor.

HSF was first identified in Drosophila as an activity that could specifically bind to the

regulatory site of the hsp70 gene. Wu ( 1984a,b, 1985) discovered a putative regulator that could

inducibly bind to the response elements in chromatin or f?ee DNA. Parker and Topo1 (1984)

uncovered an activity in heat shocked Drosophila ce11 extracts that bound the response element

of the hsp70 gene, and was active in in virro transcription assays. HSF was subsequently

purified fiom Drosophila (Wu et al., l987), yeast (Sorger and Pelharn, 1987; Weidemecht et al,,

1 988), and cultured human cells (Goldenberg et al., 1 988).

HSF in Drosophila and yeast are encoded by a single copy gene (Sorger and Pelham,

1 988; Wiederrecht et al., 1988; Clos et al., 1990). Al1 other higher eukaryotes possess multiple

HSFs; two in mouse (Sarge et al., 199 l), three in humans (Rabindran et al., 199 1 , Nakai et al.,

1997), and three in chicken and tomato (Scharf et al., 1990; Nakai and Moximoto, 1993). Of the

multiple HSFs expressed in vertebrates, HSF 1 is analogous to the inducible HSF found in yeast

and Drosophila (and fiom herein will be referred to as HSF). in yeast, HSF is an essential

protein for growth (Sorger and Peharn, 1988). In DrcosophiIa, HSF is dispensable for general

ce11 growth or viability, but it is required for oogenisis and early lamû development (Jedlicka et

al., 1 997). HSFS is activated fiom an ïnert dimer to a DNA-binding trimer during eady mouse

embryonic development, spermatogenesis, and in human erythroleukemia K562 cells exposed to

hemin (Theodorakis et al., 1989; Sistonen et al., 1992; Mezger et al., 1994; Sarge et al., 1994;

Rallu et al., 1997). Downregulation of the ubiquitin-dependent protein degradation machin-

has recently been shown to signal the activation of HSF2 DNA-binding activity (Mathew et al.,

1 998), however proteasome inhibition has also been shown to activate HSF l (Kawazoe et al.,

1998; Kim et al., 1999). HSF3 is activated in response to chemical and physiologicd stress and

has been shown to be essential for the heat shock response in avian cells (Tanabe et al., 1998).

HSF4 binds constitutively to DNA and shows a tissue-specific expression pattern in hwnan

heart, skeletal muscle, and brain (Monmoto, 1998).

Al1 HSFs have a conserved core structure composed of the DNA binding and

trimerization domains. Located in the amino-terminal portion of the protein, the DNA binding

domain of HSF consists of a winged helix-tuni-helix structure (Harrison et al., 1994). Situatecl

carboxy-terminal to the DNA binding domain is the trimerization domain, whïch consists of

three arrays of hydrophobic heptad repeats (HR-AB) (Sorger and Nelson, 1989; Clos et ai.,

1990). The hydrophobic residues located at the first and fourth position within each heptad

repeat are characteristic of a leucine zipper motif. These motifs are essential for trimerization of

HSF through the formation of a triple-stranded alpha-helical coiled-coi1 structure (Peteranderl

and Nelson, 1992; Zuo et al., 1994). A fourth conserveci hydrophobic heptad repeat (HR-C) is

located closer to the carboxy-terminus of HSF. Substitution of residues within the repeat for

non-hydrophobic amino acids results in constitutive trimer formation and DNA binding activity;

indicating that the C-terminal repeat suppresses trimerization, likely through interaction with

H R - A B (Rabindran et al., 1993).

The sequences in the C-terminal portion of the protein are widely divergent in dl HSFs

studied to date (Wu, 1995). The HSF transactivation domain has been mapped to the C-terminus

(Chen et of., 1993; Green et al., 1995; Shi et al., 1995; Zuo et al., 1995; Wisniewski et al., 1996)

and has been found to be acidic and highly potent, with the ability to fùnction even when fuseci

to a heterologous DNA binding domain (Borner et al., 1992). Sequences located between HR-

A B and HR-C negatively regulate DNA binding and transcriptional activation of HSF wieto-

Sotelo et al., 1990; Hoj and Jakobsen, 1994; Green et al., 1995; Shi et al., 1995; Zuo et al., 1995)

and it appean that this regulatory sequence alone is sufficient to sense heat stress (Newton et al.,

1996).

De Regdation of HSF Activation

The results of numerous studies show that HSF is subject to several key and complex

regulatory mechanisms (Wu, 1995). The first involves trimerization of HSF and the acquisition

of DNA-binding activity. Second, HSF acquires transcriptional cornpetence in a manner that is

separable fiom the acquisition of DNA-binding ability. This has been shown in studies using

inducers such as sodium salicylate, which activates HSF DNA-binding activity but not

transcription ( J u ~ v i c h et al., 1992; Winegarden et al., 1996; Bharadwaj et al., 1998). Third,

HSF is subject to negative regulatory mechanisms that result in deactivation or attenuation of the

response (Clos et al., 1990; Rabindran et al., 1991) and will be discussed in the section entitled,

"Negative Regulation of HSF Activity".

As previously mentioned, inactive monomeric HSF oligomerïzes to form a DNA-binding

trimer in response to stress (Westwood et al., 199 1; Bakr et al., 1993; Sarge et al., 1993;

Westwood and Wu, 1993). Intramolecular repression of the HSF monomer, resulting fiom

interaction of the N-terminal and C-terminal leucine zipper motifs (HR-NB and HR-C), is

relieved by stress without the assistance of regdatory proteins (Farkas et al-, 1998). In vitro

evidence suggests that HSF can be activated simpIy as a consequence of undergoing a

conformational change induced by stress in the extracellular environment. For exarnple, HSF

DNA-binding activity can be induced in unshocked celI extracts exposeci to elevated

temperature, low pH, hydrogen peroxide, or other protein damaging reagents (Mosser et al.,

I W O ; Zimarino et ai., 1 WOb, Becker et al., 1990; Zhong et al., 1998; Zhong et al., 1999). In

addition, Zimarino et al. ( 1 WOb) showed that polyclonal antibodies against active Drosophila

HSF result in activation of the protein. On the other hand, absolute environmental temperature

cannot be solely responsible for HSF activation. Other cellular factors must be involved. This is

indicated by the fact that human HSF expressed in Drosophila cells is 'reprogrammed' for

induction at the same temperature as Drosophila HSF; a temperature which is almost 10 OC

lower than normal. Conversely, Drosophila HSF expressed in hurnan cells is constitutively

trimeric in nature (Clos et al., 1993)

Following relief of intramolecular repression, the hydrophobic heptad repeats C-terminal

to the DNA binding domain interact in a triple-stranded coiled-coi1 structure to form active HSF

(Peteranderl and Nelson, 1992; Zuo et al., 1994). The exceptions to this rule are the budding

yeasts S. cerevisiae and K. lacfis. in these organisms HSF is found constitutively as an active,

DNA-binding trimer both in the presence and absence of heat stress (Jakobsen and Peham,

1988).

Pelham (1982) first reporteci the necessity of a 14-bp consensus sequence (5'-

CnGAAnnTTCmG-3') for the heat induced expression of the hsp70 gene in Drosophila.

Further work has shown that this enhancet-like sequence, called the heat shock element (HSE), is

highly conserved and consists of contiguous, altemating repeats of the 5-bp sequence, 5'-

nGAAn-3' (Amin et al-, 1988; Xiao and Lis, 1988). Active HSF binds at the HSE which is

found upstrearn and proximal to the promoter of al1 heat shock genes (reviewed in Lis and Wu,

1992). HSF binds with high affinity to HSEs that have from three to nine alternately oriented 5-

bp units, with each subunit of the HSF trimer binding to a single 5-bp unit (Perisic et al., 1989).

In addition, HSF trimers will bind cooperatively to adjacent HSEs (Topo1 et al., 1985; Xiao et

aL, 1991).

The phosphorylation of HSF in response to heat was first identifieci as a shifi in the

eIectrophoretic mobility of the yeast and human proteins following heat shock (Sorger and

Pelham, 1988; Larson er ai-, 1988). Sorger (1990) subsequently showed that heat shock results

in increased phosphorylation at serine and threonine residues, correlating with HSF activation.

The results from experiments with yeast suggested that HSF phosphorylation modulates the

expression of heat shock genes (Sorger and Pelham, 1988; Sorger, 1990). On the contrary, work

by Hoj and Jakobsen (1994) showed that HSF hyperphosphorylation may actually play a role in

the deactivation of HSF transcriptional activity.

In higher eukaryotes, research has now s h o w that inactive HSF is constitutively

phosphorylated and is inducibly phosphorylated in response to forms of stress such as heat or

cadmium sulfate (Sarge et al., 1993). Inducible phosphorylation is independent of DNA binding

and correlates with transcriptional activation (Cotto er al., 1996). This has been demonstrated

through the use of salicylate, whîch induces DNA binding but not transcriptional activity or

hyperp hosphorylation (Cotto et al., 1 996; Winegarden et al., 1 996). Whereas induciMe

phosphorylation is correlated with transcriptional activity, it does not appear to be required for

transcription to occur. Using the proline amino acid analogue azetidine, which does not induce

HSF phosphorylation, Sarge et al. (1993) showed that heat shock gene transcription still occurs

in treated cells- Wang and Lindquist (1 998) have found that hyperphosphorylation of HSF is not

required for hsp70 gene transcription in Drosophila embryos. Under conditions of steady state

3 3 -P IabelIing, Fritsch and Wu (1999) have demonstrated that Drosophila HSF does not show

hyperp hosphorylation upon heat shock. They did determine that there is an approximatel y qua1

increase in both phosphorylation and dephosphorylation of fiosophila HSF in response to heat,

predominantly at serine residues. Though they did not examine the role of increased

phosphorylation/dephosphorylation in regulating transcnptional activation of the heat shock

genes, Fritsch and Wu (1 999) did dernonstrate that increased phosphorylation/dephosphorylation

occumng with heat shock does not play a role in regulating the DNA binding activity of HSF.

Certain residues within the regulatory domain of hurnan HSF have now been identified as

potential sites for phosphorylation. Constitutive phosphorylation at two of these residues, senne

303 and serine 307, has been suggested to play an important role in repressing HSF activation at

control temperatures (Kiine and Morimoto, 1997). The kinases responsible for phosphorylating

these residues have been identified. Chu et ai. (1996) found that phosphorylation of serine 307

by mitogen activateci protein kinases (MAPK) of the ERIC family, primes for a secondary

phosphorylation event occurrîng at serine 303 by glycogen synthase kinase 3. Additional senne

phosphorylation occurs at residue 363 where both MAPK and rnembers of the protein kinase C

(PKC) family act, leading Chu et al. (1998) to propose that three different protein kinase

paîhways converge upon HSF to modulate transcriptional activity, including: MAPK, PKC, and

glycogen synthase kinase. Arnino acid substitution of any of these criticai serine residues,

particularly serine 307, results in the release of HSF fiom repression at control temperatures

(Knauf et al., 1 996; Xia et al., 1 998).

At least four seridthreonine residues have now been determined to be inducibly

phosphorylated in response to heat (Xia and V o e h y , 1997). inducible phosphorylation seerns

to increase the halGlife of the active trimeri possibly prolonging activity after heat shock (Xia

and Voellmy, 1997). Aside fiom these findings, more work is required to detennine the precise

role, if any, of hyperphosphorylation in the activation of HSF.

Over the past ten years there has been some debate over the subcellular localization of

inactive HSF. Results fiom numerous ce11 homogenization experiment have shown that HSF is

localized in the cytosolic fiaction of unstressed cells (Wu, 1995). This is likely explained by the

fact that inactive HSF can easily leak fiom the nucleus to the cytoplasm during homogenization

(Wu ef al., 1994). Many other studies show that HSF is always a nuclear protein, prior to and

afier stress. Westwood et al. ( 199 1 ) reveal, using indirect immunofluorescence, that HSF

difisely stains the Drosophila melanogaster polytene chromosomes under control conditions.

Following heat shock, HSF localizes at over 200 discrete sites on the chromatin, including the

nine major heat shock gene loci. Orosz et al. (1 996) have also reported the Drosophifa HSF to

be nuclear before and after heat stress. They have found that though HSF proteins lacking the

nuclear localization signal rernain in the cytoplasm, these HSFs can still show reversible heat

stress-inducible trimerization. Wang and Lindquist (1 998) have recently demonstrated that HSF

undergoes a developmentai relocalization to the nucleus in Drosophila embryos, that correlates

with the ability of the embryo to show heat-inducible hsp70 expression. HSF has also been

localized to the nucIeus pnor to heat stress in Xenopus oocytes and human cells (Mercier et al.,

1997, 1999; Jolly et al., 1999). Using indirect immunofluorescence and ce11 îransfection with

green fluorescent protein-HSF constmcts, Cotto et al. (1997) and Jolly et al. (1 997, 1999) have

investigated the subcellular localization of human HSF before and afkr heat shock. They

observed the relocalization of HSF fiom a diffuse nuclear staining pattern to fom distinct,

brightly staining HSF granules wîthin the nuclei-

E. Transcr#tional Activation of the Neot Shock Genes

In Drosophila, HSF binding to the HSE has been shown to activate hsp70 gene

transcription 200-fold (Mason and Lis, 1997). Transcriptional activation of such great

magnitude depends on cis elements in addition to the HSE at heat shock gene promoters. GAGA

factor is able to disrupt the nucleosome structure at the hsp70 promoter in an ATP-dependent

manner (Tsukiyarna et al., 1994). Shopland et al. (1995) have demonstrated that HSF access to

the hsp70 promoter depends on the ability of bound GAGA factor, TFIID, and a paused RNA

polyrnerase II (Pol II) molecule to maintain the promoter in an open configuration in the

uninduced state. This leaves the heat shock gene promoter, and the Pol II molecuIe that has

paused there after the synthesis of only 25 nucleotides (Gilmour and Lis, 1986; Rougvie and Lis,

1988; O'Brien and Lis, 1991), primed for a rapid response to stress.

HSF has now been shown to interact directly with the TBP (TATA binding protein) core

of TFIID, binding cooperatively at the hsp70 promoter (Mason and Lis, 1997). The site of

interaction between HSF and TBP is the same site that allows TBP to interact directly with the

acidic H-domain of Pol II. Mason and Lis (1997) hypothesize that HSF triggers hsp70

transcription by fieeing the paused Pol II from its association with general transcription factors

such as TFIID.

Once paused Pol II bas been released and transcription of the hsp70 gene resumes,

initiation of subsequent rounds of transcription is more rapid (O'Brien and Lis, 1991).

Sandaltzopoulos and Becker (1998) have recently discovered that this occurs because HSF

increases the reinitiation rate by helping to set up another preinitiation complex a h promoter

clearance by Pol II, In addition, Brown and Kingston (1997) suggest that HSF can direct the

disruption of downstream chrornatin in order to facilitate transcriptional elongation.

Other factors have been reporteci to bind at the hsp70 promoter, influencing basal

transcription. These include CCAAT-box-binding factor (Greene et al., 1987; Morgan et al.,

1987; L m et al., 1990) and Sp 1 (Greene et al., 1987; Morgan, 1989). In addition, a constitutive

heat shock element-binding factor, now identifieci as Ku autoantigen, has been reported to inhibit

heat-induced activation of the hsp70 gene (Liu et al., 1993; Kim et al., 1995; Yang et al., 1996a,

1996b). HSF has been shown to bind selectively to the Ku protein in vitro, and Ku-related

antigens have also been shown to associate with transcriptionally active loci in the polytene

chromosomes of Chironomus (Gorab et al., 1996; Huang et al., 1997).

With the transcriptional activation of HSF, transcription is blocked at previously active

sites and is greatl y upregulated at the heat shock gene loci in Drosophila (Ashbmer and Bonner,

1 979). This phenornenon appears to be unique to Drosophila, as the global repression of non-

heat shock gene transcription is not observed to occur to such a great degree in mamrnaiian cells

(Hyrcza and Westwood, unpublished observations). In addition to regulation at the level of

transcription, several translational controls exist for the stress response. Preexisting mRNAs are

blocked fiom translation via the inhibition of initiation and elongation, and are not degraded but

rernain stable in the nucleus (E3alinger et ai., 1983; Petersen and Lindquist, 1988; Yost et al-,

1990). In addition, polysomes are cleared of preexisting rnRNAs to facilitate the exclusive

translation of nascent heat shock mRNA (MirauIt et al., 1978). It has also been shown that hsp

rnRNAs are especially stable. Heat stress results in a 10-fold increase in the stability of hsp70

mRNA in HeLa cells (Theodorakis and Morimoto, 1987). Severe heat shock results in the

blockage of splicing (Yost and Lindquist, 1986; Yost et al., 1990). However, this blockage c m

be prevented by subjecting cells to a mild heat pretreaûnent which results in the accumulation of

hsps and thus the acquisition of thmotolerance (Yost et al., 1990; Yost and Lindquist, 1991).

These findings indicate a role for hsps in the protection of the splicing machinery and in fact

hspl04 in yeast has now been s h o w to help recover the splicing process (Yost and Lindquist,

199 1).

Upon return to normal temperatures, the preexisting mRNAs return to translation at the

same rate whereas the repression of heat shock mRNAs occurs at different rates. For example, in

Drosophila the restoration of normal protein synthesis occurs concomitantIy with the repression

of hsp70, the first heat shock mRNA to be translationally silenced. In contrat to hsp70, hsp82 is

the last heat shock mRNA to be translationally repressed (DiDomenico et al., 1982a). Upon

recovery heat shock mRNA is rapidly degraded. However, this degradation occurs only when a

certain level of heat shock protein has built up. Evidence for this cornes fiom the fact that

mRNAs are stable indefinitely when cells are treated with amino acid analogues, which result in

the production of non-fimctional hsps (DiDomenico et al., 1982a).

F. Negative Regulation ofNSF Acîivity

Exposure of the ce11 to elevated temperature for an extended period of time results in

attenuation of the heat shock response. Bound HSF is released fiom the HSE even in the

continuai presence of stimulus, thus resulting in a decrease in heat shock gene transcription

(Abravaya et al., 1991). This phenornenon can be directly observeci on Drosophila salivary

gland ce11 polytene chromosomes via indirect i~~munofluorescence. Before heat shock, HSF

exhibits a diffise staining pattem over the entire chromatin. A 15 minute heat shock results in

HSF binding at over 200 loci (Westwood et al., 199 1, personal observations). After an extended

heat shock of 120 to 1 80 minutes, the distinct banding pattern is no longer recognized, and the

diffuse staining of the chromatin rehrms, even in the presence of continual heat stress (Wu et al.,

1994; Kociuba, 1999). The return of active HSF tu the inactive monomeric form, even though

stimulus is present, indicates that HSF is under the control of some sort of negative regulatory

mechanism (Wu et al., 1994).

On the contrary to heat shock, treatrnent of cells with amino acid analogues produces

different effects with regard to attenuation. Analogues activate heat shock gene expression by

incorporation into nascent polypeptides, resulting in the production o f abnomal proteins. Under

these conditions, attenuation of the heat shock response is not observed (DiDomenico et aL,

1982b). Because amino acid incorporation also leads to the production of nonfùnctional hsps,

the finding by DiDomenico et al. (1982b) led to the suggestion that the cellular factors involveci

in deactivating HSF during attenuation are hsps themselves.

Craig and Gross (1991) have derived the "cellular therrnometer" model to explain how

hsps, in particular hsp70, might negatively regulate HSF under normal conditions. The model

follows that heat shock results in the depletion of the available pool of hsps to deal with

denatured and aggregated proteins. This results in the relief of repression of the HSF monomer

by hsp70, fieeing HSF to activate heat shock gene transcription. Once a sufficient level of hsps

have built up again within the cell, hsps repress HSF activity. In short, Craig and Gross (1991)

propose that cells sense changes in temperature as decreased levels of hsp70 within the cell.

There is a body of evidence supporting this model. Ananthan et al. (1986) and Mifflin

and Cohen (1994a) found that injection of denatured protein into unstressed Xenopus oocytes

results in the activation of HSF. The injected denatured protein would act to deplete preexisting

reserves of hsp70, leading to induction of the response. On a parallel note, Mifflin and Cohen

(1994b) found that induction could be attenuated by co-injection of hsc70. Further evidence

comes from experiments involving the artificial manipulation of intracellular hsp levels. HSF

activation following heat shock was found to be greatIy reduced in cells containing high levels of

hsp70 fiom a previous heat shock (Baler et al., 1992), or in cells overexpressing hsp70 (Mosser

et al., 1993; Baler et a/., 1996). Lastly, hsp70 has been found in a complex with active HSF in

vitro (Abravaya er a/., 1992; Mosser et a!., 1993), and with inactive HSF in HeLa cells (Baler et

al., 1996).

There is also evidence pointing against a model where the accumulation of hsp70 (or

other hsps) alone deactivates HSF. Although some assays have shown that HSF does form a

complex with hsp70, hydrodynamic studies have indicated that Drosophila HSF and human HSF

do not form stable complexes with hsp70 or other proteins (Westwood and Wu, 1993; Sistonen

et al., 1994). Rabindran et af. (1994) found hsp70 to associate with both active and inactive

HSF. However, the interaction was insufficient to suppress the activation of HSF DNA-binding

activity in vivo. Locke and Tanguay (1996) have found increased activation of HSF in type I rat

muscle fibre, which constitutively overexpresses hsp70. This particular finding goes directly

against the cellular thermometer model, which would have cells overexpressing hsp70 showing

increased HS F deactivation.

Taking d l evidence into account, the consistent result arising is that deactivation of HSF

is accelerated by increased levels of hsps in vivo (Adosser et al., 1993; Rabindran et al., 1994).

The current view is that the regulatory role of hsp70 is to deactivate active HSF. Recent evidence

supports this notion. Shi et al. (1 998) found that hsp70 and hsp4OIhdj- 1 interact directly with the

transactivation domain of HSF. Fwther, they determined that the repression of transcription

occumng with attenuation is a direct result of the association of hsp7Ohdj-1 with the HSF

activation domain, Hsp90 has recently been implicated as a negative regulator of HSF, but its

role remains uncertain as it has been reporteci to associate with both the active and inactive fonns

of HSF (Ali et al., 1998; Duina et al., 1998). Others have found an association only between the

inactive form of HSF and hsp90 (Zou et al., 1998). Lastly, a novel protein and potential negative

regulator of HSF, was discovered using a yeast two-hybrid assay. Heat shock factor binding

protein 1 (HSBPI) interacts specifically with the hydrophobie heptad repeats in the HSF

trimerization domain and has been s h o w to negatively regulate DNA-binding activity (Satyal et

al., 1998). Overexpression of HSBP 1 in mamrnalian cells represses the transactivation activity

of HSF (Satyal et al,, 1998).

The most current mode1 for the negative regdation of HSF activity incorporates al1 of the

most recent findings (reviewed in Kabakov and Gabai, 1997; Morimoto, 1998). HSF is

maintained as a monomer through transient interaction with hsp70 and hsp90. Activation of

HSF would occur following depletion of the tiee hsp pool, and diversion of hsp70 and hsp90

from the HSF monomers. During attenuation of the response, binding of hsp70 and hdj-1 to the

HSF activation domain results in repression of heat shock gene transcription. HSBP 1 binding to

HSF trimers and to hsp70/hsp90 would lead to dissociation of the trimer, and the reappearance of

H S F monomers.

G. Inducer Effects on HSF Activation

There has always been great interest on the part of heat shock researchers as to how the

extracellular stress signal is transduced to activate HSF. As describeci above, the majority of

evidence indicates that hsps might be the primary sensors of stress. From this follows an

explanation as to why so many different inducers, hctioning at normal growth temperatures,

are able to activate heat shock gene transcription. In addition to heat, inhibitors of oxidative

respiration, amino acid analogues, detergents, heavy metals and ohers are al1 proteotoxic

chernicals able to affect protein folding and to induce protein aggregation within the ce11

(Kabakov and Gabai, 1997).

However, some evidence also indicates thôt HSF can directly sense stress in the

environment. HSF translated in vitro can be activated by heat shock (Mosser et al., 1990), as can

recombinant mouse and human HSF (Goodson and Sarge, 1995; Larson et al,, 1995), and

punfied Drosophila HSF (Zhong et aL, 1998). In addition, purïfied Drosophifa HSF shows

reversible activation by oxidation with hydrogen peroxide (Zhong et al, 1998) and conditions of

low pH in the physiological range (Zhong et al., 1999). Heat stress and a nurnber of chernical

inducers cause a decrease in intracellular pH. Zhong et al. ( 1999) propose that these inducers are

able to activate HSF directly via the decrease in pH. Somehow the synthesis and/or activity of

heat shock proteins helps to reverse the effects of heat or low pH, leading to the inactivation of

HSF.

The results of the above studies make one thing clear: not every inducer activates HSF in

exactly the sarne manner. The bulk of evidence to date suggests many of the inducers converge

on a common pathway leading to unfoldeci proteins and protein aggregates within the cell. Some

inducers may be able to bypass this pathway by acting directly upon HSF.

H. Thesis Objecn'es

Westwood et al. (1991) have shown that HSF bhds at over 200 sites on Drosophilu

salivary gland polytene chromosomes following heat shock. Since the major heat shock genes

are located at only 8 of these binding sites, a long standing interest has been to detennine the

function of HSF binding at so many additional loci. Westwood et al. (199 1) speculated that some

of these sites might contain 'minor' heat shock genes that are stress-inducible, but have not yet

been identifid as heat shock genes, In accord with this ou. lab bas found Pol II to preferentiaiiy

relocate to approximately 40 sites following heat stress, including the major heat shock gene

puffs (Paraiso and Westwood, unpublished results). This fïnding correlates with the fact that the

transcription of previously active genes is repressed in Drosophila following heat stress

(reviewed in Ashbumer and Borner, f 979). We hypothesize that HSF binding induced by heat

shock results in the preferential recniitment of Pol II to the heat shock genes and subsequently,

transcriptional repression of non-heat shock genes. Alternatively, transcriptional repression

observed in response to heat could be due to a secondary effect of heat itself, with heat shock

gene transcription continuing because of some special charact eristic of the heat shock genes, or

due to some other protein factor in addition to HSF. In order to discount this second possibility,

we wanted to use an inducer other than heat to activate HSF. The proline amino acid analogue

azetidine was selected and Chapter 2 focuses on the characterization of the stress response

induced by azetidine in Drosophila melanoguster, including the activation of HSF DNA-binding

and heat s hock gene transcription.

In Chapter 3 attention was tumed to two main questions: (i) what is HSF doing at sites

other than the major heat shock gene loci in response to stress, and (ii) does activation of HSF

binding play a role in global transcriptional repression? In order to answer these questions we

examined the preferential recniitment of Pol II to the heat shock genes, and subsequent

transcriptional repression at sites other than the major heat shock gene loci in response to both

azetidine and heat. To look specifically at repression of actively transcrïbing non-heat shock

genes, the ecdysone-inducible loci 74EF and 75B were examined.

CHAPTER 2

Azetidine induces Heat Shock Factor DNA-binding

and hanscriptional activity in 60th salivary

gland cells and SL2 cells

A. ABSTRACT

In this study we have shown that the proline amino acid analogue, azetidine-2-carboxylic

acid (azetidine) (5-50 mM), induces activation of HSF DNA-binding in both Drosophila

melanoguster salivary gland cells and SL2 tissue culture cells in a manner similar to heat but on

a longer time scale. Concomitant with HSF binding, prominent heat shock gene puffs were

observed on the polytene chromosomes. Correlating with these findings, azetidine induced

transcription of the hsp70 gene in SL2 cens. Azetidine treatment resulted in an apparent increase

in HSF hyperphosphorylation. However, nurnerous hyperphosphorylated HSF isofonns were

observed making the data difficult to interpret and preventing a firm conclusion about how

azetidine affects the Drosophila HSF phosphorylation state. Finaily, hsc70 was not observeci to

localize on the polytene chromosomes following azetidine exposure, indicating that the way in

which hsc70 is targeted to the chromatin in response to heat does not function in azetidine-

treated cells. Characterization of the response to azetidine has given us a usetùl tool for isolating

the potential secondary effects that heat might exert on the stress response in Drosophila.

B. INTRODUCTION

Key experiments by Kelley and Schlesinger (1978) and Hightower (1980) first

dernonstrateci the ability of amino acid analogues to induce the synthesis and accumulation of

heat shock proteins (hsps). Given the fact that amino acid analogues result in the production of

misfolded proteins (Fowden et al., 1967), the hypothesis arose that the upregulation of hsp

synthesis fùnctioned to help the ce11 deal with the accumulation of such abnormai polypeptides

(Hightower, 1980; Goff and Goldberg, 1985; Ananthan et al., 1986).

Amino acid analogues had a significant role in early work on the heat shock response.

For example, when cells were treated with amino acid analogues Beckmann et al. (1992)

observed that the analogue-containing proteins were not released fiom their chaperones. This

was in contrast to the situation in the normal cell, where chaperone-substrate interactions are

transient. It was presumed that the analogue-incorporated proteins were not released because

they were inherently unable to fold. These findings suppon the mode1 whereby the heat shock

response is induced due to a gradua1 depletion of the intracellular hsp70 reserves, as fiee hsp70 is

used to associate with abnormal protein (Craig and Gross, 199 1).

Heat shock proteins produceci in response to analogue treatment have amino acid

analogues incorporateci themselves, rendering them nonfunctional as chaperones (Li and Laszlo,

1985). DiDomenico et al. (1 982b) observed that the stress response is constitutive in the

presence of analogue, with cells displaying a lack of attenuation. Fwther, ceils treated with

arnino acid analogues are never able to acquire themotolerance (Li and Laszlo, 1985). These

findings lend support to the theory that the response is autoregulated by the hsps themseives.

DiDomenico et al. (1982a) also found that if cells were treated with arnino acid analogues, heat

shock messages were stable indennitely. This demonstrated that degradation of heat shock gene

mRNA occurs only once a certain level of functionai hsp has built up within the cell.

In contrast to heat shock, where a response is initiated within minutes, induction by

arnino acid analogues has been found to be a slow process due to the initial period of protein

synthais required (DiDomenico et al., l982b; Thomas and Mathews, 1984). Following this

period the cellular machinery is redirected to the alrnost exclusive production of heat shock

proteins, resulting in a robust response (Thomas and Mathews, 1984). Amino acid andogues

stoichiometrically replace the corresponding amino acid during protein synthesis, dependhg on

the affinity of the analogue for the normal amino acid tRNA synthetase (Fowden et al., 1967).

Some arnino acid analogues are incorporateci to a similar degree as their normal counterparts.

For example, azetidine-2-carboxylic acid (azetidine, AzC), a proline amino acid analogue, has

been shown to replace up to 95% of proline residues in mung bean proteins (Fowden and

Richmond, 1963). In addition to the efficient nature of incorporation, the presence of azetidine

in a polypeptide will result in a turn of a-hekal structures through an angle 15" smaller than

when proline residues are present (Fowden and Richmond, 1963). This adjustment in secondary

structure results in the destruction of the tertiary form of the protein.

Previous studies using a variety of different marnmalian ceIl lines (Thomas and Mathews,

1984; Li and Laszlo, 1985; Mosser et al., 1988; Kerendian et ai., 1992; Lai et al., 1993; Sarge et

al., 1993), E-coii (Kanemori et al., 1994), and soybean seedlings (Lee et ai., 1996) have

dernonstrated hsp synthesis in response to azetidine. Heat shock gene transcription in response

to azetidine has been analyzed in HeLa cells (Thomas and Mathews, 1984; Mosser et al., 1988)

and soybean seedings (Lee et al., 1996). Azetidine treatrnent has also been demonstrated to

result in a lack of HSF hyperphosphorylation in mamalian cells (Sarge et al., 1993). This is in

contrast to heat shock which has been s h o w to induce hyperphosphorylation of yeast (Sorger

and Pelham, 1988) and human HSFs (Larson et al., 1988; Sarge et al., 1993; Cotto et al., 1996;

Xia and Voellmy, 1997). In Drosophila, Fritsch and Wu (1999) have found that HSF is not

hyperphosphorylated in response to heat, but rather undergoes approximately equal increases in

both phosphorylation and dephosphorylation on various serine residues. The effect of azetidine

on HSF in Drosophila has not yet been examined.

Recent work bas shown that azetidine will induce the formation of HSFl granules in

human cells (Cotto et al., 1997; Jolly et al., 1999). Another recent study presents results which

conflict with those acquired using azetidine in al1 other systems. Bharadwaj et al- (1998) have

shown that azetidine does not result in HSF binding to the heat shock element in Xenopus

oocytes.

In this study the ability of azetidine to induce the Dmsophila heat shock response was

examined and characterized. Azetidine (5-50 mM) was found to induce activation of HSF DNA-

binding activity in both Drosophila melanogaster salivary gland cetls and SL2 tissue culture

celis, concomitant with prominent heat shock gene puffing on the polytene chromosomes.

Correlating with HSF DNA-binding and heat shock gene puffing, azetidine induced transcription

of the hsp70 gene in SL2 ceils. Immunoblot analysis indicated an apparent increase in the level

of hyperphosphorylated HSF afier azetidine treatment. However, numerous

hyperphosphorylated HSF isoforms were observed in the azetidine-treated conditions, making

the data difficult to interpret. At this point we can not make a fim conclusion about how

azetidine treatment is affecting the phosphorylation state of Drosophila HSF.

Heat shock resuits in the colocalization of hsp70 and hsc70 on the polytene chromosomes

of Drosophila salivary gland cells. In addition, hsc70 has been found to bind chromatin in the

absence of hsp70 synthesis (Kociuba, 1999). Kociuba (1999) has suggested that both hsp70 and

hsc70 function to protect DNA-bound proteins, or the chromatin itself. Knowing that heat

induces hsc70 to Iocalize to the chromatin in the absence of hsp70, we wondered if hsc70 would

relocate to the chromatin in the presence of non-functional hsp70 resulthg fiom azetidine

treatment. Following azetidine exposure, hsc70 was not observed to accumulate on the polytene

chromosomes. This indicated that the way in which hsc70 is targeted to the chromatin in

response to heat does not fùnction in azetidine-treated cells.

Aside fiom the inability of azetidine to induce the localization of hsc7O to the chromatin,

the analogue was able to stimulate HSF DNA-binding and hsp70 transcription in a manner

similar to heat shock but on a longer time scale. Given these results, azetidine will prove to be a

usefiil inducer for ruling out potential secondary effects of heat on the DrosophiIa heat shock

response, as will be discussed in Chapter 3-

C. MATERIALS AND METHODS

Fly Stocks

Drosophila melanoguster (Oregon R) were raised on yeast-glucose medium (10% w/v

glucose, 10% w/v instant yeast, 2% w/v bacterîological agar, and 0.7%wlv p-hydroxybenzoic

acid methyl ester (Tegosept, Sigma)) covered with a thin layer of Ward's instant h o p h i l a

medium (cat. #38W0592) and instant yeast. To assist in the staging of third instar larvae, the

medium was hydrated with bromophenol blue solution (0.05% w/v in double distilled water)

which stains the gut of the larvae blue (Bainbndge and Bownes, L98 1).

Salivary Gland Treatments and Chromosome Squashes

Salivary glands were dissected fiom third instar larvae in modified TB 1 buffer (15 mM

HEPES (pH 6 .Q 80 rnM KCl, 16 rnM NaCl, 5 rnM MgC12, 1% polyethyleneglycol 6OûO

(Myohara and Okada 1988)). Glands were incubated for 1 h at 2 1 OC in a humidifiai chamber.

For chemical treatments glands were tramferrecl to a depression slide containing the appropnate

concentration of azetidine-2-carboxylic acid (Sigma), proline (Sigma), or cycloheximide (Sigma)

( 1 18 PM, Zimarino et al. ( 1990a)) dissolved in TB 1 buffer. For controls, glands were left at

roorn temperature (21 OC) in TB 1 buffer or were msferred in 100 pl TM buffer to a

microcentrifuge tube which was then submersed in a temperature-controlled circulating water

bath (Neslab RTE-21 1) set at 36.5 OC. Treated glands were fixed for 2 min in 20 pl of fixative

(50% acetic acid, 3.7% fonnaldehyde) on a siliconized (Sigrnacote, Sigma) coverslip and

squashed on a microscope slide. Preparations were fiozen in liquid nitrogen, the coverslips were

removed with a razor blade, and the slides were stored in coplin jars containing 95% ethanol.

HSF ImmunolocafiZatrOn on Polyene Chromosomes

Slides were washed in g l a s coplin jars with phosphate-buffered saline (two washes, 30

min each). Each slide was then incubated for 30 min with BTP (10% BSA, 1% Tween 20 in

PBS) in a humid chamber. Slides were incubated for 1 h with polyclonal HSF anti'body (943,

Westwood et al., 1991) diluted 1 : 1000 in BTP- Slides were then washed twice in coplin jars

containing PBS/O.Ol% Tween 20, for 10 min each wash. Preparations were incubated with a

1:200 dilution of fluorescein isothiocyanate (F1TC)-conjugated goat anti-rabbit secondary

antibody (Cappel, cat. #55655) for 30 min. The wash step was repeated with PBS/O.OI% T w e n

20 for 10 min. DNA was stained ushg Hoechst 33342 (1 pg/ml in BTP, 15 min incubation).

After a final wash in PBS/O.Ol% Tween 20, the slides were mounted in 20 pl of antifade (1

mg/ml phenylenediamine, 70% glycerol in PBS) and covered with a coverslip. The edges of the

coverdip were sealed with nailpolish, and the slides were stored at -20 OC. Images were

captured on 35 mm Fujichrome Sensia 400 ISO slide film using a Nikon Microphot fluorescence

microscope and a Nikon Plan 40X objective. Exposure tima were 6 seconds for Hoechst

staining (with neutral density filters) and fiom 22-24 seconds for FITC staining. Thirty-five

millimetre slides were digitized using an Agfa Arcus II scanner, and when necessary adjusted for

brightness and contrast using Adobe Photoshop.

Assignment of Cflological toc i

Cytological locations of heat shock genes and ecdysone-inducible genes were determined

by referring to banding patterns observai on photographic chromosome maps (Lefevre, 1976;

Lindsley and Zimm, 1992). In addition, HSF banding patterns were compared with previously

deterrnined HSF binding si tes (Westwood et al., 1 99 1).

hsc70 linmunofocaIi~*on on Polyîene Chromosomes

The procedure was the same as descn'bed for HSF immunolocalization with the following

changes: the slides were stained with 1) the monoclonal primary antiîody 3a3 ( ~ f l b i t y

Bioreagents, cat. #MM-006), which recognizes Drosophila hsc70, diluted 1 : 1 0 0 in BTP, 2)

Alexa 488 goat anti-mouse IgG (H + L), F(ab')2 fragment conjugate (Molecular Probes cat. #A-

1 10 17) diluted 1 :200 in BTP, Images were captured on Kodak Tri-X 400 ISO black and white

negative film using a Nikon Microphot fluorescence microscope and a Nikon Plan 40X

objective. Exposure times were 0.25 second with neutral density filter 4 for Hoechst staining and

22 seconds for Alexa 488 staining. Negatives were digitized using a Nikon CoolScan III scanner

and were coloured and adjusteci for brightness and contrast using Adobe Photoshop.

Tissue Culture and Cell Treatments

Schneider line 2 (SL2) celts were grown at 2 1 OC in CCM3 media (HyClone) plus 20

p g h l gentamicin (Sigma) in T-75 tissue culture flasks (Starstedt). Cells were grown to a

maximum density of 1 .O x IO' cells/ml, and pnor to experïments were aerated by shaking for a

minimum of 3 h at 170 rpm, 2 1 OC. Cells were pelleted by centriQing at 7000 rpm for 2 min at

4 OC in a table top centrifuge. Media was removed and replaceci with physiological Drosophila

saline (45 rnM potassium glutamate, 45 mM sodium glutamate, 8.7 mM MgS04, 5.0 mM Bis-

Tris, 6.8 mM CaC12-H20, 12 @itre glucose, pH 6.9). Aeration continued for at least one

additional hour. For chernical treatments, the appropriate volume of azetidine was added directly

to ce11 suspensions and aeration at 170 rpm, 21 OC was maintainecl. Room temperature sarnples

were taken fiom cells that had been aerated for 4 h or more. Cells were heat shocked by

submersing the tube in a temperature-con~olled circulating water bath set at 36.5 OC (Neslab) for

20 min.

Protein Extracts for Mobifiîy Skifi Assays

One millilitre cell samples were transfmed to a 1.5 ml microcentrifiige tube and pelleted

at 7000 rpm, 4 OC, for 2 min in a Beckman microcentrifiige. Supernatants were removed and the

ce11 pellets fiozen under liquid nitrogen and stored at -72 OC. Cells were thawed and resuspended

in five pellet volumes of lysis buffer (10 mM HEPES (pH 7.9), 0.4 M NaCl, 0.1 mM EGTA,

5.0% glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsuifonyl fluoride). After ce11 Iysis,

the mixture was centrifugeci at 1 0 000 x g for 10 min at 4 OC. Supernatants were transfmed to

new tubes and stored at -72OC.

Electrophoretic Mobiiity Shii Assays

An HSE consensus sequence (HSE3; 5'-GGG CGT CAT AGA ATA TTC TCG AAT

TCT GGG K A GG-3') was anneale. to a shorter complimentary oligonucleotide (S'-CC TGA

CCC AGA ATT CGA G-3'). The overhang was filied using 2.5 units of Klenow (New England

Biolabs), 0.1 67 mM each of dATP, dTTP and dGTf (Boehringer Mannheim), and 50 pCi of a-

3 2 ~ dCTP (3000 Ci/mmol, Amersham). HSE labelling reactions were carried out at 25 OC for 30

minutes. Following incubation 1 pl of 1 mM dCTP was added and incubated for an additional 5

minutes at 25 OC as a chase step. Reactions were stopped by adding 12 pl of Exo III stop buffer

( 1 .O % SDS, 20 mM EDTA). Unincorporated nucleotides were removed on a Sephadex G-25

(Sigma) spin column (Bio-Rad). Gel shift assays were performed essentially as described in

Zimarino and Wu (1987). 5 pl of Drosophila SL2 ce11 extract was mixed with 6.1 pl of reaction

rnix containing: 3 pl ddH20, 1 pl 10X buffer mix (100 mM HEPES pH 7.9,500 rnM NaCl, 30%

V/V glycerol), 1 pl 1OX BSNnucleotide mix (0.5 mg/ml E. coli DNA, 0.2 mg/ml poly d(&, 2

mg/ml yeast tRNA, 20 mg/ml BSA) and 0.1 pl "P labelled HSE3 (0.01 prnol). Afk a IS

minute incubation period on ice, 2 pl of 6X Ioading dye (0.25% bromophenol blue, 0.25%

xylene cyanol, 30% v/v glycerol, 3X TBE) was added to each sample. Reactions were

electrophoresed in a 1% agarose gel (Seakem ME) for 1.25-1.5 h at 82V in 0.5X TBE buffet.

Gels were blotted and dried onto Whatman DE8 1 paper and exposed to preflashed Kodak XRP-1

film with a Cronex Lightning Plus Intensiwg Screen (Du Pont) at -72 OC.

RNA Ektra~n'on

Ce11 preparations were made from SL2 cells as dacribed for electrophoretic mobility

shifl assays. RNA was extracted fiom S U cells using the RNeasy RNA extraction kit (Qiagen),

according to the manufacturer's instructions. RNA was quantified by spectrophotometry

and Azso) and used immediately for primer extension andysis.

Primer Extension Anafysis

The analysis was carried out using '*P end-labelled hsp7O oligomer (5'-CCC AGA TCG

ATT CCA ATA GCA GGC-3') and -"P end-labelled H2B oligomer (5'-GCC TTT CCA CTA

GTT TTC GGA GGC-3'). The labelling reactions were comprised of: oligomers (50 pmol), 1 pl

10X T4 buffer (New England Biolabs), 10 units T4 polynucleotide kinase (New England

Biolabs), 30 pCi y - 3 2 ~ dATP (3000 CUmrnol, Amersham), and RNAse-fiee ddHIO to bring the

volume to 10 pl. Reactions were incubated for 1 h at 25 OC and were then stopped with the

addition of 2 pl 0.5 M EDTA, 1 pl tRNA (10 &pl) and 37 pl TE. The enzyme was heat

inactivated at 65 OC for 5 min. Unincorporated nucleotide was rernoved using a Sephadex G-25

(Sigma) spin column (Bio-Rad).

For each sample, 5 pg of total RNA were lyophilized and used for analysis. RNA was

resuspended in 12.5 pl of a solution comprised of 3 pl IOX 1" strand buffer (Gibco BRL), 12

mM DIT (Gibco BRL), and 0.1 pmol of y-'Z~ dATP end-labelled primer. Al1 incubations were

performed using a Perkin Elmer 2400 GeneAmp PCR system. Primer annealing was carrieci out

at 85 OC for 5 min, followed by heating at 42 OC for 1 h. To each sample, 2.5 pl of a mixture

containing LOO units of reverse transcriptase (Superscript II RT, Gibco BRL), 10 uni& of RNA

guard (Pharmacia) and 20 mM dNTPs (5 mM dATP, 5 mM dCTP, 5 m M dTTP, and 5 m M

dGTP, Boehrïnger Mannheim) were added. Extension reactions were heated at 42 OC for 1 h.

Samples were then ethanol-precipitated ovemight with 3 pl of 2 M sodium acetate (pH 5.2) and

50 pl of 100% ethanol. Following precipitation primer extension products were dried under

vacuum for 1 h, resuspended in 10 pl fornamide loading dye (0.05% w/v bromophenol blue,

0.05% (w/v) xylene cyanol, and 20 rnM EDTA al1 dissolved in 100% formamide), and then

boiled for 2 min to denature the reaction products. Samples were electophoresed on a 6%

PAGU7 M urea gel (Sequagel) in 1 X TBE at 200 V for 50 min. Prior to electrophoresis, the gel

was pre-run for 30 min at 200 V. The gel was dried and then exposed to preflashed Kodak XRP-

1 film with a Cronex Lightning Plus intensifjing screen (Du Pont) at -72 OC. Autoradiograms

were digitized using an Agfa Arcus II scanner. Densitometry was performed using the profile

analyst feature of Molecular Analyst v. 2.1 (Bio-Rad). The hsp70 signals were first normalized

using the H2B signals. The normalized values were then used to express the amount of hsp7O

message as a percentage of the heat shock level.

Gradient Gels and Immunoblot Anabsis

Protein was extracted fiom treated samples by resuspending cells in 1X SDS sample

buffer (50 mM TrisCl pH 6.8, LOO mM DTT, 2% SDS, 10% v/v glycerol) followed by sonication

(W-220F Sonciator, Ultrasonics Lnc.). The total protein concentration of each sample was

assayed using the Bio-Rad protein assay (cat. #500-0006), according to the manufacturer' s

instructions. Heat shocked samples to be treated with phosphatase were incubated with 200 unïts

of Lambda Protein Phosphatase (New England Biolabs) as per manufacturer's directions. Protein

was Ioaded ont0 a 545% polyacrylamide gradient gel (10 pg per sample for azetidine

experiments, 5 pg per sample for CN/DNP/salicylate experiments). Gradient gels were poured

as described in Ausubel et al. (1995). Acrylamide solutions were prepared and incubated on ice

(5% solution: 5% acry1arnide:bis (30:0.8), 0.375 M Tris-CVSDS pH 8.8; 15% solution: 15%

acry1amide:bis (30:0.8), 0.375 M Tris-CVSDS p H 8.8, 5% (vh) glycerol). After loading the

solutions ont0 the gradient maker, ammonium persulfate (0.025% w/v) and TEMED (0.0008%

V/V) were added, and the gel was imrnediately poured. Gels were rtm for 18 h at a constant

current of 10 mA, and were then immediately electroblotted ont0 nitrocellulose membrane using

an Idea Scientific electroblotter (400 mA, 1 h). Prior to immunodetection of HSF, blots were

blocked in 5% powdered milk in TBST (20 mM Tris-Cl (pH 7 .9 , 137 mM NaCl, 0.1% Tween

20) for 1.5 h. Blots were incubated with the p r i m q anti-HSF antibody solution (1 :2000 rabbit

anti-HSF (943) in 2% gelatin dissolved in TBST) for 1 h at 2 1 OC. Blots were washed with

TBST and incubated with the secondary antibody (1 :2000 alkaline phosphatase conjugated-goat

a-rabbit IgG, Bio-Rad) for 45 min at 21 OC. Alkaline phosphatase detection was pwformed

using 5-bromo-4-chioro-3-indolyl phosphate p-toluidine salt/nitro blue tetrazoluim chloride

reagents as per manufacturer's instructions (Gibco BK). The percentage of HSF located in the

upper and lower bands was detennined using densitometry. The immunoblots were digitized

using an AHa Arcus II scanner. Densitometric analysis was pwformed using the profile analyst

feature of Molecular Analyst v. 2.1 (Bio-Rad).

D. RESULTS

Azetidine induces HSF DNA-binding and heat shock gene pumng in Drosopkila salivaty gland pubtene chromosomes

Drosophila saiivary gland polytene chromosomes provide a unique tool for examining

the distribution of DNA-bound proteins in vivo. For an initial characterization of HSF DNA-

binding activity in response to azetidine (AzC), treated glands were squashed and then

immunostained to visually localize HSF bound at discrete loci on the polytene chromosomes.

The specificity of the 943 anti-HSF antibody was tested using blocking experiments with

purified Drosophila HSF (J. T . Westwood, personal communication; also see Westwood et al.,

1 99 L ). The secondary antibody alone has previousl y k e n s h o w to be non-speci fic for HSF (K.

Kociuba, personal communication).

For each control or condition of azetidine treatment at a particular time point, the

experiment was replicated 3 times. Within each preparation an average of 50 chromosome

spreads were observed with HSF staining, meaning that an average of 150 chromosome spreads

were viewed for each control or time point at a particular azetidine concentration. The images

shown in Fig. 2-1 and Fig. 2-2 are representative spreads. The above is also true for al1

subsequent immunolocalization experiments in this thesis.

Salivaxy glands dissected and maintaineci at rwm temperature (2 1 OC) showed a diffise,

non-specific staining pattern for HSF on the chromatin (Fig. 2-18). Upon heat shock (36.5 OC

for 15 min) HSF was obsewed to locaiize in discrete, brightly staining bands at over 200 sites as

previously described by Westwood et al. (199 1 ) (Fig. 2-1C). At the heat shock gene loci intense

staining for HSF was observed, acwmpanied by heat shock gene puffs; decondensed regions of

the chromatin which are usually indicative of active transcription (Ritossa, 1964a; Ashburner and

Bonner, 1979). The most prominent sites of HSF staining and heat shock gene puffing were the

Figure 2-1. Five mM azetidine induces HSF binding and heat shock gene puff ig in the salivary gland poïytene chromosomes of Drosophila in a time-dependent manner.

SaIivary glands were dissected fiom D. melanuguster third instar larvae and incubated in organ

culture for 1 h pnor to treatrnent. Chromosomal squashes were prepared fiom glands that were

maintained at room temperature (21 OC) (A and B), heat shocked at 36.5 OC for 15 min (C), or

treated with 5 mM azetidine (AzC) (D-F) for the indicated times. Chromosomes were stained for

DNA using bisbenzimide (Hoechst) and for HSF using a rabbit anti-HSF primary antibody (943),

foIlowed by a goat anti-rabbit FITC-conjugated secondary antibody. Prominent heat shock gene

loci are indicated. Each scale bar represents 10 Pm. The scale bar in Panel A also applies to

Panel B. The scale bar in Panel C also applies to D-F.

87C locus; site of three copies of the hsp7O gene, and the immediately adjacent 87A locus; site of

two copies of the hsp70 gene. The loci 63BC (site of the hsp83 gene), 64F, and 67B (site of the

hsp27, hsp26, hsp23, and hsp22 genes) also displayed intense staining for HSF and distinct heat

shock gene puffs. Slightly Iowa levels of staining were observed at 33B, 70A, and 95D (site of

the hsp68 gene). Although puffing was observable at 93 D there wi?s very Little staining for HSF.

In the subsequent panels for this figure, as well as the additional figures in this thesis, oniy the

most prominent sites of heat shock gene p u f i g and HSF staining will be labelled @7C, 87A,

67B, 64F, 63BC). Althougb the 95D, 70A, and 33B sites are unlabelleci in subsequent figures,

HSF staining was still observable in d l cases.

Chromosome spreads from glands treated with 5 mM azetidine showed a general trend of

increased HSF binding and heat shock gene puff size over time. Afkr 1 h of treatment staining

for HSF was not yet observable (results not shown). Following 1.5 h of treatment, the formation

of discrete but faintly staining bands was apparent (results not shown). By 2 h of treatment the

heat shock gene loci showed staining for HSF and there was a prominent increase in the intensity

and number of other loci staining for HSF (Fig. 2- 1 D). In addition, the initial formation of a heat

shock gene puff at 87C was observed. A mal1 puff at 67B was also noticeable.

Three hours of 5 mM azetidine treatment resulted in a drarnatic increase in the size of the

heat shock gene puff at 87C (Fig. 2-1 E). An increase in puff size was also visible at 67B, and

puffs were finally apparent at 63BC and 64F as well as 9SD, 93D, 70A, and 33B which are

uniabelled in Fig. 2- 1 E. Overall, HSF staining intensity increased and the level of staining at the

3 h time point was most comparable with the heat shock control. Visual, band-by-band

inspection of the 3L and 3R chromosomes of both the heat shock control and the 3 h, 5 mM

azetidine treatment demonstrated that these two conditions result in the same nwnber of HSF

bands on the chromatin. While HSF appears to stain the heat shock gene loci with

approximately equal intensity between the heat shock control and azetidine-treated conditions,

bands of weak intensity appearing in the hûrt shock control stained with qua1 or lower intensity

in the azetidine-treated condition. Puff sizes were not always comparable between the heat shock

and azetidine-treated conditions. The puffs at 87C and 87A redting fiom 3 h of 5 m M azetidine

treatment were always larger than in the heat shock control. This difference in puff size was not

as apparent at other heat shock loci such as 67B.

By 5 h of treatrnent, heat shock gene puff size was the same as that observed at 3 h (Fig.

2-1 F). The level of HSF staining varied somewhat and was either slightiy higher or about the

sarne as that observed at 3 h. Beyond the 5 h time point, levels of HSF staining and heat shock

gene puffing were approximately the same indicating a lack of attenuation of the response

(results not shown). As a control glands were maintaineci in organ culture at 21 OC for the

duration of each azetidine treatment time point. This control dernonstrateci that HSF binding was

not induced when glands were kept in organ culture over extended periods of time (results not

shown).

To investigate the effects of treatment with higher concentrations, chromosome spreads

were prepared f?om glands treated with 50 mM azetidine. Again a general trend of increased

HSF binding and heat shock gene puff size was observed over time. However, this trend was

accelerated fiom what was observed at a concentration of 5 mM azetidine. AAer 30 min of

treatment faint staining for HSF was observed (results not shown). The first prominent increases

in both the levels of HSF DNA-binding and heat shock gene p u f i g were obswed aAer 50 min

of treatment (Fig. 2-2B); approximateiy one hour sooner than in the 5 mM condition.

Figure 2-2. Fifty mM azetidine induces beat shock gene puffig and HSF DNA-binding in polytene chromosomes at earüer t h e points than 5 aiM azetidine.

Salivary glands were dissected from D. melanoguster third instar larvae and incubated in organ

culture for 1 h pnor to treatment with 50 rnM azetidine (A-D). As controls, salivary glands were

also incubated with 50 m M proline (E) or 50 mM azetidine in the presence of 118 p M

cycloheximide (Zimarino et ai., 1990a) to inhibit protein synthesis p). Squashes were

immunostained as descnbed in Figure 2- 1. Prominent heat shock gene loci are indicated. Each

scale bar represents 10 Pm. The scale bar in Panel A also represents Panel B. The scale bar in

Panel C also represents D-F.

By 2 h of 50 mM treatment the nurnber of heat shock gene puffs and the number and

intensity of bands staining for HSF had uicreased (Fig. 2-2C) to levels approximately equal to

the heat shock control. Again, HSF appeared to stain the heat shock loci with approximately

equal intensity between the heat shock control and azetidine-treated condition, whereas bands of

weak intensity appearuig in the heat shock control stained with equal or lower intensity in the

azetidine-treated condition.

AAer 3 h of 50 m M azetidine treatment lwels of HSF staining did not change

appreciably, but the heat shock gene puffs at 87A and 87C had reached their maximum size

which was again greater than those found in the heat shock control (Fig 2-2D). Interestingly,

both 5 mM and 50mM azetidine resulted in maximal heat shock gene puff size at the same time

point. Attenuation was not observed in time points taken afier 3 h (results not shown).

Because azetidine is an amino acid analogue of proline it is incorporated during protein

synthesis, resulting in the production of abnormal proteins (Welch and Suhan, 1986). The

production of abnormal proteins in tum leads to the induction of the heat shock response. Glands

treated with 50 mM proline for 1 h showed diffuse, nonspecific staining for HSF on the

chromatin (Fig 2-2E). In addition, glands treated with 50 mM azetidine for 2 h in the presence of

cycloheximide, an inhibitor of protein synthesis, also showed diffuse, nonspecific staining for

HSF on the polytene chromosomes; much like the 2 1 OC control (Fig. 2-2F).

Together, these observations point to the fact that azetidine is able to induce a

pattern of HSF DNA-binding that is very sirnilar to heat shock, but occurs on a much longer time

scale. While heat shock and azetidine appear to result in equally intense signals for HSF binding

at the heat shock gene loci, azetidine treatment results in larger heat shock gene puffs at 87A and

87C. The HSF staining pattern induced by azetidine is a direct result of amino acid analogue

incorporation into protein, as indicated by the proline and cycloheximide controls.

Azetidine ritduces HSF DNA-bindr'ng in Drosophila SL2 cells

HSF DNA-binding activity in response to azetidine was tùrther characterized in

Drosophila Schneider Line 2 (SL2) cells using electrophoretic mobility shift assays (EMSAs),

Drosophila HSF has previously been shown to be specific for the HSE consensus sequence used

in this experiment (Zimarino and Wu, 1987). EMSAs were repeated three times for each

azetidine concentration. The results shown in Fig. 2-3 reflect typical experirnents. Again, HSF

binding to DNA in response to azetidine was found to occur in a tirne-dependent manner and

strong signals for HSF binding to the heat shock element (HSE) roughly corresponded to time

points of maximal HSF binding on polytene chromosomes. SL2 cells exposed to 5 mM azetidine

showed strong HSF binding to the HSE afier 3 h of treatment (Fig. 2-3A). Both the 3 h and 4 h

signals were slightly lower than that observed for the heat shock control. HSF binding was

accelerated in cells treated with 50 mM azetidine and occurred afier 1 h of treatment (Fig. 2-3B).

Both the 1 h and 2 h time points in the 50 m . condition showed a slightly higher signal for HSF

binding than the heat shock control. In the presence of continua1 heat stimulus, HSF binding to

HSEs begins to attenuate after 1-2 h (Kociuba, 1999). To see whether attenuation of the

response to azetidine would occur, SL2 cells were treated with both 5 rnM suid 50 mM

concentrations of azetidine for a 15 h paiod. Signals for HSF DNA-binding were obtained in

both conditions (results not shown), correlating with the results of DiDomenico et ai. (1982b)

where the arginine analogue canavanine was used. These observations fùrther support the

Figure 2-3. Azetidine activates HSF DNA-bhding to ESEs in Drosophila SL2 ceUs.

SL2 Drosophila tissue culture cells were treated with 5 mM azetidine (A) and 50 mM azetidine

(B) for the times indicated. Ce11 extracts were prepared and HSF DNA-binding activity was

analyzed using an electrophoretic mobility shifl assay (EMSA) with a radiolabelled HSE

consensus sequence as a probe. Binding reactions were run on 1 % agarose gels in 0.5 X TBE.

conclusion that azetidine can induce HSF bindïng to the heat shock element in a manner that is

time-dependent.

Azetidine induces ksp 70 gene transcr@tion in Drosophila SL2 cells

Oligomerization and DNA-binding of HSF are necessary steps proceeding the

transcriptional activation of the heat shock genes. Prirner extension analysis was used to examine

whether heat shock gene p u f i g and HSF DNA-binding observed in response to azetidine

treatment was correlated with transcriptional activity.

The analysis was perfomed on RNA extracted fiom treated SL2 cells. Hsp7O transcript

was not observed in cells maintained at room temperature, but a significant level of transcript

was observed in heat shocked cells (Fig. 2-4). Cells treated with 5 mM azetidine for 3 h

produced a signal for hsp70 transcript that was 76.5% of the signal obtained for the heat shock

control. Treatment with 50 mM azetidine produced a signai for hsp70 message that was greater

than both 5 mM azetidine treatment and heat shock, quantimg to a value that was 140 % of the

heat shock control. The experiment was repeated three times and the values shown in Fig. 2-4

are representative of a typical experiment.

Results from the primer extension analysis demonstrate that azetidine induces hsp70 gene

transcription, correlating with results fiom mobility shift assays. Shown in Fig. 2-4 are the peak

time points of hsp70 induction. Earlier time points were also taken, and the amount of hsp70

message produced was observed to increase in a tirne-dependent manner following treatrnent

with either azetidine concentration (resuits not shown). At peak times for hsp70 induction, 5

rnM azetidine appears to induce levels of DNA-binding and hsp7O gene transcription that are

Figure 2-4. Azetidine induces hsp70 gene transcription in Drosopliifa SL2 ceUs.

Total RNA extracts were prepared fiom Drosophila SL2 cells after exposure to the noted

conditions. Levels of hsp70 and histone H2B gene transcripts were analyzed by primer

extension analysis. Extension products were separated on 6% PAGW7 M urea gels. The 1eveI of

hsp7O message was expressed as a percentage of the heat shock levet using densitometry.

1 O 100 140 76.51

Hsp70 Message as a Percentage of Heat S h d (36.5 "C) Leml

lower than heat shock whereas 50 mM azetidine induces DNA-binding and transcription to

levels higher than the heat shock condition.

Azetidine results in an apparent increase in HSF liyperphosphorylation

HSF is constitutiveiy phosphorylated under control conditions and has been shown to

undergo inducible phosphorylation in response to heat and other forms of stress (Sorger and

Pelham, 1988; Lmon et al., 1988; Sarge et al., 1993; Cotto et a[., 1996 Winegarden et al., 1996;

Xia and Voellmy, 1997). Previous work has s h o w that azetidine treatrnent does not result in

inducible phosphorylation (or 'hyperphosphorylation') of HSF in mammalian cells (Sarge et al,

1993); however, a recent paper by Jolly et ai. (1999) has shown that human HSFl can be

hyperphosphorylated in response to azetidine. Fritsch and Wu (1999) have found that

Drosophila HSF does not become hyperphosphorylated in response to heat, but rather undergoes

approximately equal increases in both phosphorylation and dephosphorylation at various serine

residues. These findings prompted us to examine the phosphorylation state of HSF following

azetidine treatment in Drosophila tissue culture cells.

We used a polyacrylamide gradient gel to obtain separation between the constitutively

phosphorylated and hyperphosphorylated forms of HSF. Between the control and heat shock

conditions there appeared to be no net change in the phosphorylation state of HSF. Under

control conditions, 50.1 % of HSF was fouad in the uppermost band, which represents the

hyperphosphorylated form of HSF (Fig. 2-SA). The remainder was found in the lower band,

which represents the constitutively phosphorylated form of HSF. In the heat shock condition,

49.5 % of HSF was found in the inducibly phosphorylated form.

Under 5 m M azetidine treatrnent there appeared to be a slight increase in the amount of

hyperphosphorylated HSF at the 2 h and 3 h time points. Carefùl examination revealed that the

Figure 2-5. Azetidine results in an apparent increase in ESF hyperphosphorylation.

SL2 cells were heat shocked or treated with 5 mM or 50 mM azetidine for the indicated times

(A). Protein extracts were run on a 5-15 % potyacrylamide gradient gel. The presence of

constitutively phosphorylated and hyperphosphorylated forms of HSF were detected by Western

blot andysis with anti-HSF antibody (943). Bands corresponding to the constitutively

phosphorylated and hyperphosphorylated forms of HSF are indicated, in addition to other HSF

isoforms. The percentage o f HSF in each band was determined using densitometry. As a

cornparison to azetidine, the experiment was repeated using inhibitors of oxidative respiration to

induce the stress response (B).

, other HSF isokm#i

% hyperphosphorylated HSF 50.1 49.5 52.5 54.8 68.4 61.7

20 minutes

% slower migiab'ng HSF isoCom

hyperphosphor~kted HSF

O 0 19.8 16.7 5.8 12.4

phosphoryleted HSF

- - -

% coristitutively phosphoryiated HSF 46.4 54.0 53.1 53.6 51.8 53.6 44.1 43.8

% ~ ~ h a ~ t e d ' HSF

53.6 46.0 46.9 46.4 48.2 46.4 55.9 56.2

bands representing hyperphosphorylated HSF were actually doublets. A third band also

appeared, migrating above the hyperphosphorylated fonn of HSF. This band could account for

the decrease in constitutively phosphorylated HSF observeci. Fifty millimolar azetidine

treatment had the same effects. Under higher concentrations of analogue there appeared to be

more HSF found in the hyperphosphorylated form than in the constitutively phosphorylated

forrn.

A total of four different azetidine-treated sample sets were prepared. Sampies from each

set were run on gradient gels at least two separate tirnes. Overail, results were extrernely

variab le. In most instances, bands representing the hyperp hosphorylated and constitutively

phosphorylated foms were not clearly resolved. The results shown in Fig. 2-SA were the

clearest obtained.

As a comparison we examineci the effect of inhibitors of oxidative respiration on the

distribution of the constitutive and hyperphosphorylated forms of HSF (Fig. 2-5B). Both

cyanide and dinitrophenol resulted in a slightly lower percentage of HSF found in the

hyperphosphorylated form (ranging fiom 46.4-48.2 %). Sodium salicylate resulted in a slightly

greater percentage (55.9-56.2 %) of hyperphosphorylated HSF. Comparing the control and heat

shock conditions, the amount of hyperphosphorylated HSF decreased tiom 53.6 % in the control

to 46.0 % with heat shock. This expenment was replicated three times and Fig. 2-58 shows a

representative gel. Though the values shown in Fig. 2-SB were slightly variable fiom

experiment to expenment, they always fell within the indicated ranges.

As a control, a heat shocked sample was treated with phosphatase. This treatment

resulted in a new third faster rnigrating (lower) band representing unphosphorylated HSF (results

not shown). Further work is required to determine if the slower rnigrating bands and the

doublets representing hyperphosphorylated HSF in the azetidine-treated conditions disappear

with phosphatase treatment-

While it appears that azetidine does result in hyperphosphorylation of DrUsophifu HSF,

the appearance of nurnerous HSF isoforms makes the data difficult to interpret. At this time a

f i m conclusion can not be made regarding the effect of azetidine on the phosphorylation state of

HSF in Drosopiiila.

Azeîidine dues not resulf in the a c c u m u l . n of Arsc7O on the chrom&

Upon heat shock, hsp70 and hsc70 have been shown to colocalize at over 200 sites on the

polytene chromosomes (Kociuba, 1999). Hsc70 shows increased levels of binding to the

chromatin in salivary glands treated with cyclohexirnide, suggesting that hsc70 is able to

compensate for the absence of hsp70. Together these results indicate that hsp70 and hsc70 likely

have identical functions when bound to chromatin, possibly in the protection of DNA-bound

proteins or the chromatin itself (Kociuba, 1999).

In attempt to find out more about the fùnction of hsp70/hsc70 bound to the chromatin

during stress, we treated salivary glands with azetidine and then exarnined the disîribution of

hsc70 binding. Hsc70 locaiizes at discrete sites on the polytene chromosomes in response to heat

stress, even in the absence of hsp70 production (Kociuba, 1999). Azetidine treatment results in

the synthesis of non-functional hsps, but given the above results we would still expect to find

hsc70 binding to the chromatin.

Glands were treated with heat or azetidine, squashed, and then immunostained using an

antibody (3A3) developed against human hsp70 which also recognizes Drosophiiu hsc70

(Rabindran et al., 1994). In the 21 OC control, levels of hsc70 associated with the chromatin

were very low and no specific banding pattern was observeci (Fig. 2-6B). A heat shock for 20

Figure 2-6. Azetidine does not result in the accumulation of hsc70 on the chromath

Salivary glands were dissected corn D. melanogaster third instar larvae and incubated in organ

culture for 1 h prior to treatrnent. Chromosomal squashes were prepared fiom glands that were

maintained at room temperature (21 OC) (A and B), heat shocked at 36.5 OC for 20 min (C and

D), or treated with azetidine (AzC) (E-H) as indicated. Chromosomes were stained for DNA

using bisbenzimide (Hoechst) and for hsc70 using a monoclonal antibody against hsc70 (3a3)

followed by goat anti-mouse Alexa 488 secondary antibody. Under fluorescence microscopy

Alexa 488 normally exhibits green fluorescence. In this figure only, the original black and white

images in panels B, D, F, and H were coloured red using Adobe Photoshop. The scale bar

represents 1 O Pm.

minutes at 36.5 OC resuited in the localization of hsc70 at over 200 discrete, brightly staining

sites on the chromatin (Fig. 2-6D). Both 5 mM and 50 mM azetidine treatment did not result in

the localization of hsc70 on the chromath (Fig. 26F and Fig. 2-6H). This result was unexpected

and indicstes that the way in which hsc70 is targeted to the chromatin in response to heat does

not function in azetidine-treated cells.

E. DISCUSSION

Azetidine has previously been shown to be a strong inducer of the heat shock response in

severd mammalian and plant systems. We were interesteci in characterizhg aspects of HSF

activation in response to azetidine in Drosophila melanogaster. Azetidine treatment resulted in a

time-dependent HSF binding and heat shock gene puffing pattern. Treatment with 50 mM

azetidine resulted in initial increases in HSF binding and heat shock gene puffing at earlier time

points, however both 5 mM and 50 mM azetidine led to maximal HSF binding and heat shock

gene puffing at the same time point. These results cm be explained in terms of the fact that

azetidine can efficiently replace proline during protein synthesis (Fowden and Richmond, 1963).

It has previously been shown that one to two hours of protein synthesis is required in the

presence of analogue to induce the heat shock response (Thomas and Mathews, 1984). This

indicates that a critical concentration of abnormal protein within the ce11 is required before the

heat shock response is activated. During this initial period of synthesis, higher concentrations of

azetidine present an excess of the analogue to the ce11 and so a high proportion of al1 proteins

synthesized are abnormal due to analogue incorporation. High levels of abnormal protein in turn

result in an earlier induction of the heat shock response. Since heat shock gene puffing has

traditionally been correlateci with active transcription occumng fi-om heat shock genes (Ritossa

1 964, Ashbumer and Borner 1979), this sarne theory can be applied to explain the appearance of

heat shock gene puffs at earlier time points with higher azetidine concentrations.

DiDomenico et ai. (1982b) have shown that the quantity of hsp70 produced is related to

the severity of the heat shock. In other words, a response is induced that is proportional to the

severity of the stress. This is exemplifieci by the results of the electrophoretic mobility shift

assays. Treatment with 5 m M azetidine produced a signal for DNA-binding that was lower than

that of heat shock. In contrast 50 mM azetidine produced conditions of stress more severe than

heat shock, resulting in more HSF DNA-binding. Levels of hsp7O transcription observed in SL2

cells corresponded well with HSF binding evidence obtained by electrophoretic rnobility shift

assays. Again, a signal stronger than heat shock was obtained with 50 mM azetidine, indicating

that amino acid analogue treatment at high concentrations is a more severe f o m of stress than

heat shock.

When levels of HSF hyperphosphorylation were examinecl in SL2 cells we observed an

apparent increase in HSF hyperphosphorylation in response to azetidine. However, this result

was confounded by the fact that numerous HSF isoforms were also visible. These isoforms

could be a result of amino acid analogue incorporation into HSF, resulting in abnormal HSF

protein. Thus the doublet representing hyperphosphorylated HSF might actually include some

analogue-substituted constitutively phosphorylated HSF. The band migrating above

hyperphosphorylated HSF could be analogue-substituted hyperphosphorylated HSF. On the

surface, the increased percentage of HSF in these upper bands could mean that azetidine results

in increased phosphorylation. However, more expenments need to be done to confimi this

hypothesis.

Our results may shed some light on the conflicting reports obtained in mammalian cells.

While Sarge et al. (1993) did not observe a retardation in the mobility of HSF (which would

indicate hyperphosphorylation) in response to azetidine, Jolly er al. (1999) did observe a shift in

the mobility of human HSF. Although the change in mobility of human HSF in response to

azetidine was comparable to that induced by heat shock, the possibility can not be ruled out that

in that particular experiment there was analogue-substituted HSF present.

Corresponding to findings fiom Fritsch and Wu (1999), we did not observe an increase in

hyperphosphorylated HSF in response to heat shock but rather a balance of constitutively

phosphorylated and hyperphosphorylated HSF. The same effect was seen with inhibitors of

oxidative respiration, Although salicylate seemed to show a slight increase in HSF

hyperphosphorylation, Winegarden et al. ( 1996) have previously demonstrated that salicylate

prevents the phosphorylation of HSF in SL2 cells labelled with 32~.

Preceding the findings of Fritsch and Wu (1999), W u (1995) suggested that

phosphorylation may not be related to the regulation and fùnction of HSF, but rather may occur

as a result of changes in the balance of kinase and phosphatase activities that occur with heat

stress. We tend to follow this assertion. Although our data may hint that azetidine causes HSF

hyperphosphorylation, we do not want to place too much emphasis on the results. It seems likely

that in some cases azetidine results in analogue-substituted HSF protein, which in turn results in

decreased mobility on polyacrylamide gels.

What purpose does HS F hyperphosp hory lation serve? Xia and Voellmy ( 1 997) found

that hyperphosphorylation of HSF prolongs the haif life of the active trimer. In azetidine-treated

cells, where attenuation does not occur, there would be no need to prolong the half life of the

trimer. In accordance with this, Sarge et al. ( 1 993) suggested that HSF hyperphosphorylation is

not observed following azetidine treatment because hyperphosphoxylation of HSF is required for

attenuation to occur.

The finding that hsc70 did not localize on the chromatin in response to azetidine was

unexpected. Kociuba (1999) hypothesized that hsp70 and hsc70 play sirnilar roles bound to the

chromatin, possibly protecting DNA-bound proteins or the chromatin itself Kociuba's findings

indicate that hsc70 can bind to the chromatin even in the absence of fùnctionai hsp70. The

present results indicate that the way in which hsc70 is targeted to the chromatin in response to

heat, does not function in azetidine-treated cells. While hsp70 is not directly involved in the

localization of hsc70 to the chromatin, there could be some other protein that does play a role.

Amino acid analogue incorporation could impair the finction of such a protein, preventing the

binding of hsc70 to the chromatin.

The possibility that hsp70 and hsc70 play a protective role on the chromatin can not be

mled out, in fact, their absence under conditions of azetidine treatment coufd contribute to the

magnitude of the response observeci at high concentrations. Azetidine does not impair the

fiinction of hsc70 elsewhere in the ce11 and has been shown to enhance the expression of hsc70

as a ceIl surface antigen on tumor cel1s (Tamura et al., 1993).

Taken together, the results of this study show that azetidine can induce HSF binding and

heat shock gene transcription in a manner similar to heat shock but on a longer time scale.

Under high concentrations, azetiduie appears to act as a more severe form of stress than heat

shock. Contributing to this could be the fact that azetidine does not induce hsc70 binding on the

chrornatin. In the fuwe other experiments, such as labelling HSF with 3 ' ~ , or use of 2-D gels,

should be performed to make a firm conclusion regarding the effect of azetidine on HSF

hyperphosphorylation.

CHAPTER 3

Heat shock and azetidine cause the relocalization of

RNA polynrerase II tu the heat dock gene loci

resulting in the global repression of all other genes

A. ABSTRACT

Recent findings in our lab have shown that RNA Polymerase II preferentially relocates to

the heat shock gene loci following h a t shock in Drosophila, correlating with decreased

transcription of non-heat shock genes. HSF binds at over 200 sites on Drosophila salivary gland

polytene chromosomes following heat shock, o d y 8 of which sites are the location of heat shock

genes. We suggest that HSF binding induced by heat shock serves to divert RNA Polymerase II

fiom non-heat shock gene sites to the heat shock gene loci, resulting in transcriptional repression

of non-heat shock genes. A role for active HSF in this process was confimed using a

temperature-sensitive Drosophila mutant defective for DNA-binding at the non-permissive

temperature. Azetidine treatment resulted in the repression of non-heat shock gene transcription,

but to a slightly lesser degree than heat shock. In accordance with our hypothesis, both heat and

azetidine were found to result in repression of the transcriptionally active ecdysone-inducible

loci 74EF and 75B, concomitant with reduced immunostaining for RNA Polymerase II. Based

on these findings we suggest a possible role for the HSF activation domain in the preferential

recruitment of RNA Polymerase II to the heat shock gene loci under conditions of stress.

B. INTRODUCTION

The largest subunit of RNA Polymerase II (Pol II) carries a C-terminal domain (CTD) in

one of two forms: underphosphorylated (Pol IIA) or hyperphosphorylated (Pol 110). The CTD

plays an important part in mediating the response to transcriptional regdators by acting as a

structural fiamework for preinitiation complex formation (Dahmus, 1996). For example, the Pol

IIA CTD has been shown to contact TFIIE, TFIIF (Kang and Dahmus, 1995), and TBP (Usheva

et al., 1992). Usheva et al. ( 1992) suggest that CTD phosphorylation suppresses Pol II binding

to TBP allowing for disruption of the preinitiation complex and subsequent promoter clearance.

In contrast, Serizawa et al. (1993) f o n d that Pol IIA is able to elongate fiom a promoter

suggesting that CTD phosphoryIation is not required for promoter clearance.

Pol IIA is found paused at the promoter of the hsp70 gene indicating that CTD

phosphorylation is not necessary for elongation of Pol II to the pause, but perhaps may play a

role in stimulating the release of Pol II fiom the pause (Fernandes et ai-, 1994). Mason and Lis

(1 997) favour a cornpetition mode1 for the release of paused Pol II, where HSF and TBP interact

directly at heat shock gene promoters. They hypothesize that HSF cornpetes for binding to TBP

with Pol II to facilitate the release of Pol 11 from the pause. This particular interaction occurs at

the acidic H-domain of Pol II, just N-terminal to the CTD (Mason and Lis, 1997). Hence CTD

phosphorylation could still play a role but perhaps in facilitating elongation as opposed to the

disniption of the preinitiation complex.

Heat shock results in the reprogramming of transcription in Drosophila (Nover, 199 1).

With the onset of heat shock gene expression, transcription nom al1 other genes is rapidly

repressed (Ashbumer, 1970; Tissieres et ai., 1974; McKenzie et al., 1975; Spradling et ai-,

1975). Several studies have examineci this phenomenon. Findly and Pederson (198 1) showed

that the heat-induced expression of hsp70 coincides with transcriptionai repression of the actin

gene. Gilmour and Lis (1 985) used in vivo crosslinking to determine the relative densities of Pol

II bound to DNA under control and heat shock conditions. Their results indicated that copia

genes and cytoplasmic actin genes show a decrease in the density of bound Pol II upon heat

shock, thus indicating transcriptionai repression. Immunofluorescence studies have aiso

demonstrated that Pol II redistributes fiom sites of actively transcribing genes to the heat shock

gene loci upon heat shock (Jamrich et al-, 1977; Greenleaf et al., 1978; Bonner and Kerby, 1982;

Sass, 1982).

in light of the discovery of the IIA and I I 0 forms of Pol II, other studies have utilized

immunocytochemistry to examine the distribution of both forms of RNA polymerase KI before

and after heat shock. Weeks et al. (1993) have observed puffs at the developmental, ecdysone-

inducible loci to immunostain exclusively for Pol 110, indicating that it is the

hyperphosphorylated form of Pol II that is transcriptionally active on these and likely other non-

heat shock genes. However, at heat shock gene puffs they observed s*Aning for both Pol IIA and

Pol [IO, indicating that both forms are involved in transcribing the heat shock genes. In contrast,

results fiom our laboratory show that Pol IIA and Pol I I 0 have the same distribution at ail

actively transcribing loci on the polytene chromosomes prior to heat shock (Paraiso and

Westwood, unpublished results). Following heat shock, we have observed both Pol 1I.A and Pol

I I0 to show a preferential relocalization to the heat shock gene loci, corresponding to the initial

findings of Jamrich et al. (1 977) and Bonner and Kerby ( 1 982).

The global repression of transcription tiom non-heat shock gene sites observed in

Drosophila does not occur to near the same extent in mammalian cells. However, heat shock has

been s h o w to inhibit cytokine-inducible nitric oxide synthase gene expression (De Vera et al.,

1996% 1996b; Wong et ai., 1996), IL-if3 biosynthesis (Schmidt and AbdulIa, 1988), and tumor

necrosis factor gene expression (Snyder et al., 1 W2; Ensor et al., 1994). In addition, HSF has

been shown to act as a transcriptionai repressor, preventing the transcription of the Prointerleukin

1 P gene (Cahill et aL, 1996) and the Ras-induced transcriptional activation of the c-fos gene

(Chen et al., 1997).

Westwood et al. ( 199 1 ) have shown that HSF binds at over 200 sites on Drosophiia

salivary gland pol ytene chromosomes following heat s hock. Major heat shock genes, e n d h g

hsps, are located at ody 8 of these sites and so the question arises as to the firnction of the

additional HSF binding. Westwood et al. (1991) suggested that some of the HSF binding sites

could be 'minor' stress-inducible genes still to be identified, or genes whose transcription

continues during heat shock such as the histone genes. Included among the non-heat shock gene

sites at which HSF binds are 74EF and 7SB, the major ecdysone-inducible developmental loci. Ln

mid-late third instar Drosophila larvae, ecdysone titers increase, resut ting in visible pu ffs at

74EF and 75B. The E75 gene (at 75B) codes for the production of two steroid receptor

transcription factors (Segraves and Hogness, 1990) and the E74 gene (at 74EF) codes for two

ets-related transcription factors (Burtis and Thummel, 1990)- These transcription factors are

involved in the induction of later genes, which in tum are involved in tissue rnorphogenesis in

late larval and pupal stages (Woodard et al., 1994; also reviewed in Russell, 1996). Upon heat

shock Westwood et al. (199 1 ) observed that in addition to HSF binding, puff size had regressed

at 74EF and 75B. This result suggested that HSF binding could cause a shut down of

developmental programs under conditions of stress, and that in essence the transcriptiond

activator could also act as a repressor (Westwood et al., 199 1 ).

We hypothesize that HSF binding induced by heat shock plays a role in the divasiun of

Pol II from actively transcribîng genes to the heat shock gene loci. This would result in the

repression of active transcription fiom non-heat shock genes such as those located at 74EF and

75B. Altematively the phenornenon of Pol II relocalization and transcriptional repression could

be due to a secondary effect of heat, with other protein factors in addition to HSF facilitating the

sole transcription of the heat shock genes during heat shock. In order to help rule out this second

possibility we used the amino acïd analogue azetidine to induce HSF activity. Previous studies

have focussed mainly on repression effects observed with heat shock. Observations of Pol II

relocalization and transcriptiond repression i n response to azetidine would indicate that the

effects observed with heat were due to active HSF itself.

Treatrnent with azetidine resulted in a decrease in transcription and Pol II binding

indicating that Pol II relocalization is not completely due to secondary effects of heaî. We used a

temperature-sensitive HSF mutant defective for DNA-binding at the non-permissive temperature

to confinn that the relocalization of Pol II was due directty to HSF binding. Pol II was observed

to relocalize at the heat shock gene loci in response to azetidine treatrnent at the permissive

temperature, but not at the non-permissive temperature, confirming the necessity for active

DNA-bound HSF. Both heat and azetidine resulted in a decrease in transcription and Pol II

staining at 74EF and 75B foilowing ecdysone addition, indicating that active HSF is successful

in shutting down transcriptionally active non-heat shock genes. HSF contains a potent activation

domain, comparable in nature to the VP16 transcriptional activator. We suggest a possible role

for the HSF activation domain in the redistribution of Pol II to the heat shock genes under

conditions of stress.

C. MATERIALS AND METHODS

Fiy Stocks

Drosophila melanogaster (Oregon R) and hsf mutants (Car1 Wu, NIH; Jedlicka et al.,

1 997) were raised as previously descriied in Chaptw 2.

lir Vivo Heat Treatments

Late third instar larvae with very faint blue guts were heat shocked inside

microcentrifuge tubes containing a small piece of Kimwipe saturateci with water. Tubes were

subrnersed in a temperature-controlled circulating water bath (Neslab RTE-21 1) set at 36.5 OC

for 25 min.

Salivary Gland Treatments in Organ Culture

Salivary glands were dissected fiom third instar larvae in modified Ti3 1 buffer (15 mM

HEPES (pH 6.8), 80 mM KCl, 16 rnM NaCI, 5 mM MgC12, 1% polyethyleneglycol 6000

(Myohara and Okada, 1988)). Glands were incubated for 1 h at 21 OC in a humidified chamber.

For chernical treatments glands were transferred to a depression slide containing the appropriate

concentration of azetidine (Sigma), 1 p M 20-hydroxyecdysone (ecdysone, Sigma), or azetidine

in the presence of ecdysone (see Results section), al1 dissolved in TB 1 buffer and incubated for

the appropriate time period. The optimal ecdysone concentration of 1 ph4 was chosen as per

Karim and Thummel (199 1). For controls, glands were left at room temperature (21 OC) in TB 1

buffer or were transferred in 100 pl TB1 buffer to a microcentrifuge tube which was then

submersed in a temperature-controlled circulating water bath (Neslab RTE-21 1) set at 36.5 OC

for 20 min.

Br UTP Labelling

Nascent transcripts were labelled following a procedure modifieci fiom Haukenes et aL

(1997). DOTAP reagent (Boehnnger Mannheim) was mixed with 10 mM BrUTP (Sigma) to a

standard volume of 50 pl. If azetidine or ecdysone treated glands were to be labelled, these

chernicals were also included in the labelling mix. The DOTAP was allowed to cornplex with

the BrUTP in a g l a s depression slide in a humidified chamber at least 10 min pnor to labelling.

Labelling in the presence of azetidine andlor ecdysone, or under controi conditions was carried

out in the glas depression slide. For labelling under conditions of heat shock, the mix was

transferred to a microcentnfuge tube to be submersed in a water bath. Two pairs of glands were

used per 50 pl of mix, and the labelling was carrieci out for 20 min.

Chromosome Squaslies

Please refer to Chapter 2.

HSF Immunolocufi~ation on Polyene Chromosomes (Fig 3-5 on&)

HSF immunolocalization was carried out as described in Chapter 2 with the following

exceptions: (i) preparations were incubated with a 1:200 dilution of Rhodamine Red-X-

conjugated F(ab')., fragment goat anti-rabbit IgG (H + L) secondary antibody (Jackson

ImmunoResearch Laboratories, Inc., cat. #111-296-003) for 30 min, and (ii) images were

captured on 35 mm Fujichrome Sensia 400 ISO slide film using a 45 second exposure time for

Rhodamine Red-X staining.

Dual Immunolocali~tion for HSF and BrUTP on Polytene Chromosomes

Staining for HSF was performed first, and was as described in Chapter 2 with the

following changes: 1) the HSF antibody was diluted 1:500 in BTP; 2) the Rhodamine Red-X

conjugated goat anti-rabbit secondary antibody was diluted 1: 100 in BTP. To stain for BrUTP

incorporation, the slides were incubated for 1 h with a 1 : 1 0 0 dilution in BTP of a monoclonai

antibody against bromodeoxyurïdine (Chernicon, cat. #MAB 1467). This was followed by hvo

washes, for 10 min each wash, with PBS/O.OI% Tween 20. The slides were then incubated for

30 min with an FITC-conjugated F(ab')~ hgment goat anti-mouse IgG (H + L) secondary

antibody (Jackson ImmunoResearch Laboratories, Inc., cat. #) diluted 1:200 in BTP. The

staining protocol was then completed as previously described. images were captwed on Kodak

Tri-X 400 ISO black and white negative film using a Nikon Microphot fluorescence microscope

and a Nikon Plan 40X objective. Exposure times were 0.25 second with neutral density filter 4

for Hoechst staining, 22-24 seconds for FITC staining, and 14- 16 seconds for Rhodamine Red-X

staining. Negatives were digitized using a Nikon CoolScan III scanner and were coloured and

adjusted for brightness and contrast using Adobe Photoshop.

Dual ImmunolacaIizadon for HSF and RNA Polynrerrrse 11 on Polytene Chtomosornes

Staining for RNA Polymerase II was performed f h t . Slides were incubated for 1 h with

monoclonal CC-3 antibody (Michel Vincent, Université Laval; also see Thibodeau and Vincent

(1991)), which recognizes the hyperphosphorylated form of HSF, diluted 1:400 in BTP. Alexa

594 goat anti-mouse IgG (H + L), F(ab')z fragment conjugate (Molecular Probes, cat. # A 4 1020)

diluted 1 : 100 in BTP were used as the secondary antibody. Following the wash step as described

above, the rest of the staining procedure was perfonned as previously described, with the

following changes: 1) the HSF antibody was diluted 1:lOûO; 2) the FITC goat anti-rabbit

secondary antibody (Cappel, cat. #55655) was used, diluted 1:200 in BTP. Images were

photographed as described in the section above.

Assigtament of C'ogrèul Loci

Please refer to Chapter 2.

D. RESULTS

Azetidine induces the advation of heat shock gene îranscn'ption and the global repression of transcription from other genes in a concentration dependent manner

We took advantage of a technique originally devised by Wansink et al. (1993) for

labelling nascent RNA transcripts with the uridine S'-triphosphate analogue, 5-bromouridine 5'-

triphosphate (BrUTP). Labeiled transcripts are detected with anti'bodies against bromo-

deoxyuridine (anti-BrdU), which also recognize BrUTP. To deliver BrUTP into the ceIl we used

DOTAP, a cationic liposomal transfection agent (Haukenes et al., 1997). Under fluorescence

microscopy the sites of active transcription c m then be colocalized with bound HSF or other

proteins.

This method has become a useful tool for looking at overall patterns of gene transcription

in salivary gland cells exposed to various forms of stress. Previous studies have shown that non-

heat shock gene transcription is repressed during heat stress while the transcription of the heat

shock genes is greatly upregulated (Ashburner and Borner, 1979). The ovemding question is

whether or not this phenomenon occurs due to some effect of heat, or due to the activation of

HSF itself To address this question we wanted to examine the level of transcription occurring

inside the ce11 foIlowing treatment with an inducer other than heat. Azetidine was selected, as it

is able to induce a response in a manner similar to heat, but without the presence of heat itself.

Salivary glands were treated in organ culture with 5 mM or 50 mM azetidine for the

indicated times. Following treatment glands were squashed and then immunostained using

antibodies against both HSF and BrUTP. Nascent transcript patterns on the polytene

chromosomes were then compared between azetidine, heat shock, and control conditions.

In the 21 OC controi approximately 200-250 distinct, brightly staining bands were

observed representing nascent gene transcripts (Fig. 3-18). Following a 20 minute heat shock at

36.5 OC the nurnber of sites of transcription were greatly reduced to approximately 35-45 (Fig. 3-

ID). Chromosome 3 was visually inspected, in this and subsequent chromosome spreads, to

determine the percentage of remaining sites of transcription correlateci with HSF staining.

Ninety-four percent of sites stahing for BrUTP incorporation during heat shock were found to

correlate with HSF binding sites (Fig. 3-lC), comparable to previous results nom our lab

(Paraiso and Westwood, unpub lished results).

Treatrnent with 5 mM azetidine for 4 h resulted in a pattern of HSF binding and heat

shock gene puffing very similar to what was previously observed (compare Fig. 2- ID and Fig. 3-

LE). Following treatment the nurnber of sites of transcription was reduced compared to the 2 1

OC control, with approximately 100- 1 10 bands staining for BrUTP incorporation (Fig. 3- 1 F). The

reduction in overall transcription was not as great as in the heat shock condition but an average

of 95 % of sites of transcription were observed to correlate with HSF staining.

Under 50 mM azetidine treatment for 2 h there was a great reduction in transcription

from previously active sites; however, the level of transcriptional repression observed was still

slightly less than the heat shock control with 50-60 sites staining for BrUTP incorporation. Of

the remaining sites of transcription, approximately 91 % were also observed to correlate with

HSF staining. Again, the pattern of HSF binding and heat shock gene p u f i g was similar to

what was previously observed (compare Fig. 2-2C and Fig. 3- I G).

As a controf, cells were treated with DOTAP in the absence of BrUTP. The results in

Fig. 3-11 show that the reagent alone does not induce HSF binding. I f cells are labeiled with

BrUTP alone, the incorporation rate into nascent RNA decreases to around 10 % as compared to

when DOTAP and BrUTP are used together (Chang and Westwood, manuscript in preparation).

Figure 3-1. Azetidine induces the activation of heat shock gene transcription and the giobpl repression of transcription from other genes-

Salivary glands were dissected fiom D. mefanogaster third instar larvae and incubated in organ

culture for 1 h prior to treatment. Salivary glands were maintained at room temperature (21 OC)

(A and B), heat shocked at 36.5 OC for 20 min (C and D), or treated with azetidine (AzC) (E-H)

as indicated. Salivary gland cells were labelled with BrUTP (see Materials and Methods)

concomitant with heat shock, or during the last 20 minutes of azetidine treatment. Control

glands were labelled for 20 minutes at 21 OC. Chromosome squashes were irnmunostained for

HSF using a rabbit anti-HSF pnmary antibody (943) followed by a goat anti-rabbit Rhodamine

Red-X-conjugated secondary antibody (A, C, E, and G). To stain for BrUTP incorporation a

monoclonal antibody against bromodeoxyuridine was used followed by a goat anti-mouse FITC-

conjugated secondary antibody (B, D, F. and H). Prominent heat shock gene loci are indicated.

The scale bar represents 10 Pm.

These r e d t s indicate that azetidine has an effect on the global repression of

transcription. The severity of the stress correlates with the level of transcriptional repression

observed, as higher concentrations of azetidine resulted in a greater amount of overdl repression

at earlier time points.

Azetidine treatment causes hyperphosphotykted RNA Poïymerase Il to red imfute to the keat shock gene loci

We next wanted to confirm that the decrease in global transcription observed in response

to azetidine was due to the redistribution of RNA polymerase II on the chromatin. FoIlowing

heat or azetidine treatment, sdivary glands were squashed and immunostained with antibodies

against HSF and the hyperphosphorylated form of RNA Polymerase iI (CC-3 antibody), HSF

and Po1 II could then be colocalized and their distributions comparecl under control, heat shock,

and azetidine-treated conditions. Unfortunately hyperphosphorylated Pol II and nascent RNA

couid not be colocalized, as both antibodies used to detect these molecules were mouse

monoclonals.

In the 21 OC control 190-200 bands were observed to stain for Pol II (Fig. 3-2B). The

bands were distinct and stained with varying intensities. Staining for HSF showed a diffise

pattern, typical for control conditions. Heat shock at 36.5 OC for 20 minutes resulted in a

dramatic decrease in the number of bands o b s e d to stain for Pol II. Approximately 30 bands

were detectable. The brightest bands mapped to the major heat shock gene puffs, some of which

are labelled in Fig. 3-2D. HSF was found to colocalize with Pol II at these sites (Fig. 3-2C).

Compared to the 21 OC control, the number of bands observed in the 5 mM azetidine-

treated condition decreased to 120- 130 (Fig. 3-2F). Of the sites staining for Pol II, on average

Figure 3-2. Azetidine treatment causes hyperphosphorylated RNA Polymerase II to redistribute to the heat shock gene loci,

Salivary glands were dissected fiom D. melanogasfer third instar larvae and incubated in organ

culture for 1 h pnor to treatment. Chromosomal squashes were prepared h m glands that were

maintained at room temperature (21 OC) (A and B), heat shocked at 36.5 OC for 20 min (C and

D), or treated with azetidine (A&) as indicated (D-F). Chromosomes were stained for HSF

using a rabbit anti-HSF primary antibody (943), followed by a goat anti-rabbit FITC-conjugated

secondary antibody (A, C, E, and G). Hyperphosphorylated RNA polymerase II was stained for

using monocIonal CC-3 antibody followed &y Alexa 594 goat anti-mouse secondary antibody

that fluoresces red (B, D, F, and H). Prominent heat shock gene loci are indicated. The scaie bar

represents 10 Pm.

92 % were found to correlate with HSF staining. Fifty millimolar azetidine treatmeut resulted in

a m e r decrease in the number of bands staining for Pol II, with approximately 60-70 bright,

distinct bands visible (Fig. 3-2H). On average, 97 % of the bands staining for Pol 11 were found

to correlate with WSF staining-

While azetidine did not result in as great a decrease in Pol II staining as heat shock, we

again observed a concentration dependent effect. interestingly, we oaly saw a decrease in the

number of sites with Pol II during azetidine treatment if large heat shock gene puffs were present

at 87C and 87A, accompanied by high levels of HSF staining (result not shown). Results fkorn

this experiment closely paralleled the results f?om the BrUTP incorporation experiment (Fig. 3-

1). Previous results from our Iaboratory show an exact correlation between sites of transcription

and sites of Pol II binding on the same spread, in both control and heat shock conditions (Paraiso

and Westwood, unpublished results). Any spread to spread variation between the number of

sites of transcription and the number of sites of Pol II staining observed could be due to the

concentrations of the antibodies used to stain the preparations. For example, if the antibody used

to stain for BrUTP incorporation had been slightly more concentrated perhaps there would have

been a few more bands observed to stain for BrUTP incorporation, or perhaps the staining

intensity of very weak bands would have increased.

Azetidine induces the redistribution of RNA Polymerase 11 to heat shock gene loci, and subsequent transcriptional repression in hsf mutants

Given that heat and azetidine can induce the redistribution of Pol 11 to the heat shock

gene loci, we wanted to confirm that active HSF binding has a role in this process. We used the

temperature-sensitive Drosophila HSF mutant, hsf, which contains HSF with a single amino

acid mutation in the DNA-binding dornain rendering the protein unable to bhd HSEs at the non-

permissive temperature (>33 OC) (Jedlicka et al, 1997). If we observe the redistribution of Pol II

to heat shock gene loci and subsequent transcriptional repression following azetidine treatment at

the permissive temperature, but not at the non-permissive temperature, this will be suggestive of

a role for activated HSF in the redistribution process.

In both the control and heat shock conditions we observed approximately 160 sites of Pol

II binduig (Fig. 3-3B, Fig. 3-3D). There was no change in the distribution of Pol iI when

switched to the non-permissive temperature, however it should be noted that a greater arnount of

Pol II was observed in the control condition in wild type larvae (1 90-200 bands, Fig. 3-2B).

Treatment with 50 rnM azetidine resulted in a significant decrease in Pol II staining to 70-80

bands (Fig. 3-3F), similar to wild type larvae. Of the remaining bands staining for Pol II, on

average 9 1 % were correlated with HSF staining.

Treated glands were labelled with BrUTP, squashed and then immunostained with anti-

HSF and anti-BrdU antibodies. In the 21°C control a diffuse staining pattern for HSF was

obswved, comparable to that observed in wild type flies (Fig. M A ) . Under control conditions

transcription was abundant, with 190-200 bright and distinct bands staining for nascent

transcripts (Fig. 34B). In the heat shock control distinct bands of HSF staining were not

observed, as was expected (Fig. 3-4C). However, HSF still seemed to be loosely associated with

the chromatin, following the pattern of DNA banding. Comparable tu the 2 1 OC control, 200-2 1 O

discrete, bright bands representing nascent transaipts were observeci (Fig. 34D). While 50 mM

azetidine induced a pattern of HSF binding and heat shock gene puffing similar to that observed

in wild type larvae (compare Fig. 3-4E to Fig. 2-ZC), the degree of global transcriptional

Figure 3-3. Azetidine induces the redistribution of RNA Polymerase II to heat shock gene loci in hsf mutants.

Salivary glands were dissected from D. melanogaster hsf mutant third instar larvae and

incubated in organ culture for 1 h pnor to treatment. Chromosomal squashes were prepared from

glands that were maintained at room temperature (2 1 OC) (A and B), heat shocked at 36.5 OC for

20 min (C and D), or treated with 50 mM azetidine (A&) (E and F) for the indicated times.

Treatment with 50 mM aetidine was selected for use in this and subsequent experiments as this

particular concentration was observed to induce the most significant repression of non-heat

shock gene transcription and relocalization of RNA Polymerase II to heat shock gene loci in

Figures 3-1 and 3-2. Immunolocalization for HSF (A, C, and E) and RNA Polymerase II (B, D,

and F) was performed as descnbed in the legend to Figure 3-2. Prominent heat shock gene loci

are indicated. The scale bar represents 10 Pm-

Figure 3-4. Azetidine induces the activation of heat shock gene transcription and the repression of non-heat shock gene transcription in hsf mutants.

Salivary glands were dissected from D. melanogasfer hsf mutant third instar larvae and

incubated in organ culture for 1 h prior to treatment. Chromosomal squashes were prepared fiom

glands that were maintained at room temperature (2 1 OC) (A and B), heat shocked at 36.5 OC for

20 min (C and D), or treated with 50 mM azetidine (AzC) (E and F) for the indicated times.

Immunolocalization for HSF (A, C, and E) and BrUTP (B, D, and F) incorporation was

performed as described in the legend to Figure 3-1. Prominent heat shock gene loci are

indicated. The scale bar represents 10 Pm-

repression was not as great. One hundred fifty to one hundred sixty bands representing active

sites of transcription were observed (Fig. 34F)- Of the bands staining for BrUTP incorporation,

approximately 95 % were correlated with HSF staining.

HSF binds to major devdopmental loci follow'ng in vivo heat shock

Arnong the abundant number of non-heat shock gene sites where HSF binds following

heat shock are the developmentally important loci 74EF and 75% (Westwood et al., 199 1). Large

puffs, signiwng transcription, form at these loci in response to the mouiting homone ecdysone

(reviewed in Russellz 1996). Westwood et al. (199 t ) noticed regression in puff size in addition

to HSF binding at 74EF and 75B following heat shock. They suggested that HSF binding to

regulatory loci functions to shut down developmental programs until environmental conditions

become more favorable for growth and development.

Figure 3-5 shows the results of a re-creation of the experiment initially pertOrmed by

Westwood et al. (1991). Very late third instar larvae were selected (those with alrnost no blue

gut) in order to be able to observe the natural ecdysone puffs at 74EF and 75B. In the 21 OC

control large puffs are visible at 74EF and 7SB as rounded and somewhat elongated regions of

decondensed chromatin (Fig. 3-5A). Following heat shock of the whole larva, puffs at 74EF

and 758 were shorter in length, less rounded, and more condensed as indicated by the opacity of

the cluomatin in this region (Fig. 3-SC). Three intensely staining HSF bands were observed to

localize at 74EF and 758 following heat shock (Fig. 3-5D).

Ecdysone puff regression and repression of transcripîbn occur a? 74EF and 75B in response to heat

If our hypothesis holds mie, that HSF binding results in the repression of non-heat shock

genes, then a decrease in transcription fiom ecdysone-inducible genes should be observable. Our

Figure 3-5. HSF binds to major developrnental loci foîiowing in vivo heat shock.

To examine natural ecdysone puffs, late third instar Drosophiia Iarvae were maintaineci at room

temperature (21 OC) (A and B) or heat shocked (36.5 OC) for 25 minutes (C and D). Salivary

glands were then dissected out and the distribution of HSF on the chromatin was examined using

irnmunocytochemistry with the 943 antibody against HSF and goat anti-rabbit Rhodamine Red-

X-conjugated secondary antibody (B and D). Puff sizes at the ecdysone-inducible loci were

exarnined by staining the DNA with Hoechst (A and C). The ecdysone-inducible loci 74EF and

75B are indicated. The scale bar represents 10 Fm.

lab has unsuccessfblly attempted to use molecular techniques such as RT-PCR and primer

extension to prove that ecdysone-inducible gene transcription is repressed by heat shock. We

decided to use the BrUTP incorporation technique to see if we could observe a decreased signal

for nascent transcripts at 74EF and 75B following heat stress.

To perfonn these experiments we needed to be able to control the appearance of the

ecdysone puffs. Therefore, we performed d l ecdysone and heat treatments in organ culture.

Following BrUTP incorporation glands were squashed and subsequently immunostained using

anti-BrdU and anti-HSF antibodies.

To determine the level of transcription occurring at 74EF and 75B in response to

ecdysone, glands were treated with ecdysone for 1 h at 2 1 OC. Brightly staining, intense and

sizable bands were observed, localized within large puffs at the ecdysone-inducible loci (Fig. 3-

6B). To test the effect of heat shock on transcription fiom the 74EF and 75B loci, glands were

treated to one of three different conditions. The first two conditions involved heat pretreatment

to induce HSF binding before or concomitant with ecdysone exposure. The third condition

involved pretreatment with ecdysone, to induce the ecdysone-responsive genes, followed by heat

shock in the presence of ecdysone, to induce HSF.

In glands pretreated with a 20 minute heat shock at 36.5 OC followed by exposure to

ecdysone for 1 h, BrUTP incorporation at 74EF and 75B was much lower than what was

observed in the control condition (compare Fig. 3-6D to Fig. 3-6B). HSF binding was observed

along with very slight puffs at these loci (Fig. 3-6C). In glands heat shocked for 20 minutes at

36.5 OC in the presence of ecdysone, followed by an additional 40 minutes in ecdysone alone,

BrUTP incorporation at 74EF and 75B was again observed to be much Iower than in the

Figure 3-6. Ecdysone puff regression and repression of transcription occur at 74EF and 7SB in response to heat.

Salivary glands were dissected fiom D. melanoguster third instar larvae and incubated in organ

culture for 1 h prior to treatment. Chromosomal squashes were prepared fiom glands that were:

treated with 1 pM ecdysone for 1 h (A and B), pretreated with a 20 minute heat shock at 36.5 OC

followed by exposure to 1 pM ecdysone for 1 h in the presence of heat (C and D), heat shocked

for 20 minutes in the presence of ecdysone followed by an additional 40 minutes in the presence

of ecdysone done (E and F), or pretreated with ecdysone for 1 h followed by heat shock for 20

minutes in the presence of ecdysone (G and H). Squashes were immunostained for HSF (A, C,

E, and G) and BrUTP incorporation (B, D, F, and H) as describeci in the legend to Figure 3- 1.

Prominent heat shock gene loci are indicated. The scale bar represents 10 Pm.

ecdysone alone control condition (compare Fig. 3-6F to Fig. 3-6B). Slightly higher

incorporation at the ecdysone-inducible loci was observeci in this condition as compared to the

heat shock pretreatment. HSF binding and a reduction in puff size as compared to the ecdysone

control, were also observed (Fig. 36E). When glands were pretreated with ecdysone for 1 h

followed by a heat shock at 36.5 OC for 20 minutes in the presence of ecdysone, transcription at

the 74EF and 75B loci was no Ionger observed (Fig. 3-6H). Even though transcription was

cornpletely repressed, puff size was much larger than observed in the heat shock pretreatment

(Fig. 3-6G).

Regardless of whether glands were exposed to heat shock pnor to or following ecdysone

exposure, a decrease in transcription at 74EF and 75B was consistently observed. Taking into

account our previous observations, that Pol II redistributes to the heat shock gene loci following

heat stress, we next wanted to examine Pol II staining at the ecdysone-inducible loci. When

glands were treated with ecdysone for 1 h large puffs were observed at 74EF and 75B with

strong signals for Pol II staining localized within the puffs (Fig. 3-7B). When glands were

pretreated with a 20 minute heat shock at 36.5 OC the ecdysone puffs had regressed in size and

strong HSF binding was observed (Fig. 3-7C), similar to what was seen in Fig. 3-6. However, a

fairly intense signal for Pol II binding that had an intensity equal to or slightly less than the

control, remained at these loci (Fig. 3-7D). Heat shock treatment concurrent with ecdysone

exposure yielded a very similar result (Fig. 3-7E and Fig. 3-7F). When glands were pretreated

with ecdysone and followed by heat shock in the presence of ecdysone, both puffing and HSF

binding were observed at 74EF and 758 (Fig. 3-7G). The level of Pol II staining was much

lower than the control and heat shock pretreated conditions (Fig. 3-7H).

Figure 3-7. Ecdysone puff regression and decreased RNA Polymerase LI staining are obsewed at 74EF and 7SB in response to heat.

Salivary glands were dissecteci fkom D. melanogaster third instar larvae and incubated in organ

culture for 1 h pnor to treatment. Chromosomal squashes were prepared €rom glands that were:

treated with 1 p M ecdysone for I h (A and B), pretreated with a 20 minute heat shock at 36.5 OC

followed by exposure to 1 p M ecdysone for 1 h in the presence of heat (C and D), heat shocked

for 20 minutes in the presence of ecdysone followed by an additional 40 minutes in the presence

of ecdysone alone (E and F), or pretreated with ecdysone for 1 h followed by heat shock for 20

minutes in the presence of ecdysone (G and H). Squashes were irnmunostained for HSF (A, C,

E, and G) and RNA Polymerase II (B, D, F, and H) as described in the legend to Figure 3-2.

Prominent heat shock gene loci are indicated. The scale bar represents 10 pm.

Azetidine fieutment resulks in ttanscr@tr'onaf repressiioi, ut 74EF and 7SB foflowing ecdysone induction

Since the initial observation by Westwood et al. (199 1) that heat shock causes ecdysone-

induced puffs to regress, the remaining question has been whether or not puff regression is due to

an effect of heat itself or to active HSF. We wanted to rule out the effects of heat by using a

non-heat shock inducer. Again we selected the proline amino acid analogue azetidine. When

glands were pretreated with 50 mM azetidine for 2 h followed by ecdysone treatment in the

presence of azetidine for 1 h, fairly intense staining for both BrUTP incorporation (Fig. 3-88)

and Pol II (Fig. 3-8D) was observed at 74EF and 75B. Though a significant reduction in

transcription was not observed, the puffs did appear to have regressed in size as compared to the

controls in Fig. 3-6B and Fig. 3-88. Strong signals for HSF binding at 74EF and 75B were also

observed (Fig. 3-8A and Fig. 3-8C).

When glands were pretreated with ecdysone for 1 h followed by treatment with 50 mM

azetidine for 2 h in the presence of ecdysone, HSF binding was apparent but there was no puffing

discemible at 74EF and 758 (Fig. 3-9A and Fig. 3-9C). A control experiment demonstrated that

puffing did not regress when glands were treated with ecdysone alone for 3 h (results not

shown). Staining for BrUTP incorporation at the ecdysone-inducible loci was reduced compared

to both the control and the azetidine pretreated conditions (Fig. 3-98). The level of staining for

Pol II was also very low (Fig. 3-9D).

Figure 3-8. Azetidine pretreatment shows only a modest effect on transcriptional repression at 74EF and 75B.

Salivary glands were dissected tiom D. melamgaster third instar larvae and incubated in organ

culture for 1 h prior to treatment. ChromosomaI squashes were prepared fiom glands that were

pretreated with 50 rnM azetidine for 2 h followed by exposure to 1 p M ecdysone for 1 h in the

presence of azetidine. Squashes were immunostained for HSF (A and C), BrUTP incorporation

(B), and RNA Polyrnerase II (D) as described in the legends to Figure 3-1 and Figure 3-2.

Prominent heat shock gene loci are indicated. Each scale bar represents 10 Pm. The scale bar in

Panel A also represents Panel B. The scale bar in Panel C also represents Panel D.

Figure 3-9. Azetidine treatment results in transcriptional repression at 74EF and 758 following ecdysone induction.

Salivary gIands were dissected from D. melanogaster third instar Iarvae and incubated in organ

culture for 1 h prior to treatment. Chromosornai squashes were prepared from glands that were

pretreated with 1 p M ecdysone for 1 h followed by treatment with 50 m . azetidine for 2 h in the

presence of ecdysone. Squashes were immunostained for HSF (A and C), BrUTP incorporation

(B), and RNA Polymerase II (D) as described in the legends to Figure 3- 1 and Figure 3-2.

Prominent heat shock gene loci are indicated. The scale bar represents 10 Pm.

E. DISCUSSION

Changes in transcriptional activity in response to heat stress have been well documented

in Drosophila. While transcription of the heat shock genes is greatly increased, the synthesis of

most other messenger RNAs is suppressed and pre-existing mRNAs are stabilized within the

nucleus (Ashburner, 1970; Tissieres et aL, 1974; McKenzie et al., 1975; Sprading et a[., 1975;

Yost et ai., 1990). in addition, nomal protein synthesis ceases, accompanied by a rapid

disappearance of poiysomes ( M c K e ~ e et al-, 1975). Much of the litmature therefore niggests

that in addition to transcriptional conbol, the heat shock response is also largely regulated at the

level of translation (Lindquist, 1980, 198 1 ; DiDomenico et al., 1 982b; Ballinger and Pardue,

1983; Lindquist, 1986). The repression of normal protein synthesis observed in Drosophila cells

following heat shock is not observeci to near the same degree in mammalian cells, as can be

observed when cells are labelled with 35~-methionine following heat shock (Westwwd and

Steinhardt, 1989). To date, heat shock has been shown to inhibit the expression of only a

handfùl of genes in mammalian cells, including cytokine-inducible nitric oxide synthase (De

Vera et al., l996a, 1 996b; Wong et al., 1 W6), IL- 1 B (Schmidt and Abdulla, 1988; Cahill et al.,

1 996), tumor necrosis factor (Snyder et al., 1 992; Ensor et al., 1 994), and c-fos (Chen et al.,

1997).

One question that has interested us is whether the transcriptional repression that occurs in

Drosophila is a secondary effect of heat itself. Most other studies examining the repression of

transcription in Drosophila have utilized heat to induce the stress response. To answer this

question we decided to look at the effeçts of the proline amino acid analogue azetidine on

transcription during the stress response. While we observed transcriptional activation of the heat

shock genes, we also saw a concentration dependent decrease in transcription of most other

genes. Accompanying this was the relocalization of RNA polymerase II to the heat shock gene

loci. This indicated to us that the repression of transcription obsewed was due to the

relocalization of Pol II to the heat shock genes, and M e r îhat this was likely an effect of

activating the stress response itself.

Temperature-dependent changes of gene expression have previously been observed in

Drosophila. Rising ternperature can be viewed as a hyperthennic gradient, with the optimal

synthesis of certain hsps occurring at certain points dong the gradient. For example in

Drosophila, the optimal synthesis of hsp83 occurs at 33 OC, whereas the optimal synthesis of

hsp70 occurs at 37 OC (Lindquist, 1980). Spradling et al. (1977) demonstrated the accumulation

of specific heat shock mRNAs at different ternperatures. Correlated with this, the various heat

shock gene puffs show characteristic temperatures of induction and exhiiit increasing puff size

with increasing temperature (Ashbumer and Bonner, 1979).

Given that certain temperatures induce the optimal synthesis of certain hsps, it seems

inherent that the repression of gene transcription should also occur dong a gradient. Indeed,

when Drosophila tissue culture cells are heat shocked at various ternperatures and then labelled

with "s-methionine or 'H-leucine, non-heat shock protein synthesis is observed to be gradually

repressed with each increase in ternperature (Lindquist, 1980; Ballinger and Pardue, 1983;

Westwood and Steinhardt, 1989). Lindquist (1980) has observed that the levels of certain

proteins norrnally synthesized at 25 OC slightly increase and then decrease with rising

temperature. For example, between 23-3 1 OC, 3~-leucine incorporation inmeases. At 33 OC

incorporation falls to 50-75 % of the maximum and at 37 OC incorporation M e r decreases to 2-

10 % of the maximum. Spradling et al. (1977) have shown that there is a gradual repression in

previousl y active gene transcription with increasing ternperature in Drosophila cells. Evidence

from our lab has shown that when whole larvae are heat shocked at 36.5 OC approximately 40

sites of transcription are visible, including sites at the major heat shock gene puffs.

Approximately 90 % of these remaining sites of transcription show a correlation with HSF

staining, indicating that these additionai transcriptionally active sites are also stress-inducible

genes. We designate these 'minor' heat shock genes as they are yet to be identified. On the

other hand, severe heat shock at 38 OC will completely repress transcription fkom these minor

heat shock genes, leaving only the major heat shock loci active (Paraiso and Westwood,

unpublished results). This also indicates an effect of temperature on gene expression.

In the current work, we observed transcription to be repressed in a manner comeiating

with the severity of the stress. Higher azetidine concentrations resulted in a higher degree of

global transcriptional repression and loss of Pol II staining. In contrast to heat, azetidine appears

to have less of an effect on the repression of the minor heat shock genes that are still observecl to

be active at 36.5 OC. After 2 h of 50 m M azetidine treatment there were 50-60 sites of BrUTP

incorporation and 60-70 sites of Pol II staining observed. At least 90 % of the time both sites of

BrUTP incorporation and sites of Pol II binding were observed to correlate with HSF staining.

After 4 h of 5 mM azetidine treatment there were 100-1 10 sites of nascent transcription and 120-

130 sites of Pol II staining, again correlating with HSF staining at least 90 % of the time. Given

the presence of HSF staining correlating with sites of active transcription following azetidine

treatment it is possible that the remaining sites are minor heat shock genes, a subset of which are

the minor heat shock genes observed to be active following 36.5 OC heat shock. ïhis would be

in accord with the original hypothesis of Westwood et al. (199 1 ), that many of the sites of HSF

binding following stress are actually minor heat shock genes.

I f we were to think of transcriptional repression occurring in a hierarchical manner,

whether it be in response to heat or arnino acid analogue treatment, one could imagine non-heat

shock genes being repressed first. At temperatures of 36.5 OC or greater, repression occurs very

rapidly, whereas with amino acid analogue treatment repression takes longer due to the initial

period of protein synthesis required to induce the response. While heat and azetidîne both appear

to induce the repression of transcription from non-heat shock genes, both inducers also result in

activation of transcription of the heat shock genes, Judging fkom the results of inducing salivary

gland cells with 5 mM azetidine, there are potentially up to 100 minor stress-inducible genes that

are yet to be identified. It also appears that a certain subset of the minor heat shock genes

induced by 5 mM azetidine are repressed by higher concentrations of analogue and also heat

treatment. Moving dong the gradient, it is apparent that a certain subset of the minor heat shock

genes remaining afier 50 mM azetidine treatment are repressed by 36.5 OC heat treatment. The

minor heat shock genes remaining afier a 36.5 OC heat shock appear to be repressed only by

more severe heat shock at 38 OC. The genes remaining active after this type of treatment are the

major heat shock genes. HSF binding appears to rernain even afier repression of the minor heat

shock genes occurs. More work will be required to determine what allows certain heat shock

genes to remain active while others are turned off.

While it was apparent that the relocalization of Pol II to the heat shock genes following

stress results in the repression of al1 other genes, we still wanted to confirm the involvement of

HSF binding to the chromatin. To do this we used the temperature-sensitive Drosophila HSF

mutant, hsf . Redistribution of Pol II and repression of gene transcription following azetidine

treatment at the permissive temperature, but not at the non-permissive temperature, would be

suggestive of a role for active HSF in the redistribution process. There was no change in the

nurnber of bands staining for Pol II between the control (permissive temperature) and heat shock

(non-permissive temperature) conditions. Treatment with 50 mM azetidine resulted in a

significant decrease in Pol II staining, confinning that DNA-bound HSF does play a role in the

relocalization of Pol 11 in response to stress. Lending support to this conclusion, Pol II staining

was only observed to decrease in response to azetidine when large puffs at the heat shock gene

loci were observable. indirectly this hints at the requirement for active HSF in the relocalization

process, as heat shock gene puffing usually correlates with active transcription and thus active

HSF.

With regard to transcription, both the control and heat shock conditions showed a sirnilar

staining pattern in the hsf mutants whereas 50 m M azetidine treatment did not result in as great

a decrease as would have been expected, compared to the level of Pol II staining observeci in Fig.

3-3F. The high level of staining might again be attributable to the concentration of antibody

used. If the antibody were more concentrated than usual, bands that would normally stain with

low intensity would appear brighter, resulting in an apparent overall increase in staining.

Previous work by Westwood et al. (1 99 1) demonstrateci HSF binding at the ecdysone-

inducible developrnental loci 74EF and 75B in response to heat. They suggested that HSF

binding at these loci fbnctions to shut down developmental programs until growth conditions

become more favorable. Greenleaf er al. (1978) and Shopland and Lis (1996) have both

observed a loss of RNA polymerase II staining at these loci in response to heat shock. In

keeping with Our hypothesis, transcription and Pol II staining at the ecdysone-inducible, non-heat

shock gene loci should decrease following heat and azetidine treatment; a direct result of Pol 11

being drawn from these genes to the heat shock gene loci.

When glands were pretreated with heat to induce HSF binding, followed by ecdysone

treatment to induce the ecdysone genes, we consistently observed a low signal for transcription

and Pol II staining at 74EF and 75B. This can be explained by the simple fact that the response

had begun to attenuate during the ecdysone induction period. In contrast, when glands were

pretreated with ecdysone to induce the ecdysone genes, followed by heat shock in the presence

of ecdysone to induce HSF, there was no signal apparent for transcription or Pol II binding. This

indicated that HSF binding is best able to repress active non-heat shock gene transcription while

the heat shock response is tùlly induced. A slight arnount of puffing was still observable in the

ecdysone pretreated condition, even in the absence of transcription. Ja-ch et al. (1977) have

previously observed that puffs do not imrnediately regress after Pol II binding ceases. In

addition, puffing does not always necessarily signifi transcription (Winegarden et al., 1996).

When these expenments were repeateà using azetidine instead of heat, the effects of

azetidine were not as strong as those of heat. When glands were pretreated with azetidine to

induce HSF binding, followed by ecdysone treatment to induce the ecdysone genes, fairly strong

signals for transcription and Pol II binding at 74EF and 75B were observable. These signals were

stronger than when heat was used, likely because cells exposed to amino acid analogue do not

exhibit attenuation of the stress response. In contrast, when cells were pretreated with ecdysone,

followed by azetidine treatment in the presence of ecdysone, alrnost undetectable signals for

transcription and Pol II binding at 74EF and 75B were observed. During the sarne time period, in

the presence of ecdysone alone, the ecdysone puffs do not attenuate, indicating that the

transcriptional repression observed must be due to HSF binding induced by azetidine.

The results of this study indicate that HSF binding to the polytene chromosomes in

response to stress Iikely has a role in the preferential recruitment of Pol II to the heat shock gene

loci and the resulting decrease in non-heat shock gene transcription. How might active HSF

accomplish this? The HSF activation domain is as strong as, i f not stronger than, the VP16

activation domain (Borner et al., 1992; Newton et al., 1996)- When HSF binds to major and

minor heat shock gens upon stress, perhaps the activation domain plays a role in attracting Pol

II exclusively to the heat shock gene sites. This is an wticing possibility, however more work

will be required to c o n h this.

CHAPTER 4

General Discussion

A. Possible funmkn(s) of HSF binding & sites otker than the major keat dock gene loci

Westwood et ai. (1991) fïrst observed HSF binding at over 200 loci on polytene

chromosomes of Drosophila meianogaster following heat shock. included among these sites

were those of the major heat shock gene pues; however, HSF staining of intemediate intensity

was also observed at approximately 156 other sites on the chromatin. In explanation of HSF

binding to so many loci Westwood et al. (1991) suggested that some sites, such as 48E and

88EF, could be accounted for as k i n g previously documented minor heat shock genes. For

example, 88EF contains the heat shock cognate gene hsc4. Interestingly, Westwood et ai. (1 99 1)

observed HSF to bind at five out of six sites containing heat shock cognate genes Messenger

RNAs and cDNAs fiom heat shocked Drosophila cells have previously been shown to hybridize

with nurnerous sites on polytene chromosomes (Spradling et ai., 1 977; Lis et aL, 198 1) although

Westwood et al. (1991) documented HSF binding at only 9 of these sites. Westwood et ai.

( 199 1) also suggested that some sites could be those of genes which continue to transcribe during

heat shock, such as the histone genes, or they could simply be random accessible binding sites

occurring statistically in the genome. HSF binding was also mapped to the ecdysone-inducible

loci at 74EF and 75B, plus an additional 47 out of 125 loci documented by Ashburner (1972) to

puff on the last day of larval life. In this case, Westwood et al. (1 99 1 ) hypothesized that HSF

binding was fùnctioning to repress transcription from developmental genes under conditions of

stress.

In the current study, azetidine induced HSF binding on polytene chromosomes in a

pattern very simiIar to that induced by heat, resulting in approximately 200 HSF binding sites

folIowing the optimal time of treatment for a given concentration, With low azetidine

concentration (5 mM) around 100 sites of Pol IVtranscription were observed, showing at least 90

% correlation with HSF binding. With high azetidine concentration (50 mM) around 50 sites of

Pol II/transcription were observed, again showing at least 90 % correlation with HSF binding.

Previous findings fiom Our lab have demonstrated approximately 40 sites of Pol II staining

correlated with HSF binding following heat shock at 36.5 OC. Given the colocalization of Pol II

and HSF, the sites remaining following 36.5 OC heat shock or azetidine treatment are likely those

of minor heat shock genes plus the major heat shock genes already known. Mapping of

cytological loci will be required to determine if the same sites remain active following treatment

with both heat and azetidine. The fact that following 5 m M azetidine treatment approximately

100 transcriptionally active sites correlate with HSF binding, raises the interesting possibility

that there are more uncharacterized minor heat shock genes than previously thought. This will

be eiaborated upon in the following section.

If there are on the order of 100 heat shock genes, both major and minor, there are still up

to 100 other HSF binding sites to account for. They could be developmental loci, or random

binding sites as suggested by Westwood et al. (199 1). The possibilities remain that HSF is not

specifically bound at these sites but rather is aggregated on the chromatin, or even that it is Pol II

attracting HSF to the chromatin. An experiment to prove that HSF is actually bound to the

chromatin would involve the construction of BrUTP IabeIled probes for HSEs. Following

irnmunostaining, the nurnber of possible HSF binding sites on the chromatin could be counted.

B. HSF binding plays a role in transcn'ptional repression

In the current work azetidine was observed to activate transcription of major, and most

likely minor heat shock genes. Azetidine treatment also resulted in non-heat shock gene

repression. In addition, a repression effect was observed on the minor heat shock genes. That is,

far fewer minor heat genes were observed to be active in the 50 mM azetidine-treated condition

as compared to the 5 inM condition.

We hypothesize that HSF binding at heat shock gene loci results in transcriptional

repression of non-heat shock genes because Pol II is drawn away fiom the non-heat shock gene

sites by active HSF. In other words, there is a preferentiai recniitrnent of Pol II to the heat shock

gene loci due to HSF binding. As discussed in Chapter 3, we were able to confirm the role of

HSF binding by treating Drosophifa hsf mutants with azetidine-

With regard to the repression effect observed on the minor heat shock genes, we suggest

that these genes are repressed in a hierarchical rnanner. That is, as treatment with azetidine

continues, or as conditions become more stressful (e-g. higher azetidine concentrations or higher

ternperatures), minor heat shock genes begin to be repressed. The rninor heat shock genes

remaining active under high stress conditions would be repressed only by severe conditions (e.g.

38 OC heat shock), and henceforth only the major heat shock genes would remain active as

discussed in Chapter 3. Even after transcriptional repression has occwed at a particular site,

HSF appears to remain bound to the chrumatin. Cytological mapping will be required to c o n h

this at specific sites.

This leads to the question of whether most of the HSF binding sites observed after heat

shock or maximal induction with azetidine, were actually stress-induced at some point.

Westwood et al. (1991) initially counted 156 sites of intermediate staining intensity (not

including 8 of the 9 major heat shock puffs), and 39 sites of weak staining intensity. The 39

weakiy staining sites were suggested to be random sites of HSF binding, however the additional

156 loci al1 have the potential to be sites of minor stress-inducible genes. Five millimolar

azetidine treatment for 4 h resulted in around 100 sites of active transcription correlateci with

HSF binding. It could be possible that at a point earlier than this there were achially

approximately 1 50- 160 sites of active transcription colocalized with HSF binding, accounting for

the 156 HSF binding sites observeci by Westwood et al. (1991). As treatment time progressed,

polyrnerase was recniited from minor heat shock genes in a hierarchical manner. Even though 2

h of 50 mM azetidine treatment appeared to result in a higher level of repression than 4 h of 5

mM azetidine treatment, a longer time of treatment with the lower concentration would most

likely result in an equivalent level of repression Given that there are au estimami 12 000-14

000 genes in the Drosophila genome (Miklos and Rubin, 1996), the possibility of there being

upwards of 100 minor stress-inducible genes does not seem unlikely.

How is active HSF, bound at the heat shock genes, able to draw Pol II away fiom non-

heat shock genes leadïng to transcriptional repression? Our explanation cornes by way of

comparing HSF to the transcriptional activator VP16. VP16 will compete with Pol II for

interaction with TBP, as will HSF. However, HSF is able to compete with both VP16 and Pol II

for binding to TBP (Mason and Lis, 1997). In addition to this, the HSF activation domain has

been shown to be as strong as or stronger than the VP 16 activation domain, which itself is a

strong acidic transcriptionai activator (Borner et al., 1992; Newton et aL, 1996). The fact that the

HSF activation domain is so potent leads us to think it is able to attract Pol II away fkom active

non-heat shock gene sites, preferentially recruiting Pol II to the heat shock gene loci.

We have also discussed the possibility that following activation minor heat shock genes

are repressed in a hierarchical manner based on the exposure t h e or severity of the stress. What

causes certain minor heat shock genes to be repressed before others? Related to this question,

what allows the major heat shock genes to keep transcribing, even under severe conditions of

stress (e.g. 38 OC)? There m u t be something special about the heat shock genes themselves.

Perhaps it is the architecture of the chromaiin, the number of heat shock elernents present, or

maybe other protein factors bind at heat shock gene promoters allowing for transcription to

continue during stress. Any of these factors could Vary between minor heat shock genes or

groups of minor heat shock genes. As the severity of stress inmeases, or induction time

lengthens, any of these factors could become compromised. Depending on the characteristics of

the various minor heat shock gene(s), certain genes rnight then be repressed before others. This

same reasoning could be applied to explain why only the major heat shock genes remain active

afier 38 OC heat shock.

At this point we also can not exclude the possibility that higher temperatures inhibit the

phosphorylation of Pol IIA, thus inhibiting the transcription of non-heat shock and minor heat

shock genes. This could also explain why we do not see complete transcriptional repression of

minor heat shock genes at high azetidine concentrations. Results fiom our lab have indicated

that there is slightly less staining for Pol 11 (using CC-3 antibody) in hsf mutants afier 38 OC

heat shock as compared to 36.5 OC heat shock. in these particular mutants heat should have no

eflect on HSF binding and hence transcriptionai repression (Paraiso and Westwood, unpublished

observations). The slight decrease in transcription observed could therefore be due to an

inhibiting effect of heat itself on Pol II.

To Our knowledge, the mass binding of a transcriptional activator to DNA, resulting in

the widespread repression of transcription has not been previously reported. There are certainly

numerous examples o f transcriptional activators which also tùnction as transcriptional

repressors, albeit of specific genes. These include p53 (reviewed in Ko and Prives, 1996), Sp3

(Ihn and Trojanowska, I997), c-myc (Lee et al., 1996; Lee et al., 1997), and YY 1 (Bushmeyer et

al., 1995).

Transcriptional repressors c m act by one of several mechanisms. For example, a

repressor might use an active and direct mechanism, binding directly to basal transcription

factors or RNA polymerase II. Altematively, repressors can act through a passive mechanism,

competing with Pol II for binding to DNA or other proteins such as TBP (Hanna-Rose and

Hansen, 1996). Tt seems that HSF acts as a repressor of non-heat shock gene transcription

primarily through an indirect mechanism. By binding to heat shock genes, HSF appears to create

a cornpetition with other g e n s for Pol II. Having such a strong transcriptional activation

domain, HSF wins out over other transcriptional activators and Pol II is preferentially recniited

to the heat shock genes.

C. HSF bhding at ecdysone-inducible loci

We had originally hypothesized that HSF could be binding in the promoter andor other

gene elements of the genes at 74EF and 7SB, blocking movement of the polymerase, or maybe

even disrupting the ecdysone receptor-ultraspiracle heterodimer required for ecdysone gene

transcription (Russell, 1996). In marnrnalian cells, HSF has been found to bind to a heat shock

element located withui the promoter of the Prointerleukin 1 p gene (Cahill et a/., 1996). Such an

interaction would block transcription of the gene by Pol II. HSF has also been shown to repress

the transcription of the c-fos gene. In this instance HSF does not act by binding in the promoter,

but likely through an interaction with an upstream signal transduction component or CO-activator

(Chen et al., 1997).

Tt now appears that transcriptional repression in Drosophila does not occur by the same

mechanism as in mammalian cells. According to our hypothesis the g e n s at 74EF and 75B,

being non-heat shock genes, should be repressed as Pol II is attracted away and towards the heat

shock genes. This was hdicated by the cytological evidence obtained in the current study. It is

interesting to think that the repression of specific gene transcription by HSF in Drosophila does

not occur per se, but rather happens as a result of the transcriptional downreguiation of the entire

complement of non-heat shock genes in response to stress.

The question rernains as to why HSF binding occurs at 74EF and 75B. It wuld be

suggested that Pol II is what is attracting HSF to these sites, but HSF is observed to bind at 74EF

and 75B even when the genes are inactive. HSF staining at these loci is fairIy bnght, indicating

that there may be multiple HSEs present. Thus random binding at these sites does not seern a

Iikely answer either. More work will be required to answer this question.

D. 1s there a universaï inducimg signal of the stress response?

Our main goal in using azetidine as an inducer of the heat shock response in Drosophila

was to help rule out secondary effects that heat might exert on transcriptional repression

occurring with ce11 stress. Use of different inducers has been important in the past for attempting

to detemine the way in which the stress signal is transduced by the cell. That is, do al1 induca

generate a single cornrnon signal or do multiple signals feed into a common pathway? Only by

the study of different inducers are we able to gain insight into this question.

Many of the known inducers generate unfoldeci or abnormal proteins. In fact some work

now shows that most if not al1 inducers of the heat shock response including heat, are capable of

triggering thiol oxidation. In turn this leads to cross-linking between and within proteins

resulting in protein unfolding (Zoe et ai., 1998; Freeman et a[., 1999). However, evidence is

mounting that in addition to causing protein unfolding certain inducers have the ability to

activate HSF directly. This was first indicated when HSF binding to DNA was induced by low

pH in crude human and Drosophifa ce11 extracts (Mosser et al., 1990; Zimarino et al., 1990b).

Heat and hydrogen peroxide have now been shown to induce the trimerization and DNA-binding

of purified Drosophifa HSF, indicating that these inducers are able to act directiy on HSF (Zhong

et ai., 1998). Low pH has also been demonstrated to activate purified Drosophifa HSF directly,

and can synergize with the actions of heat and oxidation (Zhong et al., 1999). Zhong et a L

(1999) hypothesize that in vivo, heat stress and other inducers can activate HSF due to moderate

intracellular acidification. The effects of low intracellular pH could involve changes in salt

bridges or hydrogen bonding leading to destabilization of the monomeric form of HSF and hus

trimerization. Alternately, changes in sait bridges or hydrogen bonding could lead to the

stabilization of the HSF trimer (Zhong et al., 1999). There is also the possibility that heat alone

could have a slight effect on the conformation of active HSF, resulting in a more

therrnod ynamicall y stable structure at increased temperatures.

In conflict with these findings, I)nimmond et ai. (1986) found that though heat shock

resulted in a decrease in intracellular pH in Drosophifa, this condition was neither sufficient nor

required for activation of the heat shock response. Many inducers such as amino acid analogues,

heavy metals, and arsenite do not have the effect of lowering pH (Zhong et al-, 1999). lnstead

these inducers act through indirect mechanisms, the most obvious being the accumulation of

abnormal protein. Thus the effect of lowered pH within the ce11 could be to contribute to the

generation of abnormal proteins (Mosser et al., 1990).

While our investigations with azetidine did not provide any m e r information as to how

the stress signal is transduced, a potential effèct of heat on HSF may have been observed. Fi@

millimolar azetidine treatment induced almost the sarne level of transcriptional repression as 36.5

OC heat shock. It is possibte that a longer time of treatment with azetidine would have resulted in

a Ievei of repression equivalent with heat shock, with Pol II localized at exactly the same loci on

the chromatin in each condition. However, there also remains the possibility that heat has a

direct effect on HSF itself, resulting in stronger transcriptional repressive effects.

E. Future Directions

Future directions for this work will involve c o ~ a t i o n of the involvernent of the HSF

activation domain in the preferential r&tment of RNA polymerase U from non-k t shock

gene sites to the heat shock gene loci. To accomplish this, several transgenic fly lines will be

constructed. Into a wild type or hsf (nul1 mutant) (Jedlicka et al., 1997) background, each one

of the following wiil be inserted (among others): (i) Drosophila HSF with the activation domain

deleted, (ii) the GAL4 DNA-binding domain joined to the Drosophila HSF activation domain,

and (iii) the HSF DNA-binding domain joined to the VP 16 activation domain. Salivary glands

will be dissected fiom each of these lines and treateci with heat or azetidine. Similar to the

current study, salivary gland squashes will be performed and irnrnunostained for transcription,

Pol II, and HSF.

Each one of these transgenic fly lines will give us an important piece of information.

Foremost, we would not expect to see repression of transcription at non-heat shock gene loci or

the recruitment of Pol II to the heat shock genes in flies containing HSF with the activation

domain deleted. Fusing the HSF activation domain to the GAL4 DNA-binding domain should

tell us if the activation domain of HSF is sufficient for the repression effeçts observed. Fusions

of the HSF DNA-binding domain ta the VP 16 activation domain have previously been shown to

exhibit activity much like intact HSF (Borner et al., 1992) and so we would expect to see the

stress-induced repression of non-heat shock gene transcription in this fly line.

Another important experïment will involve the use of Drosophila 'gene chips', which

have thousands of cDNAs arrayed ont0 a microscope slide, Using Cy-3 or Cy-5 labelleci cDNA

fiom control, heat shocked, or azetidine-treated Drosophila cells, we would be able to look for

repressed genes at the genomic level. This would provide an easy metiiod for iden t img the

minor heat shock genes.

In the current work we have taken a stride towards understanding why non-heat shock

gene transcription is repressed during ce11 stress. RNA polymerase II was observed to reloçalize

at the heat shock gene loci in response to an inducer other than heat, proving that this effect is

due to the activation of HSF. In addition, it now seerns that the heat shock response is largely

regulated at the Ievel of transcription. Determining exactly how HSF mediates the preferential

recruitment of RNA polymerase iI fiom non-heat shock gene sites to the heat shock gene loci

will add yet another layer of complexity to the regulatory processes already known to govern

HSF in Drosophila.

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