Function and regulation of human and soybean heat shock transcription factors expressed in yeast and HeLa cells

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Function and regulation of human and soybean heat shock transcription factors expressed in yeast and HeLa cells
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Yuan, Chao-Xing, 1962-
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FUNCTION AND REGULATION OF HUMAN AND SOYBEAN HEAT SHOCK TRANSCRIPTION FACTORS EXPRESSED IN YEAST AND HELA CELLS By CHAO-XING YUAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1996

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1 ACKNOWLEDGMENTS The author is deeply grateful to his mentor. Dr. William B. Gurley, for his valuable advice, guidance and support throughout the course of this research. Thanks are due to Dr. Francis C. Davis, Dr. Robert J. Ferl, Dr. Charles L. Guy and Dr. Donald R. McCarty for their advice and guidance while serving as members of the advisory committee. The author would like to thank Drs Karen Koch for the initial training in her laboratory. The author also like to thank Dr. Eva Czarnecka-Verner and Don Baldwin for their helpful discussions as well as for providing biological materials and expertise. The author wishes to extend especial thanks to Brian J. O'Donnell, Dr. Stephen G. Zam, Dr. Samuel R. Farrah, Drs. Sally L. Mackay and Drs. Barbara A. Torres for their expertise in mammalian cell tissue culture. Special thanks are due to Dr. James B. Flanegan for precious HeLa cell nuclei. The author would like to thank Dr. Roy A. Jensen and his students as well for use of their thermocycler ii

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TABLE OF CONTENTS ACKNOWLEDGEMENTS ii ABSTRACT X INTRODUCTION 1 REVIEW OF LITERATURE 8 Heat Shock Response and Heat Shock Proteins 8 Function, Structure and Regulation of Heat Shock Transcription Factors 9 Function of HSFs and DNA Binding 9 Characteristics of the DNA Binding Domain 10 Properties of the Oligomerizat ion Domain 12 Attributes of Activation Domain 14 Role of Masking in Yeast HSFs 16 Trimerizat ion 19 Regulation of HSFs by Cellular Proteins 20 Role of HspVO 21 Phosphorylation of HSFs 23 The Steps of HSF Regulation 24 Mechanism of Transcriptional Activation 26 Transcriptional Activation and Promoter Structure 26 Models of Transcriptional Activation by Activators .... 27 Synergism of Transcriptional Activation 29 Transcriptional Initiation: A Key Step for the Regulation of Transcriptional Activation 31 Transcriptional Elongation: in Special Cases Expected to Achieve Transcriptional Activation .... 33 Targets of Activators in Transcriptional Activation 35 TBP and TFIID in Transcriptional Activation 35 TAFs of TFIID in Transcriptional Activation 39 Involvement of TFIIB in Transcriptional Activation .... 42 Positive Cofactors in Transcriptional Activation 44 MATERIALS AND METHODS 48 Yeast Strains and Growth 48 Yeast Vectors and their Derivatives 48 Construction of HSF Mutants in Yeast Vectors 52 Yeast Two-Hybrid System Screening 54 Site-Directed Mutagenesis 55 PCR-Mediated Random Mutagenesis 55 Heat Shock Treatment and (3-gal Assay in Yeast System 56 Western Blot Analysis of GmHSF in Yeast 56 Production of Antibodies against GmHSF34 57 GST-Fusions and Pull-down Assay 58 iii

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HeLa Cell Transfection and Heat Shock Treatment 62 HeLa Cell Nuclear Extract and Far-Western 64 The Squelch-Rescue Assay for Analysis of in vivo Interactions 64 Co-immunoprecipitation 67 Pull-down Assay of hTBP Deletions Transiently Expressed in HeLa Cells 68 hTFIID Complex Pull-down by GST-fusions of hHSFl 70 RESULTS 71 The Function and Structure Studies of HSFs from Human and Soybean 71 Mapping the Transcriptional Activation Domains of hHSFl and hHSF2 in Yeast and HeLa Cells 71 Identification of Subdomains within the Transcriptional Activation Domain of hHSFl 79 Attempts at Domain Mapping of Soybean HSF34 and HSFS in Yeast 87 Substitution of scHSF with HSFs from Human and Soybean Cells 91 Activity of hHSFl and 2 in Yeast under Sustained Heat Shock 94 The Functional Targets of hHSFl in Transcription 96 Strategies Used to Characterize Protein-Protein Interactions in vitro and in vivo 96 In vitro and in vivo interactions between hHSFl and hTBP 100 Interactions between hHSF and hTFIIB 110 In vitro and in vivo interaction between hHSFl and hTAF32, 55 and PC4 113 Use of the Yeast Two-hybrid System to Screen for Proteins Interacting with hHSFl 118 A Coupled GST-Pull-Down and Far-Western Analysis of Nuclear Proteins Capable of in vitro Interactions with the CTAl of hHSFl 119 Interaction between TFIID and Negative Regulation Domain 121 Repressor Function of the Negative Regulation Domain 123 DISCUSSION 126 Mechanism of Basal Repression 126 Negative Regulation of Transcriptional Activation Domains 129 HSF2 Expression 130 Activation Domains in HSFs 131 Mechanism of Transcriptional Activation of hHSFl 136 The Role of the Interaction between hTBP and hHSFl 139 Model for Heat Inducible Regulation of HSF 140 Basis for Future Studies in Plants 143 SUMMARY AND CONCLUSIONS 144 LIST OF REFERENCES 14 9 iv

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BIOGRAPHICAL SKETCH 168 1 I 1 V

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LIST OF VECTORS Vectors page 1. pYDA (derived from pPC97 and pPC86) is a yeast expression vector used in the studies of basal repression of HSFs 50 2 pYGAL2 and pYDBD22 are yeast expression vectors with the GAL1,10 promoter used for the substitution of scHSF with HSFs from human and soybean 51 3. pALeu and pDTrp are yeast expression vectors used in yeast two-hybrid system for cDNA screening 53 4. pGEX-SB is a GST fusion protein vector for expression in E. coli 59 5. p24-SB is a E. coli expression vector with the T7epitope 60 6. pNG-SB and pNeo-NB are mammalian expression vectors 63 7. pT7-NLS is a mammalian expression vector with T7tag and nuclear localization signal 66 8. pNT7-SB is a mammalian expression vector with the T7-tag 69 vi

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LIST OF ABBREVIATIONS aa Amino acid ADl (aa 401-420) The first activation domain of hHSFl AD2 (aa 430-529) The second activation domain of hHSFl P-gal p-galactosidase bp base pair CEl, 2 Two short conserved elements of yHSF CMV Cytomegalovirus CTAl (aa 422-529) C-terminal activator 1 of hHSFl CTA2 (aa 397-536) C-terminal activator 2 of hHSF2 CTAl-Plus (aa 382-529) CTAl plus HR-C of hHSFl CTD C-terminal domain of pol II DBD DNA binding domain dHSF Drosophila HSF DMP Dimethylpimelimidate ECL Enhanced chemiluminescense 5-FOA 5-f luoroorot ic acid GAGA Drosophila transcription factor GAL4 Galactose regulated transact ivator from yeast GAL4-DBD GAL4 DNA binding domain GmHSFs HSFs from Glysine max L. cv. Williams GST Glutathione S-transf erase GTF General transcription factor hGH Human growth hormone vii

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hHSF Human HSF HR-A Hydrophobic heptapeptide repeat A HR-B Hydrophobic heptapeptide repeat B HR-C (aa 382-422) Hydrophobic heptapeptide repeat C hr hour hs Heat shock HSE Heat shock element HSFs Heat shock factors Hsp Heat shock protein klHSF K. lactis HSF luc Lucif erase LpHSF HSF of tomato (Lycopericon peruvianum) mHSF Mouse HSF NLS Nuclear localization signal NR Negative regulation domain (aa 212-310) of hHSFl OD Oligomerization domain PCs Positive cofactors PIC Pre-init iation complex pol II RNA polymerase II pXGH5 Human growth hormone vector with mMT-1 promoter scHSF S. cerevisiae HSF TAFs TBP-associated factors TBP TATA binding protein TFIIA Transcription factor A for pol II TFIIB Transcription factor B for pol II TFIID Transcription factor D for pol II VP16 Herpes viral protein 16 viii

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yHSF Yeast HSF ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy FUNCTION AND REGULATION OF HUMAN AND SOYBEAN HEAT SHOCK TRANSCRIPTION FACTORS EXPRESSED IN YEAST AND HELA CELLS By CHAO-XING YUAN December, 1996 Chairman: Dr. William B. Gurley Major Department: Plant Molecular and Cellular Biology Program Activities of human and soybean heat shock transcription factors (HSFs) were characterized in HeLa and yeast cells. Transcriptional activation domains were mapped to the Ctermini of human HSFl and HSF2 using GAL4 DNA binding domain fusions. The most C-terminal activation domain of human HSFl was composed of multiple subdomains that seem to function synergist ically No direct evidence of transcriptional activity was observed for GmHSF34 and GmHSF5 from soybean. A general conservation in mechanism exists between humans, plants and yeast, since both human HSFs and soybean GmHSFS were able to substitute in yeast for the endogenous HSF Similar activity patterns for human HSFs were observed under heat shock and basal conditions in HeLa and yeast, except for

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7 slight differences in the kinetics of the heat shock response Protein-protein interactions between HSFl and general transcription factors (TFIIB, TBP, TAF32, TAF55 and PC4) were characterized in order to identify potential targets of contact in the transcriptional preinitiat ion complex. These contacts represent one of the final steps in heat stress induced transcription of heat shock genes TATA binding protein (TBP) and transcription factor IIB (TFIIB) were identified as major targets for HSFl transcriptional activation domains based on in vitro interaction assays. Coimmunoprecipitat ion of transiently expressed proteins in HeLa cells and squelch-rescue assays confirmed predictions based on in vitro results that interactions between HSFl activation domains and TBP and TFIIB can occur in vivo. A negative regulatory region (NR) of HSFl was shown to bind TFIID in nuclear extracts through contacts that probably involve TATA associated proteins (TAFs) These results suggest a model for transcriptional regulation by HSFl that involves a shift in the equilibrium between formation of dysfunctional TFIID complexes with the NR and functionally competent complexes with the C-terminal activation domains. xi

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INTRODUCTION In most organisms a sudden elevation in temperature of approximately 10C results in a major shift in transcriptional expression. Expression of most normal genes is attenuated to varying degrees and a subset of genes known as heat shock genes are activated (Ashburner and Bonner, 1979; Craig and Gross, 1991) In soybean as many as 19,000 copies of transcripts per cell encoding low molecular weight heat shock proteins (Hsps) accumulate over a 2 hour (hr) period (Schoffl and Key, 1982) The protein that mediates this massive induction of transcription is the heat shock transcription factor (HSF) In eukaryotes other than yeast, activation of HSF is thought to involve a conformational change of pre-existing HSF accompanied by trimerizat ion of identical subunits converting the protein from a non-DNA binding form to one that binds the heat shock consensus element (HSE) located in the 5 '-flanking region of heat shock genes (Westwood et al., 1991) The HSE is required for heat induced activation of the promoter and is comprised of at least three tandem repeats of a 5 base pair (bp) sequence ( 5 -nGAAn-3 ) arranged in alternating orientations (Amin et al 1988; Barros et al., 1992; Pelham, 1982; Xiao et al 1991). Maintenance of HSF in a repressed state under non-heat shock conditions (basal repression) is thought to involve interactions between

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2 HSF and cellular factors such as Hsp70 (Kim et al., 1995; Rabindran et al 1994), and/or Hsp90 (Nadeau et al., 1993). More than twenty HSFs from different species including yeast, Drosophila, mouse, HeLa cell, tomato, soybean, maize and Arabldopsis have been cloned as cDNAs and sequenced (Czarnecka-Verner et al., 1995; Gagliardi et al 1995; Rabindran et al 1991; Sarge et al 1993; Sarge et al 1991; Scharf et al 1990; Schuetz et al 1991) (M. D. Barros et al., unpublished) In animals there exist more than three major classes of HSFs with distinct lineages, while in plants two major classes with sub-groups in each | class are known (Czarnecka-Verner et al 1995; Nover et al 1996) Class A in plants is transcriptionally active, however, class B is inactive except LpHSF24 (CzarneckaVerner, unpublished observation) Across kingdoms each class is distinct in amino acid sequence structure indicating a complexity in lineage and, perhaps, specialization in HSFs among eukaryotes. The various HSFs show relatively little overall amino acid identity, but are considerably conserved in the DNA binding (DBD) and oligomerizat ion domains (OD) The DBD comprises a three helix cluster typical of helix-turn-helix proteins, and the OD consists of a series of 4, 3(abcdefg) ^ pattern hydrophobic heptad repeats (HR-A and HR-B) forming a coiled-coil during trimerizat ion (Sorger and Nelson, 1989) Activation domains of the Saccharomyces cerevisiae HSF '{ (scHSF) are located in both the N-terminal and C-terminal I

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3 regions of the protein (Chen et al 1993; Jakobsen and Pelham, 1991; Nieto-Sotelo et al 1990). The C-terminal activation domain is the strongest and best characterized and appears to be required for the transient heat shock response as well as sustained growth at high temperatures (Chen et al., 1993; Nieto-Sotelo et al., 1990). In tomato three HSFs have been cloned and a tryptophan motif (Trp-repeat) located C-terminal to the OD has been associated with transcriptional activity in transient assays using tobacco protoplasts (Treuter et al., 1993) The activation domains of mouse HSFl (aa 425-471) and HSF2 (aa 473-517) are C-terminal to the third hydrophobic repeat (C-terminus) and possess an overall negative charge (Shi et al., 1995) The activation domain of Drosophila HSF is also located in the C-terminus and rich in acidic and hydrophobic amino acid residues (Wisniewski et al. 1996) The activation and repression domains of hHSFl have become some of the best characterized in higher eukaryotes (Green et al., 1995; Newton et al 1996). The activation domains (ADs) of hHSFl consist of two direct tandem domains at the C-terminus. Activation domain 1 (ADl) is rich in acidic and hydrophobic residues and is predicted to form an a-helix similar to a typical acidic activator. However, AD2 is a negative charge-, prolineand serine-rich activator without any predictable secondary structure. The activation and regulation domains of hHSF2 remain largely

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4 uncharacterized It is also unknown whether the regulation mechanism of HSFs across kingdoms is conserved. The initial objective of this research is to characterize expression of soybean HSFs in yeast cells to evaluate the degree of conservation in mechanisms of basal repression and activation between plants and fungi. Similar experiments were conducted in parallel with human HSFs in yeast and HeLa cells as a form of positive control using well characterized HSFs, since the soybean HSFs were recently cloned and not characterized. For functional domain mapping of HSFs, the GAL4 DNA binding domain (GAL4-DBD) was fused to the N-terminus of the HSFs and the reporter genes for monitoring HSF activity were the GAL promoter joined to lacZ (Chevray and Nathans, 1992) for the yeast assay system and the GAL4-binding-site-TATA promoter fused with the luciferase (luc) gene for the HeLa assay system. This strategy eliminated background activity from endogenous HSFs and allowed deletion of the natural DBD and nuclear localization signal (NLS) since these were provided by the GAL4-DBD. Although use of GAL4-DBD fusions provided advantages for functional domain mapping, it was not suitable for addressing questions regarding basal repression of HSF activity and overall compatibility of the plant and human HSFs with yeast. For this purpose, substitutions of human and soybean HSFs in yeast were employed to monitor the heat shock response of heterologous HSFs in yeast using an HSE containing promoter

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5 to drive the reporter gene. The endogenous plasmid-borne scHSF was eliminated by growth on 5-FOA plates. After functional domain analysis of the HSFs in both heterologous and homologous systems under heat shock and nonheat shock conditions, a natural extension of these studies was to more directly address questions of mechanism in the activation of HSFs by identifying targets of contact for HSFs in the transcriptional preinit iat ion complex (PIC) Transcription has at least three steps: initiation, elongation and termination. Transcriptional initiation by RNA pol II seems to require the ordered assembly of general transcription factors (TFIID, TFIIB, TFIIA, TFIIE, TFIIF, TFIIG/TFIIJ, TFIIH/BTF2 and pol II) into the PIC (Buratowski, 1994; Conaway and Conaway, 1993; Roeder, 1991; Zawel and Reinberg, 1993) It is believed that the interaction of transcriptional activators with any component of the PIC is required for transcriptional activation. In some cases, the binding affinities of transcriptional activators to some protein members of the PIC are often strongly correlated with transcriptional activating potentials of activators (Wu et al., 1996) Therefore, the first step in studying the mechanism of activation by transcriptional activators is the identification of potential targets proteins within the PIC followed by an evaluation of the functional relevance of the interactions in vivo. As the second major objective of this dissertation research, several kinds of strategies were employed to identify functional targets of hHSFl. First,

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6 various in vivo and in vitro protein-protein interaction techniques were used to test the affinity of hHSFl with general transcription factors and cofactors including hTBP, hTFIIB, PC4, hTAF32 and hTAF55 Assays involving immobilized GST-fusion proteins (GST-pull-down assays) were used to detect the protein-protein interactions in vitro allowing precise mapping of the interaction sites. Since interactions observed in the in vitro assay may be due to the relatively high concentrations of proteins used and the high sensitivity of detection methods, not all interactions observed in vitro may be biologically significant in living cells. To reduce the enforced nature of in vitro assay, co-immunoprecipitation of co-expressed proteins was adopted to further test the validity of interactions observed from GST-pull-down assays. Proof that the observed interactions are specific and biologically relevant finally requires a functional assay in vivo. An assay based on the squelching of activator-mediated transcription (squelch-rescue assay) was used to confirm that potential interaction can occur in vivo and have functional roles in transcriptional activation. All of the strategies discussed above depend on the availability of the proteins known to be components of the PIC, and are based on the assumption that these are the only possible targets. To compensate this drawback, the yeast two-hybrid system and far-western approaches were used to select possible unknown target proteins. Through a series of tests, two general

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transcription factors emerged as likely targets of contact for hHSFl C-terminal activation domains, hTBP and hTFIIB,

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8 LITERATURE REVIEW Heat Shock Response and Heat Shock Proteins Living cells can sense adverse environmental stimuli and display a rapid molecular response. Under heat shock conditions, the expression of most genes is greatly attenuated and a relatively small subset of genes are highly induced at the transcriptional level. These heat inducible genes encode several classes of molecular chaperones known as heat shock proteins (Hsp) The spectrum of Hsps synthesized in different organisms show notable similarities. Hsps are highly conserved between diverse organisms are classified into related groups according to their average molecular weights: HsplOO, Hsp90, Hsp70, Hsp 60, and small Hsps (14 to 27 kDa) The Hsp70 family contains the most highly conserved proteins (Roberts and Key, 1991) and its members have diverse functions (Pelham, 1990) including renaturation of denatured and aggregated proteins (Pelham, 1986) folding and assembly of nascent proteins (Beckmann et al 1990; Craig, 1993), and facilitation of protein translocation (Deshaies et al 1988) The Hsp60 family is localized to the mitochondria and is also involved in proper folding and assembly of proteins (Craig, 1993; Ellis and van der Vies, 1991; Nadeau et al 1993) The Hsp90 family, often in complexes with Hsp70

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9 and/or Hsp60, function as regulators by interacting with various cellular proteins such as hormone receptors (Renoir et al., 1986), kinases (Oppermann et al., 1981), actin and tubulin (Mager and Kruijff, 1995) Members of the HsplOO family are involved the acquisition of stress tolerance (Parsell et al., 1994) Small Hsp are thought to protect cytoplasmic proteins from forming irreversible aggregates at high temperature (Mager and Kruijff, 1995; Mansfield and Key, 1987) In plants this group proteins are highly expressed upon heat stress (Helm et al., 1990). Function, Structure and Regulation of Heat Shock Transcrip tion Factors Function of HSFs and DNA Binding The analysis of inducible gene expression has provided many insights into the molecular mechanisms of transcriptional control. Activation of heat shock (hs) genes in response to elevated temperature and other environmental stresses has been studied extensively as a paradigm for inducible gene expression (Ashburner and Bonner, 1979; Craig and Gross, 1991; Schoffl and Key, 1982) These studies have demonstrated that induction of hs genes in eukaryotes is mediated by the activation of preexisting heat shock transcription factors (HSFs) which in turn, bind to specific cis-elements within heat inducible promoters. These upstream heat shock elements (HSEs) are comprised of three to nine

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10 repeats of a 5-bp unit, nGAAn, arranged in inverted orientation (Amin et al 1988; Barros et al 1992; Pelham, 1982; Xiao et al 1991). Each of these 5-bp sites is able to bind a subunit of the HSF oligomer. When higher eukaryotes are exposed to hs, non-DNA-binding HSFs (monomers) are converted into active trimers (Westwood et al 1991), Trimeric HSF binding to HSE is highly cooperative (Topol et al., 1985; Xiao et al 1991) so that promoters containing multiple HSEs serve as platforms for high stable HSF-DNA complexes (Xiao et al 1991). Exploitation of cooperativity | I in the numbers, and arrangement of the 5-bp units and the degree of match with the consensus may provide a mechanism for the the differential binding of HSFs to hs promoters resulting in a selective heat shock response (Lindquist, 1980) This theme of differential binding of HSF for selective gene expression is further expanded by the observation that mHSFl and mHSF2 show large differences in the degree that cooperativity influence DNA binding (Kroeger et al., 1993) This may be one of the reasons why the highly cooperative HSFl is involved in the heat shock response and HSF2 which does not show cooperativity plays a role in developmentally regulated HSF expression. Characteristics of the DNA Binding Domain DNA sequence and deduced aa sequence of HSFs indicate that HSFs are not highly conserved in overall sequence; i i however, all HSFs contain isolated domains which have been -I

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highly conserved during evolution. The functional domains identified within HSFs are the DNA binding domain (DBD) the oligomerization domain (OD) and the transcriptional activation domain (AD) (Nover et al 1996). The phenomenon of highly conserved HSEs located upstream of all hs genes strongly suggests that all HSFs share a very similar structure in terms of the DBD, and function in a similar manner. Amino acid sequences located near the Nterminus of HSFs are the most highly conserved within the protein and constitute the DBD which consists of four antiparallel (i-sheets and three-helix clusters typical of a helix-turn motif (Damberger et al., 1995; Harrison et al., 1994) The DBD is required for specific interaction with the HSE and may also function in the repression of transcriptional activity during normal growth, at least in yeast. For example, a single point mutation (M232V) in the scHSF (HSF from S. cerevisiae) DBD (Wiederrecht et al 1988) results in constitutive transcriptional activity (Bonner et al., 1992) The ancient origins of the DBD are reflected in the conservation of 26 aa with the putative recognition helix of bacterial sigma factors. The motif is conserved in dHSF (HSF from Drosophila) scHSF, klHSF (HSF from K. lactis ), LpHSF24 (HSF from tomato) and bacterial sigma 32 (hs sigma) and sigma 70 factors (Clos et al 1990; Sarge et al 1991). Sigma 32 is required for transient activation and sigma 24 for permanent activation under severe hs conditions in E. coli

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12 Plant DBDs are unique in that all plant HSFs (soybean, tomato, Arabidopsis and maize) seem to have a deletion of 11 aa residues between p3and P4-strands meaning these two strands are connected by a turn of 4 aa residues instead of the much larger loop present in animal and yeast HSFs (Nover et al., 1996). In addition, parsimony analysis of DBDs suggest that plant HSFs can be classified into two major groups, A and B, containing multiple members with representatives in every species (Czarnecka-Verner et al., 1995; Nover et al., 1996). There is no apparent correlation between the lineage groups delineated in plants and functional classes present in mammalian and avian species, or close relatedness to the HSFs of yeast (Nover et al 1996) Properties of the Oligomerizat ion Domain The 00, consisting of two blocks of hydrophobic heptad repeats (HR-A and HR-B) is involved directly in proteinprotein interactions in the formation of the trimeric form of HSF which is then able to efficiently bind DNA (Sarge et al 1993; Westwood and Wu, 1993) In addition to its role in trimerizat ion, the 00, along with the HR-C in the C-terminal region of HSFs, is required for sequestration of HSFs in the cytosol under non-heat shock conditions; deletion of either of these two regions results in constitutive nuclear localization of hHSF2 (Sheldon and Kingston, 1993) The OD is located C-terminal to the DBD and shows a high degree of conservation in its overall pattern of hydrophobic residues

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13 (Peteranderl and Nelson, 1992; Sorger and Nelson, 1989), but little conservation in the specific aa sequence varying from 41% between yeast and human HSFl, to 7 9% between HSFs and Drosophila HSF (Rabindran et al., 1991). The hydrophobic region takes the form of a heptad repeat characterized by the occurrence of a bulky hydrophobic aa, commonly Leu, lis, or Val, in every first position ("a" position) and at every fourth position ("d" position) within the seven residue repeat (abcdefg) typical of bZip proteins. These amino acids form a hydrophobic surface that provides the region of contact between paired helices (Sorger and Nelson, 1989) Although hydrophobic interactions between a and d positions in different strands are the major stabilizing force in triple-stranded coiled-coils the specificity of trimerizat ion is thought to be conferred by charged residues at the e and g positions (Alber, 1992) If the inactive form of HSF is maintained in a metastable state by various molecular forces, for instance, by a combination of hydrophobic, charged, and polar interactions, then the perturbation of a subset of these forces by any one inducer of the stress response could be sufficient to initiate the trimerizat ion The ODs of plant HSFs show a similar overall pattern of heptad repeats as that found in other eukaryotic organisms; however, plant HR-As are shorter, frequently have a hydrophobic amino acid other than leucine at the d position of the fourth and fifth heptads, and often have zipper

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14 destabilizing residues at the a position. All these differences are predicted to make the interactions in plant HR-A region weaker and the trimer of plant HSFs less stable (Czarnecka-Verner et al 1995). The ODs of plant HSFs appear to have two types: type I including animal and yeast HSFs in addition to AtHSFl, LpHSFS, LpHSF30 as well as GmHSF21; type II unique to plants, consisting of LpHSF24, GmHSFS, GmHSF29, GmHSF31 and GmHSF34 (Czarnecka-Verner et al., 1995). The two classes of ODs correspond exactly with the plant HSF groups A and B derived from parsimony analysis of the DBD (Group A = type I, Group B = type II) The type I OD is demonstrated by the inclusion of a glutamine-rich region between HR-A and HR-B, but the HRA and HR-B of type II are adjacent. The other difference between the two types is the spacing between the OD and the DBD. In type I, the separation is only 12 to 26 aa, but in type II the spacing is much larger, 50 to 74 aa (CzarneckaVerner et al., 1995) The functional and evolutionary significance of these two major classes of plant HSFs is still unclear; however, a preponderance of the Group B HSFs (type II OD) have no transcriptional activity in transient assays (Czarnecka-Verner et al, unpublished) Attributes of Activation Domain Activation domains (ADs) are frequently located at the C-termini of HSFs (Hoj and Jakobsen, 1994; Nakai and Morimoto, 1993; Newton et al., 1996; Nieto-Sotelo et al

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15 1990; Rabindran et al 1993; Shi et al., 1995; Zuo et al 1995) Yeast HSF (yHSF) contains at least two domains that are capable of functioning as transcriptional activators. The two domains located in the N-terminus (CEl) and Cterminus (C-terminal domain) of the HSF, can provide either constitutive, or unregulated transcriptional activity, respectively. Transcriptional activity of the C-terminal domain is much more stronger than that of CEl (Chen et al 1993; Jakobsen and Pelham, 1991; Nieto-Sotelo et al 1990). In both mHSF and dHSF the C-terminal activation domains show little amino sequence conservation and are rich in hydrophobic and acidic residues (Shi et al., 1995; Wisniewski et al., 1996) hHSFl seems to have two separable tandem ADs (ADl and AD2) at the C-terminus (Green et al., 1995; Newton et al., 1996) ADl contains only 20 aa residues embedded in HR-C; AD2 is located in the extreme C-terminal region corresponding to the CTAl (C-terminal activator 1) originally defined in this dissertation research. The natural fusion of the HR-C and CTAl forms the CTAl-Plus domain encompassing all of ADl and AD2. ADl/HR-C and AD2/CTA1 show distinct differences in terms of aa composition and predicted secondary structure. ADl/HR-C is rich in acidic and hydrophobic residues and predicted to form an amphipathic ahelix similar to those typical of acidic activators such as GAL4, VP16. Although AD2/CTA1 is acidic (13% negative) and rich in hydrophobic aa (13% Leu) similar to ADl/HR-C, it also contains high percentages of proline (18%), serine (24%) and

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16 glycine (8%) and seems unable to form either an a-helix or P-strand. A comparison of aa compositions of typical proline-rich activators with AD2/CTA1 is presented in table 3. Based on the aa composition and the low probability of forming secondary structures, it seems more appropriate to define the AD2/CTA1 as a proline-rich activator instead of acidic activator. Therefore, hHSFl seems to contain two distinct activation domains at the C-terminus, one acidic and the other proline-rich. The close apposition of the ADl/HR-C and AD2/CTA1 makes it likely that these two activation domains function synergistically to activate transcription as j shown for TFE3 activator domains (Artandi et al 1995) In tomato three HSFs have been cloned and a trp-repeat located C-terminal to the OD has been associated with transcriptional activity in transient assays using tobacco protoplasts (Treuter et al., 1993). Mutagenesis analysis of the repeat indicated that trp is not important for activator activities per se However, the short peptide motifs rich in aromatic, large hydrophobic and acidic aa residues (AHA motif) seems related with transcription (Never et al., 1996). Role of Masking in Yeast HSFs In the masking model of HSF regulation, it is proposed that the N-terminal masking domain interacts with the Cterminal transcriptional activator to block its function. The critical experiments that support this hypothesis are a series of functional domain mapping and domain swapping

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17 studies of yHSF indicating the importance of global protein structure in the regulation of transcriptional activity (Bonner et al., 1992; Jakobsen and Pelham, 1991; Nieto-Sotelo et al., 1990; Sorger, 1990). The most crucial experimental result which severed as the original basis for proposing that HSF activation domains were masked under basal conditions was the gain of constitutive activity that occurred when the Nterminal 146 aa region of yHSF was deleted (Sorger, 1990) An HSF monomer was envisioned to be essentially folded in half in such a way that the activation domains are inaccessible and not functional. Another variation of the masking model requires that the masking of the AD is the result of interactions with a non-HSF protein ( intermolecular masking) In this scheme, masking is alleviated by dissociation of the second protein resulting from a conformational change in the HSF. Regions of yHSF that are potentially involved in masking are the two conserved elements (CEl and CE2) the DBD and the OD (Bonner et al 1992; Jakobsen and Pelham, 1991; Nieto-Sotelo et al., 1990; Sorger, 1990) Since mutations within the OD cause relief of repression of HSF activity under non-heat shock conditions, repression of the AD seems to require the normal oligomerization state of the HSF molecules (Chen et al., 1993) Mutations within the DBD can also initiate the loss of repression of HSF activity (Bonner et al., 1992). This may indicate that global structure is important for masking and complete repression of transcriptional activity. CE2 is

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18 crucial for masking and repression since its deletion causes yHSF to be const itutively active (Jakobsen and Pelham, 1991), presumably through releasing the C-terminus and permitting it to interact with the transcription initiation complex. The first 8 aa residues of CE2 are believed to bind either to the structural core of HSF, or to another polypeptide to maintain the AD in an inactive conformation (Jakobsen and Pelham, 1991) CE2 seems to function by stabilization of the hypothetically folded structure of the repressed form of yHSF. CEl also seems to contribute to repression, but is subsidiary to CE2 in its role (Jakobsen and Pelham, 1991) The DBD is also essential for repression of yHSF. Conversion of Met 232 to Val in the DBD has a very dramatic effect, releasing repression almost completely (Bonner et al 1992) This suggests that Met 232 is intimately involved in the maintenance of the repressed (masked) state of yHSF, although the exact role of this residue is not clear. The location of Met 232 within the DBD indicates that this region may be bif unctional, involved in both protein-DNA interactions and protein-protein interactions. The intramolecular masking model of HSF seems unsuitable to mammalian HSFs Functional domain mapping and domain swapping experiments from mHSFl and hHSFl indicated that global protein structure is not required for basal repression. The DBD and HR-A deletions of mHSFl had no effects on basal repression, or heat shock induction of transcriptional activity (Shi et al 1995), and complete

PAGE 30

deletion of the DBD and the OD (HR-A and HR-B) of hHSFl had no effect on heat inducibility of ADl (Newton et al., 1996) Furthermore, the entire mechanism of heat inducibility seems to reside in the negative regulation domain (NR) which encompassed only 90 aa The VP16 activator was rendered heat inducible after joining a GAL4-DBD-NR fusion to its Nterminus (Newton et al 1996). The precise mechanism whereby the NR confers heat inducible regulation to ADl and the heterologous VP16 is still an intriguing question. It may be that a modified version of the intermolecular masking model still applies, but only involves the NR and a Cterminally located activation domain (s). Trimerization Trimerization is essential for high affinity binding of HSFs to HSEs (Lis and Wu, 1993; Sorger and Nelson, 1989; Westwood et al 1991; Westwood and Wu, 1993), since both the monomeric and dimeric forms of HSF have low affinity for HSEs (Sarge et al 1993) HSF binding in eukaryotes other than yeast is regulated by a change in the oligomerization state upon hs HSF binding to HSEs is highly synergistic at two distinct levels: between subunits of the HSF trimer, and between trimers (Xiao et al 1991) The binding of one dHSF trimer to an HSE with six nGAAn repeats enhances the binding of a second trimer by over 2,000-fold through protein interactions between the DBDs (or with the DBD plus the HR-A of the OD) (Wyman et al., 1995; Xiao et al., 1991). This

PAGE 31

20 cooperativity is particularly important for the highmagnitude and promptness of the response. Why is HSF trimeric? In addition to structural requirements for DNA binding, a second reason may be that control of oligomerizat ion plays a role in the regulation of the hs response via interaction with Hsp70 (Clos et al 1990) Regulation of HSFs by Cellular Proteins Posttranslational modifications including phosphorylation may have the potential to affect the multimeric state of HSFs. Purified HSFs can be activated in vitro by heat (Larson et al., 1995) which suggests that the preexistent, inactive form of HSFs can assume the active conformation without biochemical modification of protein structure. Current thinking on HSF regulation suggests that a complex of molecular chaperones (Hsps) interact with HSF in the basal state to achieve masking and cytosolic localization. Maintenance of the inert state seems to occur through association with either constitutive (p72/hsc70) or inducible (HspVO) members of the Hsp70 family (Kim et al., 1995; Mosser et al 1993; Nunes and Calderwood, 1995). However, the interaction between heat shock factors and Hsp70 alone may not be sufficient to mask the activity of HSFs (Rabindran et al 1994). Since Hsp90 has also been shown to associate with HSF by coimmunoprecipitat ion studies (Nadeau et al., 1993), it may have a role in the regulation of HSF activity as well. General support for models of HSF

PAGE 32

21 regulation involving inhibitory molecules is seen in the following two observations. First, hHSFl is activated below its normal temperature when expressed in heterologous cells such as Drosophila, tobacco protoplasts, or frog oocytes which implies that additional cellular factors are involved in regulation (Clos et al 1993; Treuter et al 1993). Second, recombinant HSFs from HeLa, Drosophila and tomato exhibit constitutive HSE binding activity when expressed in E.coli (Clos et al., 1990; Rabindran et al., 1991; Scharf et al 1990; Schuetz et al 1991), presumably due to the lacks of regulatory proteins that would normally repress its activity under non-hs conditions Role of HRp70 There are several studies suggesting a regulatory connection between Hsp70 levels and the transcription of hs genes in eukaryotes (Craig and Gross, 1991; DiDomenico et al., 1982; Kim et al 1995; Morimoto et al 1990; Mosser et al., 1993; Nunes and Calderwood, 1995; Stone and Craig, 1990) Exposure of HeLa cells to hs results in transient activation of HSF; its DNA-binding activity increases rapidly, plateaus and attenuates, during which the intracellular levels of HspVO increase (Abravaya et al., 1991) Injection of denatured protein into unstressed Xenopus oocytes stimulates the activation of HSFs (Mifflin and Cohen, 1994a) and co-injection with Hsp70 can antagonize this induction (Mifflin and Cohen, 1994b) Several studies

PAGE 33

22 strongly suggest that Hsp regulates its own synthesis by directly interacting with HSF, although not supported by hydrodynamic studies (Sistonen et al., 1994; Westwood and Wu, 1993) HspVO has been shown to be able to bind activated HSFs in vivo using non-denaturing gel electrophoresis and coiitununoprecipitat ion techniques (Abravaya et al., 1992; Baler et al., 1992; Rabindran et al., 1994). It has also been shown in vitro that HspVO can block the activation of HSFs from a non-DNA binding state to a DNA-binding form (Abravaya et al., 1992; Clos et al., 1990; Sorger, 1991). This blocking effect is abolished by the addition of ATP (Abravaya et al 1992) It has been proposed that Hsp70 acts the cellular temperature sensor (Craig and Gross, 1991) The pattern of Hsp70 expression suggests that HspVO can interact with HSFs at normal temperatures and repress HSF activation. Damaged proteins resulting from partial thermal denaturation compete for HspVO, thus depleting the pool of HspVO and allowing HSFs to escape basal repression (Ananthan et al., 1986; Baler et al 1992) Repression will be restored once the ratio of denatured protein substrates to HspVO has been returned to normal, either by the production of sufficient HspVO, or by removal, or refolding of the substrates. Continuous exposure of yeast to high temperature stress reveals that an increase in the severity of the heat stress eventually abolishes the attenuation phase of HSF activation and alters the pattern of hs gene expression from transient to sustained (Chen et al

PAGE 34

23 1993; Sorger, 1990) One interpretation of this phenomenon is that during prolonged hs, the continuous demand on Hsp70 for binding to damaged proteins depletes the pool of Hsp70. Hence, the negative regulation of Hsp70 is removed and the activity of stress-inducible HSFs remains constant without an attenuation phase. However, this view fails to explain why HSF is not eventually attenuated by the continuous accumulation of Hsp70. An alternative hypothesis is that severe hs decreases the Hsp70 affinity to HSFs dramatically, resulting in no Hsp70 binding to HSFs, even when Hsp70 is present. The affinity change is postulated to result from conformational changes of HSFs or Hsp70, or be conferred by perturbation of ATP levels (Benjamin et al., 1992). Phosphorylation of HSFs There is a strong correlation between HSF activation and HSF phosphorylation (Craig and Gross, 1991; Sorger, 1990; Sorger, 1991; Sorger and Pelham, 1988), but the role of HSF phosphorylation has remained elusive. hHSFl is hyperphosphorylated during hs, but this phosphorylation is not required for either oligomerizat ion or DNA binding (Larson et al 1988; Larson et al 1995). The transcriptional activation of hHSFl also has no phosphorylation requirement (Newton et al 1996). Removal of all possible phosphorylation sites in the ADl of hHSFl (Newton et al 1996), as well as in the CE2 of scHSF (Jakobsen and Pelham, 1991), did not affect transcription.

PAGE 35

24 In yeast, hs-induced phosphorylation is not necessary for the activation, but is required for HSF deactivation (Hoj and Jakobsen, 1994) The lack of a requirement for phosphorylation of the ADs is consistent with experiments where the hs response remained normal after replacement of the ADl and AD2 of hHSFl with the VP 16 activator (Newton et al., 1996). In addition, structural changes of HSFs can be observed in the absence of phosphorylation. In vitro, hs can convert the purified human and mouse HSFs from the non-DNA binding form into an active DNA-binding form (Goodson and Sarge, 1995; Larson et al 1995). This transition does not depend upon phosphorylation of HSFs, since a single aa mutation (K298A) in the NR, which alone is sufficient for heat-inducibility, destroyed the heat activation (Newton et al 1996) Taken together, it seems that phosphorylation of HSFs is not essential for transcriptional activation of HSFs. The Steps of HSF Regulation The finding of multiple HSFs in several animals and in plants (Nover et al 1996), and of tissue-specific expression of different HSFs (Forenza et al 1995; Goodson et al., 1995), raises the possibility that HSFs may be specialized for different stresses, or different functions in development (Sistonen et al 1994; Treuter et al., 1993), and that they may be regulated differentially. For HSFs primarily responsible for the hs response instead of developmental processes, induction of gene expression relies

PAGE 36

25 on an activation of preexisting HSFs. This activation process is composed of at least three separable steps. The first is thought to involve unmasking of the OD and a nuclear localization signal (NLS) resulting in trimerization and nuclear localization (Westwood et al., 1991; Westwood and Wu, 1993) The exposure of the OD for trimerization and the NLS for transportation into nuclei may be facilitated by removal of negative regulators such as Hsps from HSFs. The second step in activation involves cooperat ivity in binding of HSF to hs gene promoters (Xiao et al 1991) This process may require auxiliary factors such as the GAGA factor in Drosophila or AT-rich element binding factors in plants, for keeping the promoter in an "open configuration" (Czarnecka et al., 1992; Shopland et al 1995). The final activation step occurs after DNA binding by the HSF trimer. Binding to promoter DNA is not sufficient for transcriptional activation as shown by the lack of transcriptional activity after treatment with salicylic acid or other anti-inflammatory agents even though HSFl was shown to be localized in the nucleus and bound to the promoter (Jurivich et al., 1992) At present the mechanism for the final activation step remains largely speculative, but attention has been focused on the possibility of conformational change, since phosphorylation appears to have been ruled out. The discussion above reviewed the regulation of HSFs activity without consideration of the large question regarding the mechanisms of transcriptional activation in

PAGE 37

26 general. At present, these is very little known regarding HSF interactions with other components of the transcriptional apparatus. General pathways of transcriptional activation and possible mechanisms of HSF-mediated transcriptional activation will be discussed in next section of the Literature Review. Mechanism of Transcriptional Activation Transcriptional Activation and Promoter Structure For RNA polymerase II promoters, transcription occurs at two levels, basal and activated. Basal transcription is directed by a core promoter and a minimum number of general transcription factors (GTFs) Activated transcription requires transcriptional activator proteins, activatorbinding sites upstream of the core promoter, and all of the GTFs required for basal transcription, plus coactivators in some cases (Ge et al 1994; Kretzschmar et al 1994a; Parvin et al 1994). A core promoter is sufficient for basal transcription, but not for activated, since it lacks sites for upstream activators to bind. Core-promoters are highly variable in DNA sequence, yet all contain one or two recognizable sequence elements, the TATA box and/or initiator element (Inr) Most core-promoters contain TATA boxes including the core-promoters of most plant hs genes (Czarnecka et al 1992; Gurley et al 1992). Other core promoters have an Inr but lack a TATA box (Struhl, 1989), as

PAGE 38

27 is the case for a petunia Hsp70 gene (Winter et al 1988) A small numbers of genes contain both elements, or neither (Emami et al 1995) The requirement of a TATA box for basal and hs-inducible expression of most hs genes has been shown in plant and animals (Czarnecka et al., 1989; Williams and Morimoto, 1990) For TATA-less hs promoters, some other DNA element, for example, a Inr, must be present to substitute the role of TATA box (Winter et al 1988). The promoter structures upstream of core promoters may be complex depending on the particular gene. For example, in many plant hs gene promoters, the region upstream of the TATA box contains multiple AT-rich elements including AT-composite and AT proximal elements, and several HSEs (Barros et al 1992; Czarnecka et al., 1992; Gurley et al., 1992). The upstream elements can be organized in clusters to form enhancers, or exist as individual DNA binding sites for specific transcriptional activators (Jones, 1994; Struhl, 1989) The modular nature of cis elements means that promoter expression can be dictated simply by grafting specific elements into existing promoters, or minimal promoters. This strategy is often employed in the design of reporter genes useful in the characterization of activator proteins (Bonner et al 1992; Patel et al., 1995) Models of Transcriptional Activation by Activators Many activators have been shown to make physical contact with GTFs in a process that leads to transcriptional

PAGE 39

28 activation. The recruitment of GTFs by activators is thought to be the key mechanism for activator-mediated transcriptional activation. Activators have been shown to recruit their targets to the promoter and thereby, enhance the assembly of the PIC (Barberis et al 1995; Roberts et al., 1995). Stabilization of the PIC, in turn, increases the rate of initiation and/or the processivity of transcriptional elongation (Yankulov et al 1994). When activators are bound far from the transcriptional initiation site, the distance problem of recruitment can be overcome by the flexibility of DNA in looping. For instance, distant HSE sites can be more than 1Kb upstream of the start site, and the separation between two HSEs can be several hundred bp apart (Wyman et al 1995). In this case, HSF has been shown in vitro to bind to these distant sites and bring the two clusters of HSE together by DNA looping (Wyman et al 1995). DNA looping also allows HSF to bring its targets into close proximity with the PIC, efficiently to recruit GTFs needed for transcriptional activation. This ability of DNA to loop provides opportunities for protein-protein interactions between activators, and between the activators and their targets to synergist ically activate transcription. During the recruitment process, conformational changes of the activators and their targets, as well as topological re-organization of the protein-promoter complex may occur (Hori and Carey, 1994; Oelgeschlager et al., 1996; Roberts and Green, 1994) There may be two consequences of

PAGE 40

29 recruitment. If the conformational changes are productive, they may result in transcriptional activation; otherwise, nonproductive recruitment may lead to transcriptional repression. This hypothesis is supported by several experimental observations. One example involves the thyroid hormone receptor which can function both as an activator in the presence of thyroid hormones and as a repressor in the absence of the hormones. Unliganded thyroid hormone receptor alpha can recruit TBP in such a way that it inhibits the ^ subsequent steps of PIC formation, and addition of thyroid hormone can turn this repression into activation (Fondell et al., 1995; Fondell et al., 1996). In this case, recruitment leads either to repression, or activation. Synergism of Transcriptional A ctivation In some genes, activators can bind to multiple binding sites and enhance transcription synergist ically (Sauer et al., 1995a). The level of transcriptional activation by Bicoid (an activator from Drosophila) is correlated in a cooperative fashion with the number of the DNA binding sites (Sauer et al 1995a) Some activators such as the activation domain of p65 NF-kB, VP16 and ZEBRA (a non-acidic activator) can interact with multiple targets to show a synergistic effect on transcriptional activation (Chi et al 1995; Schmitz et al., 1995) presumably via cooperative recruitment of GTFs In theory, HSFs have great potential to activate transcription in a synergistic manner. Since

PAGE 41

30 typical hs promoters usually bind from six to twelve HSF monomer units (two to four trimers) (Czarnecka-Verner et al 1994), cooperativity in target recruitment is very likely. The potential for synergism should be even greater if each HSF monomer has the capacity to recruit more than one type of target protein. When different activators function on a same promoter, their combination may also produce synergistic effects on transcriptional activation (Chi et al 1995; Sauer et al 1995b) through interacting distinct components (such as several TAFs) of the PIC (Sauer et al 1995a; Tjian and Maniatis, 1994) The two isolated activation domains (one is glutamine-rich; the other is alanine-rich) from Biocoid can synergist ically stimulate transcription when they are fused to the hunchback (HB) DNA binding domain (Sauer et al 1995a) A similar synergism has been demonstrated in the natural fusion of the two activation domains that exists in the Bicoid protein (Sauer et al., 1995a). The activator TFE3 contains two activation domains, one acidic and the other proline-rich, which also show cooperativity in the activation of transcription (Artandi et al., 1995). These examples illustrate the synergistic potential of activators consisting of multiple activation domains in transcriptional activation. In the activation of hs genes, HSFs may utilize several strategies to activate transcription synergist ically First, hs gene promoters can provide a substrate for the binding of multiple HSFs (Czarnecka et al 1992; Mager and Kruijff,

PAGE 42

1995; Xiao et al 1991). The synergism inherent in the binding of multiple HSF trimers is supported by the in vitro binding studies (Xiao et al 1991), and the observations that a single HSE in animal, plants and yeast hs promoters is not sufficient for efficient transcription of hs genes (Dudler and Travers, 1984; Gurley and Key, 1991; Schoffl et al., 1989; Wei et al 1986). Another possibility is that HSFs, such as hHSFl, may contain multiple activation domains contributing synergist ically to transcriptional activation. Functional domain mapping of hHSFl reported here will shed light on the number of targets and types of activation domains present in this HSF Transcriptional Initiation: A Key Step for the Regulation of Transcriptional Activation One model for transcriptional initiation by pol II requires the ordered assembly of general transcription factors (TFIID, TFIIB, TFIIA, TFIIE, TFIIF, TFIIG/TFIIJ, TFIIH/BTF2 and pol II) into the PIC (Buratowski, 1994; Conaway and Conaway, 1993; Roeder, 1991; Zawel and Reinberg, 1993) The initial and rate-limiting step of this assembly process is the binding of TFIID to the TATA box (Klein and Struhl, 1994) The facilitated recruitment of TBP to nucleosomal DNA in vivo is shown to be critical in the regulation of eukaryotic gene expression (Imbalzano et al 1994)

PAGE 43

32 One simple model of transcriptional activation suggests that a few key components of the PIC, such as TBP/TFIID and/or TFIIB, are rate-limiting; the simple recruitment of these key factors by transcriptional activators can result in transcriptional activation (Chatterjee and Struhl, 1995; Klages and Strubin, 1995; Klein and Struhl, 1994) For example, kinetic studies of TBP using specificity-altered TATA-promoter and mutated-TBP assay system (Klein and Struhl, 1994) suggest that the recruitment of TBP is rate-limiting in vivo (Klein and Struhl, 1994) The direct fusion of TBP with the LexA DNA binding domain results in the activation of transcription in the absence of transcriptional activators (Chatterjee and Struhl, 1995) indicating that the recruitment of TBP is the key step for transcriptional activation. This recruitment model is not inconsistent with the RNA polymerase II holoenzyme idea (Barberis et al., 1995; Ossipow et al., 1995; Thompson and Young, 1995) According to this hypothesis, RNA polymerase II holoenzyme is a titanic protein complex including many GTFs and cofactors which are preformed before the complex binds to the promoter. The number of proteins thought to form the holoenzyme becomes larger as more proteins are continuously shown to be associated with the complex (Emili and Ingles, 1995; Ossipow et al., 1995). Activator-mediated recruitment of a single transcription factor, or even a mega-protein complex such as the holoenzyme, can facilitate transcriptional initiation and lead to transcriptional activation (Barberis et al., 1995).

PAGE 44

33 Although other pathways to activation exist, such as keeping the chromatin template open (Paranjape et al 1994), and stripping off the repressors (Kraus et al 1994) for activator-mediated transcriptional activation; these routes seem to be limited to particular situations, not generalized mechanisms for a broad-spectrum of activator-mediated transcriptional activation. For HSF mediated transcriptional activation, it seems reasonable that at a minimum, HSF must have affinity for at least one GTF and recruit it to the PIC; j however, this and other mechanisms of activation remain to be demonstrated. i Transcriptional Elongation: In Special Cases Expected to Achieve Transc riptional Activation The complete assembly of a PIC on a given promoter is not always sufficient for productive and efficient transcription. In the case of some genes such as HIV, Drosophila HsplO and human c-myc, the pivotal step of activation is elongation, a step where some activators may exert their influence (Bentley and Groudine, 1986; Cullen, 1993; Lis and Wu, 1993) Control of gene expression through the regulation of elongation has the potential advantage of achieving a quick response to activation because the PIC has been pre-assembled Some activators such as Tat, VP16 and Ela, in addition to the facilitation of TATA-box binding by TFIID (Yankulov et al 1994), can increase the processivity of transcriptional elongation (Shopland et al 1995;

PAGE 45

34 Yankulov et al 1994). The degree of transcriptional processivity enhanced by acidic activators depends on the quantity, other than the quality, of activation modules, and the number of DNA binding sites (Blair et al 1996) For example, GAL4 DNA binding domain fused with a trimer of acidic activation modules (AAM3) resulted in ~5-fold increase of processivity compared with that induced by a dimeric module fusion (Blair et al., 1996). Acidic activators such as VP16 and Rel A are individually sufficient for the stimulation of elongation (Blair et al., 1996), but in the Drosophila Hsp70 promoter, the stimulation of processivity by HSF requires the cooperation of the GAGA factor; dHSF alone has no effect on elongation (Shopland et al 1995) Other activators, like the GAGA factor (Shopland et al., 1995), have a negative effect on processivity of transcription simply by stimulating the basal rate of initiation without affecting the elongation process. One model for gene activation suggests that in cells there are two kinds of transcriptional elongation complexes, nonprocessive and processive forms (Bentley, 1995; Yankulov et al 1994). In the absence of activators, the nonprocessive form is dominant; however, in the presence of activators, processive complexes of elongation become dominant The balance between the two forms is governed by the promoter elements, including the TATA-box and enhancers, and activators bound to these elements. This model may only be useful in special cases when applied to genes that exhibit a significant level of

PAGE 46

35 aborted transcriptional elongation. Overall, it appears for most genes that the key regulatory mechanism for transcriptional activation involves the control of transcriptional initiation in which GTFs are recruited to the PIC through protein-protein interactions with activator proteins Targets of Activators in Transcriptional Activation TBP and TFIID in Transcriptional Activation According to the multistep assembly model, TBP/TFIID facilitates formation of the PIC by recognizing and binding the TATA box which serves as the nucleation point for building the PIC (Buratowski 1994; Conaway and Conaway, 1993; Roeder, 1991; Zawel and Reinberg, 1993) TBP is a relatively small protein with a unique saddle shape and has been shown to bend DNA upon binding (Nikolov et al 1992). TBP is believed to contain determinants for protein-protein interactions with pol II, certain TAFs and various sets of activators and repressors (Tang et al 1996). TBP, a subunit of TFIID, was first cloned from yeast, and then from Drosophila and human cells (Hahn et al 1989; Hoey et al 1990; Hoffman et al 1990). Although TBP was isolated as a single polypeptide from yeast, subsequent studies with Drosophila and human TBP showed that in these organisms TBP is tightly associated with multiple proteins called TAFs (TBP associated factors)

PAGE 47

36 TFIID/TBP binding to the chromatin template is rate limiting in gene expression, and the kinetics of this process are subject to regulation by activators in vivo (Klages and Strubin, 1995; Klein and Struhl, 1994), and by SWI/SNFs (SWTs, yeast genes controlling gene the .SMitching process; SNFs, yeast genes controlling sucrose non^ermentation) (Imbalzano et al 1994). Activators bound to upstream regions of the promoter have been shown in vivo to increase recruitment of TBP to the TATA box (Klages and Strubin, 1995) When TBP was translat ionally fused with a DNA binding domain such as LexA, elevated levels of transcription occurred in the absence of activator protein (Chatterjee and Struhl, 1995) This artificial fusion connection mimics the interaction that normally occurs between a transcriptional activator and TBP or TFIID, and the recruitment of TBP/TFIID leading to transcriptional activation. Since TBP is a small protein, it is surprisingly found that TBP can interact directly with numerous activators such as VP16, Tat, Tax-1, ElA, Zta, ICP4, v-Rel, c-Rel, NF-KB, p53, E2F-1, cFos, c-Jun, c-Myc, PU-1, Spl and SSAP (Defalco and Childs, 1996; Schmitz et al 1995). In many cases, the strength of act ivator-TBP interactions correlates well with the ability of activators to stimulate transcription both in vitro and in vivo (Liu and Berk, 1995; Melcher and Johnson, 1995; Schmitz et al 1995; Song et al., 1995; Wu et al 1996) However, there are notable examples that seem to indicate little relevance between the ability of binding to

PAGE 48

37 TBP and the strength of activation (Tansey and Herr, 1995) There are many possible explanations for this apparent discrepancy. First, the interactions detected between activators and TBP in vitro may be irrelevant in living cells due to the forced nature of interactions under nonphysiological conditions. Second, some bindings, which are very inefficient in vitro, may still be sufficient for transcriptional activation in vivo. A third possibility is that activators may be capable of activating transcription through multiple pathways in vivo where the redundancy in interactions may mask the effects of a single interaction on transcriptional activation. The multiple pathway scenario is especially attractive due to numerous examples of activator binding to TAFs A set of TBP mutants obtained from genetic screening by Lee and St ruhl (Lee and Struhl, 1995) are defective in responding to acidic activators, but appear normal for constitutive transcription in vivo. This set of mutants shows loss of TATA box binding ability, but seem to interact normally with VP16 and TFIIB in vitro. These results imply that the concave surface of TBP not only recognizes and binds the TATA element, but certain undefined aspects of this interaction strongly influence activated versus basal expression. Similar mutants that disrupt DNA binding and are defective in activated, but not basal transcription have been characterized (Kim et al 1994). These mutants have been shown to either disrupt VP16 binding, or prevent association

PAGE 49

38 between TBP and TFIIA or TFIIB (Kim et al 1994) From these results it seems likely that perturbations in the TATA binding surface may also effect a change in conformation affecting potential interactions with activators, TAFs and other components of the PIC. Other explanations have been offered to account for the behaviors of activation-specific mutants located on the DNA binding surface of TBP (Lee and Struhl, 1995) For example, activated transcription may require the TBP molecular to remain bound at TATA for a certain length of time, a property that might be affected by the mutant. Another possibility is that the TBP/DNA complex with the mutant TBP may not present a conformation that is responsive to activators even though activator binding itself is not effected. Interestingly, TFIID can form homodimers through selfassociation of the TBP subunit in vivo (Taggart and Pugh, 1996) which is consistent with the dimeric crystal structure of TBP (Nikolov et al 1992). The dimerized form of TFIID is inefficient in binding the TATA-box and is transcriptionally inactive. It has been suggested that an equilibrium exists between the monomeric DNA-binding form of TFIID and the dimeric non-DNA-binding TFIID that is subject to regulation by a battery of activators and repressors (Taggart and Pugh, 1996) This proposed mode of activation is highly speculative, since it seems to go beyond the simple recruitment model of TFIID to the promoter, suggesting that

PAGE 50

regulatory proteins also directly influence the ratio of active versus inactive forms of TFIID. TAFs of TFIID in Transcriptional Activation In TAF-f acilitated transcriptional activation, TAFs enhance activator-mediated recruitment of the TFIID complex to the promoter just by providing surfaces of contact for direct interactions between the TAFs and the activators. However, the complete mechanism by which TAFs function in the transcriptional activation is still unknown. Since some TAFs are able to interact with activators and GTFs such as TFIIA, TFIIB, TFIIF, pol II (Goodrich et al., 1993; Hisatake et al 1995; Klemm et al., 1995; Ruppert and Tjian, 1995), the simultaneous interactions of TAFs with transcriptional activators and GTFs provide a way for cooperative binding to greatly facilitate the PIC formation and stability. In this role, TAFs function as a bridging factor between activators and GTFs It has been suggested that different TAFs may connect distinct subsets of activators to the transcriptional machinery resulting in different types of activated transcription (Chiang and Roeder, 1995) In other words, different activators, such as acidic, glutamine-rich, proline-rich, or isoleucine-rich activators, may interact with distinct TAFs in a single TFIID complex to modulate transcription in a specific way. For example, some types of activators in their contact with the PIC may influence the

PAGE 51

40 initiation rate more than processivity, and other activators may contact other components of the PIC that produce the opposite effect. Experiments that argue against the idea that the degree of processivity is regulated by specific TAFs was obtained by studying the relationship between the number of activation domains (either within the same activator, or resulting from multiple activator binding sites in the promoter) and synergism of activation and processivity (Blair et al 1996). The degree of processivity (type of transcription) was strictly dependent on the overall activity of the activator; low numbers of DNA binding sites, or activation domains resulted in relatively weak activation that showed little processivity. Activators show very specific patterns of affinity with regard to TAFs and other components of the PIC. In Drosophila, glutamine-rich activators require contact with dTAFllO (Hoey et al 1993), and acidic activators interact with dTAF40 (Goodrich et al 1993; Thut et al., 1995). In humans, Spl (glutamine-rich activator) and VP16 (a typical acidic activator) activators require hTAF250 for transcriptional activation (Wang and Tjian, 1994) The estrogen receptor, but not VP16, interacts with hTAF30 (noknown Drosophila counterpart) (Jacq et al., 1994). Acidic activators also interact with hTAF31/32 which is the homolog of dTAF40 (Klemm et al 1995). Multiple activators including Spl, YYl, USF, CTF (proline-rich) ElA (acidic) and Tat can interact with hTAF55 (no-known homologue in

PAGE 52

41 Drosophila) (Chiang and Roeder, 1995) Since hHSFl contain both an acidicand proline-rich activation domains, likely candidates for interactions include hTAF32 and hTAF55 based on the studies cited above. Although TAFs have been shown to be essential in numerous examples of activated transcription, and serve as direct targets of many transcription activators in higher eukaryotes, a contrary view of the importance of TAFs in transcriptional activation has emerged recently in studies conducted in yeast (Moqtaderi et al 1996; Walker et al 1996) The complete inactivation of six yTAFs (TAF145, 90, 68, 60, 47 and 30) and the conditional shut-down of four yTAFs (TAF145, 90, 60 and 19) (Moqtaderi et al 1996; Walker et al., 1996) had no effect on the transcriptional activation of several genes including HspVO. Since yeast TAFs (yTAFs) are not the obligatory targets of transcriptional activators, other components in PIC, for example yeast TBP (yTBP) or yeast TFIIB (yTFIIB) may be the only universal targets of transcriptional activators. Because of the TAFs are highly conserved from yeast to humans, they must serve some essential function, perhaps in the cell cycle. One interpretation of the yeast finding is that perhaps TBP and TFIIB are major targets for activators in all eukaryotes, but in animals, the TAFs also serve in this capacity in addition to their other more essential roles. i 'I

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42 Involvement of T FIIB in Transcriptional Activation TFIIB has been cloned from archaebacteria, yeast, humans, Xenopus, and plants with aa identities ranging from 32-99% (Baldwin and Gurley, 1996; Pinto et al 1992; Wampler et al., 1992). The core domain of human TFIIB (112-316) is highly conserved and is located in the C-terminal half of TFIIB. The core resembles cyclin A and interacts with the Cterminal stirrup of TBP through protein-protein interactions (Bagby et al., 1995). In addition. X-ray crystallography has demonstrated contacts between the core and the phosphoribose backbone of the TATA-box through protein-DNA interactions (Bagby et al., 1995; Nikolov et al 1995). Alanine scanning analysis indicated that Core TFIIB also interacts with DNA upstream and downstream of TATA-box (Tang et al 1996) The N-terminal domain of core TFIIB forms the downstream surface of the ternary complex where it may determine the transcription start site (Nikolov et al., 1995). The remaining surface of TFIIB, especially the N-terminus of TFIIB, as well as TBP, provides a stable platform for PIC assembly and interactions with TAFs, GTFs, activators and coactivators The N-terminus of TFIIB (1-106), dispensable for TBP binding, is essential for recruitment of pol II to the DNA template and the interaction with TFIIF (Ha et al., 1993) Although the zinc finger is not important for interactions of TFIIB with other GTFs, it may provide a target surface for binding activator proteins For example.

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43 the N-terminus of TFIIB, including a putative zinc finger and an adjacent charged region is required for f t zQ-mediated transcription activation (Colgan et al., 1995). Because of TFIIB 's central role in PIC assembly and transcriptional regulation, it serves as a point of communication among activators, TAFs, coactivators, and GTFs The binding of TFIIB to the TFIID-TFIIA complex on promoters results in further stabilizing the PIC and provides a surface for the attachment of the pol II/TFIIF complex (Buratowski and Zhou, 1993; Choy and Green, 1993; Ha et al., 1993). Acidic activators, proline-rich and glutamine-rich activators can stimulate transcription by targeting TFIIB to the PIC in vitro and in vivo (Colgan et al 1995; Colgan et al., 1993; Defalco and Childs, 1996; Hori et al., 1995; Kim and Roeder, 1994; Lin and Green, 1991; Roberts et al 1993; Xiao et al 1994) TFIIB mutants defective in activated, but not basal transcription, have been identified (Roberts et al 1993). Affinities of TFIIB for activators show a strong correlation with transcription activating potentials (Wu et al 1996) which suggest that TFIIB is a bona fide target for recruitment by transcription activators. Activator interactions cause a dynamic conformational change of TFIIB. Structural and functional studies have shown that TFIIB has two domains: the C-terminal core domain (192 aa) containing two imperfect direct repeats capable of interacting with VP16, and an N-terminal domain (124 aa) containing a putative Zn-finger and possessing the ability to

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44 interact with the C-terminal domain intramolecularly (Roberts and Green, 1994) In native TFIIB, the Nand C-terminal domains are engaged in an intramolecular interaction which can be disrupted by VP16. It has been postulated that acidic activators like VP16 function in a capacity beyond simple recruitment (Roberts and Green, 1994) Their disruption of the folded state of TFIIB in binding to the core may initiate a conformational change that exposes binding sites for other GTFs to facilitate the ordered assembly of the PIC. Positive Cofactors in Transcriptional Activation Besides the requirement for GTFs, transcriptional activation, in some cases, needs positive coactivators (PCs) as well. PCs contribute significantly to transcriptional activation in reconstituted mammalian transcription systems. Several members of a family of mammalian cofactors have been identified such as PCI, PC2, PC3/DR2/Topo I and PC4/pl5. Of these, only PC3/DR2/Topo I and PC4 have been cloned (Ge and Roeder, 1994; Kretzschmar et al., 1993; Merino et al 1993). A few positive cofactors have also been found in yeast (Berger et al 1992; Swaffield et al 1995). Among these, some appear to be SRB (^uppressers of the CTD deletion of ENA polymerase R; B = II) proteins tightly associated with the CTD (£-t.erminal domain) of the largest subunit of pol II (Koleske and Young, 1994) The CTD contains up to 52 repeats of the heptapeptide YSPTSPS in human (Young, 1991)

PAGE 56

45 PC2, a 500 kDa protein complex isolated from a HeLa cell cofactor fraction, is specifically required for activation by the artificial acidic activator GAL4-HA (Kretzschmar et al 1994b) In the absence of activators, PC3/DR2/Topo I represses basal transcription in vitro, a condition that can be overcome by addition of TFIIA (Kretzschmar et al 1993; Merino et al., 1993) This observation is led to the hypothesis that a function of TFIIA is to strip away repressors present in the TFIID (Cortes et al 1992). However, in the presence of activators including acidic-, proline-, and glutamine-rich activators, it increases activator-mediated transcription to levels higher than these expected to result simply from the removal of repressors (Kretzschmar et al 1993; Merino et al 1993). Both the activation and inhibitory effects of PC3 are specific for TATA-element-containing promoters (Merino et al 1993). Since PC3 can be copurified with hTFIID fraction, and its direct interaction with hTBP has been demonstrated by farwestern analysis, PC3 may function as a positive cofactor through direct interaction with TBP (Merino et al 1993). It also contains DNA topoisomerase I activity which is dispensable for transcriptional repression, activation and elongation (Merino et al 1993). PC4 substantially increases the amplitude of activatordependent activation with representatives of all types of transcription activators including acidic (GAL4), proline-

PAGE 57

rich (CTF) glutamine-rich (Spl) activators indicating its general function in transcriptional activation (Ge and Roeder, 1994) The N-terminus of PC4 contains a SEAC motif, a £L£.rine-rich and an aiiidic aa-rich region which is essential for its coactivator function (Kaiser et al., 1995). It also has phosphorylation sites for casein kinase II (CKII) which is a protein that represses cofactor activity (Kaiser et al 1995; Kretzschmar et al., 1994a). The phosphorylation of PC4 by CKII can abolish interactions of PC4 with the TBP-TFIIApromoter complex, activators, and disrupt its cofactor function (Ge et al., 1994; Kretzschmar et al 1994a). PC4 can interact with VP16 and TFIIA simultaneously (Ge et al., 1994) This bridging function may serve to recruit TFIIA for stabilization of TFIID binding. The C-terminal domain has single-strand and double-strand DNA binding abilities whose precise roles are unclear. However, its double strand DNA binding ability correlates with its capacity to facilitate activation (Kaiser et al 1995). PC4 contains a lysine-rich region that is responsible for nonspecific DNA binding. In summary, regardless of the exact mechanism of activator-mediated transcription, physical contact between activators and components of the PIC is a critical early step in this process. Since TBP, TFIIB, TAF32, TAF55 and PC4 are very important targets for various transcriptional activators, it seems worthwhile to characterize potential interactions between HSFs and these four components of the

PAGE 58

47 PIC in vitro, as well as test the functional significance of these interactions in vivo.

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48 MATERIALS AND METHODS Yeast Stra ins and Growth Yeast strain PCY2 (MATa gal4 gal80 URA3 : : GAL-lacZ lys2801 his-200 trpl-63 leu2 ade2-101) was used to assess Pgalactosidase (P-gal) activity of all constructs containing the GAL4-DBD fused to the N-terminus of the mutant HSFs (Chevray and Nathans, 1992). Strain YJB3A1 (a/trpl-289 [TRPl HSE-lacZ] ura3-52 leu2-3, 112 his31/his3HindIII met2/MET2 hls4-519/HIS4 adel-lOO/ADEl hsflgal~ /gal''' [YEpHSF^^SuRA] ) contains the scHSF on a YEplac vector and was used to substitute the heterologous HSFs from soybean and humans (Bonner et al 1992) This strain also contains an integrated HSE-lacZ gene for monitoring HSF activity. The plasmid pBS16M232v was grown in strain YJB341 (designated 341/BS16m232v) and contained a VP16 fusion with the N-terminus of scHSFm232v iri a pDBD vector (Bonner, 1991) The point mutation of methionine at residue 232 makes the HSF constitutive (Bonner et al 1992). Yeast Vectors an d their Derivatives Plasmid pPC97, a GAL4-DBD fusion vector (Vector 1), is an improved version of the vector pPC62 (personal communication; P. M. Chevray), and differs only in the

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49 polylinker sequence which is identical to that in pPC86 (Chevray and Nathans, 1992) Plasmid pPC86 was used as template to PCR-amplify the GAL4 activation domain (GAL4-AD, aa 768-881) via specific primers. The upstream primer contained a Bgl II site and the downstream primer a Not I site. The PGR product corresponding to the GAL4-AD was subcloned into pPC97 between Bgl II and Not I sites to generate vector plasmid pYDA (Vector 1) containing both the GAL4-DBD (N-terminal) and the GAL4-AD (C-terminal) with a site for insertion of modified HSF sequences in between. The open reading frame was maintained in the GAL4-DBD-AD fusion protein of pYDA. Plasmids derived from YCpGAL2 (without VP16) and pDBD22 (VP16 C-terminal fusion vector) were used to shuttle heterologous HSFs into yeast for HSF substitution experiments (Bonner, 1991) The polylinkers of these two plasmids were modified to facilitate the cloning of HSFs by the substitution of Sma I and Eco RI sites with Sal 1 and Sma 1 sites by insertion of a synthetic oligonucleotide to create plasmids pYGAL2 (no VP16) and pYDBD22 (VP16 fusion) (Vector 2) The sequences of the new polylinkers are: 5'AA,GCT,TCG,TCG,ACC,CGG,GAG,ATC,TAA,C,TAA,G,TAG-3' for pYGAL2; and 5'-AA, GCT,TCG,TCG,ACC,CGG,GAG,ATC,TGG,GCC,CCC-3' for pYDBD22. Yeast strain PCY2, and vector plasmids pPC97 and pPC86 were kindly provided by Dr. P. M. Chevray (The Johns Hopkins Univ.). Yeast strain YJB341, vector YCpGAL2 and pDBD22 were kindly provided by Dr. J. J. Bonner (Indiana Univ. )

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Vector 1 pYDA is a yeast expression vector used in the studies of basal repression of HSFs. The vector was derived from pPC86 and pPC97 (Chevray and Nathans, 1992)

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51 H Sm E Bg H Sm E Bg i Hind III and Bgl II digestions, Insert synthetic oligo H SaSmBg Hind Sail pYGAL2:iA!GCT,TCG,TCG,ACC,CGG,GAG,ATC.TAAcTAAgTAG Bgl II T r Smal Hind III r n r Sail pYDBD22:AA,GCT,TCG,TCG,ACC,CGG,GAG,ATG,TGG,GCC,CCC I I Smal VP16 Vector 2 pYGAL2 and pYDBD22 are yeast expression vectors with the GALl, 10 promoter used for the substitution of scHSF with HSFs from human and soybean. Both vectors were derived from YCpGAL2 and pDBD22 (Bonner, 1991)

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52 Construction of HSF Mutants in Yeast Vectors The cDNA clones containing hHSFl and hHSF2 were kindly provided by Dr. Carol Wu and Dr. Robert Kingston. Nested deletions of hHSFl, hHSF2, GmHSFS, and GmHSF34 were generated by PGR using Vent DNA polymerase (New England Biolabs) according to the manufacturer's instructions. A series of specific downstream and upstream primers with added Sal I and Bgl II sites, respectively, were synthesized and used in PCRmediated amplification reactions. The PCR products were digested with Sal I and Bgl II and subcloned direct ionally into yeast expression vectors pPC97, pPC86, pYDA, pYGAL2 and pYDBD22. Modified HSFs cloned into vectors pPC97 (GAL4-DBD fusions) and pYDA (GAL4-AD fusions) were transformed (Ausubel et al., 1991b) into yeast strain PCY2, and those in vectors pYGAL2 or pYDBD22 were transformed into strain YJB341. Transf ormants from strain YJB341 were further selected on 5fluoroorotic acid (5-FOA) plates which ensured loss of the endogenous scHSF contained on the URA3 vector plasmid. Yeast strain YJB341 was grown in yeast medium supplemented with 2% galactose, and PCY2 was grown in medium containing 2% glucose. For the two-hybrid system assay, two constructs in pPC86 and pPC97 were co-transformed into PCY2 and selected on Leu and Trp double drop-out (Leu and Trp minus) medium.

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53 Vector 3. pALeu and pDTrp are yeast expression vectors used in yeast two-hybrid system for cDNA screening. Both vectors were derived from vector pPC86 and pPC97 (Chevray and Nathans, 1992)

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54 Yeast Two-Hybrid System Screening All manipulations of the yeast two-hybrid system screening were done according to the protocol supplied by Clontech. The HeLa S3 matchmaker cDNA library (Clontech) in yeast expression vector pGAD-GH (GAL4-AD fusion vector with Leu2 selection marker) was amplified and the DNA purified by CsCl ultracentrif ugat ion A C-terminal deletion construct (CTAl-deletion, aa 1-422) of hHSFl was cloned into the Sal 1 and Bgl II sites of yeast expression vector pDTrp (Vector 3) (GAL4-DBD fusion vector with Trpl selection marker) derived i from pPC97 and pPC86. This plasmid (the bait vector) and the cDNA library were co-transformed into yeast strain HF7c and selected on His-drop-out (His minus) plates supplemented with 20 mM 3-amino-triazole (3-AT) Positive clones were reselected by filter assay of P-gal according to the protocol supplied by Clontech. Clones passing through the double selections were grown on Leu-drop-out medium to maintain the vector plasmid containing the cDNA insert and lose the bait plasmid. DNA of the cDNA clones were isolated and cotransformed with the bait plasmid to eliminate false positives. Only the clones that required the bait plasmid for P-gal expression were selected for further characterization. Clones passing these tests were subcloned into pUC19 and DNA sequenced (ICBR sequencing core of UF)

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55 Site-Directe d Mutagenesis For site-directed mutagenesis and internal deletion of GmHSF34, the full length GmHSF34 was subcloned into the Sal I and Bam HI sites of phage M13mpl8. Mutagenesis was conducted using the Muta-gene M13 in vitro mutagenesis kit (Bio-Rad) except that Klenow enzyme was used to replace the T4 DNA polymerase in the synthesis of the second strand. The mutants were selected by restriction enzyme digestion at a site supplied by the primer, and the sequence of the mutants was confirmed by DNA sequencing. The modified GmHSF34 coding regions were subcloned into pYDA and transformed into yeast PCY2 The p-gal activities of the mutant HSFs were determined at both basal (25C) and heat shock (37C) conditions PCR-Mediated Ra ndom Mutagenesis Low fidelity PGR random mutagenesis (Kassenbrock et al., 1993) was used to generate random mutants of the C-terminal region (AD2, aa residues 464 to 529) of hHSFl. An upstream primer with a Sal 1 site and a downstream primer with a Bgl II site were used to PCR-amplify aa residues 464 to 529 of AD2 for random mutagenesis. The PGR products were digested with Sal 1 and Bgl II, and subcloned into pPC97 The mixed population of mutants were transformed into yeast strain PCY2 to form a mutant library. Mutations that affected transcriptional activity were screened either by a filter

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56 assay, or a liquid assay of P— gal according to the yeast twohybrid system protocol (Clontech) All mutant yeast colonies were pooled and their DNAs were isolated and transformed into E.coli strain DHSa. Each mutant was reisolated and reintroduced into yeast strain PCY2 Their reduced level of transcriptional activity was reconfirmed by the ^gal liquid assay. All the confirmed mutants were sequenced by using a specific primer located in the GAL4-DBD just upstream of the polylinker Heat Shock Treatment and [3-gal Assay in Yeast System Temperatures of 25C (non-heat shock) 37C and 40C were used to stress the yeast cultures for periods of time from zero to 20 hr after the OD600 of the cultures reached approximately 0.5. After temperature treatments, P-gal assays were performed. These experiments were repeated three times. In order to test the survival of exogenous-HSFsubstituted strains under hs and cold stress conditions, the transformants selected from 5-FOA plates were spread on Leu drop-out and galactose-containing yeast medium and incubated at 15C, 30C and 37C. Western blot Analysis of GmHSF in Yeast Cells from 1.25 ml cultures (ODgQQ =1.0) were pelleted and lysed by addition of 160 |Xl of fresh 1 85 M NaOH/7.4% 2mercaptoethanol and kept on ice for 10 min Proteins were precipitated by addition of 160 [11 of 50% (wt/vol)

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57 trichloroacetic acid and kept on ice for 10 min The proteins were collected by spinning 2 min in an Eppendorf centrifuge and washed with 1 ml of ice-cold acetone. The air-dry proteins were re-extracted at 94C for 5 min with 150 ^.1 of 2x SDS loading buffer (Ausubel et al 1991a). The soluble proteins (ca. 40 |i.g/lane) were separated by 8% SDSPAGE, and the resolved proteins were transferred to a nitrocellulose membrane by electrophoresis. The ProtoBlot Western blot AP System from Promega was used to detect the expressed GmHSF34 in yeast. A 250-fold dilution of the GmHSF34 polyclonal antibody serum was used to probe the western blot Production of Antibodies against GmHSF34 The full length clone of the soybean HSF, GmHSF34, was subcloned into the pETlSb vector (Novagen, Inc.). The clone was transformed into E. coli BL21-DE3, induced with 1 mM isopropyl P-D-thiogalactopyranoside (IPTG) and grown at 37C for 3 hr. The GmHSF34 protein was extracted from inclusion bodies with 1.5% N-lauroyl sarcosine plus 1 mM ethylenediaminetetraacitic acid (EDTA) and purified by Ni column chromatography (Frankel et al., 1991; Hochuli et al 1987) Protocols for preparation of inclusion bodies and Ni column purification of histidine tagged proteins were provided by Novagen. Recombinant proteins were further purified by 8% SDS-PAGE with the band envisioned by precipitation in the gel with cold 0.25 M KCl. Recovered

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58 bands containing 300 \lq of GmHSF34 protein were lyophilized for 2 days and pulverized for injection into mice. For antibody production, two mice received triple injections at 50 |i.g of protein per injection. GST-Fusions and the Pull-down Assay Glutathione-S-transferase (GST) fusion vector pGEX-SB was derived from pGEX-KG (Guan and Dixon, 1991) by filling the EcoR I site and inserting a Sal I and Bgl II linker between SaJ I and Hind III sites. All fusions including hHSFl and 2, hTBP, hTFIIB, as well as their deletions, were constructed between SaJ I and Bgl II sites in expression vector pGEX-SB (Vector 4) and expressed in E.coli strain BL21. Expression of the fusion proteins were induced by 0.1 mM IPTG at room temperature for 3 hr and purified by Glutathione Sepharose 4-B beads according to the Pharmacia protocol (commercial supplier) The purified fusion proteins were stored in Buffer A(IOO) without BSA. Buffer A(IOO) contained 1.25 ml of IM HEPES, pH 7.5, 0.25 ml of 1 M MgCl2, 12.5 |Xl of 0.4 M EDTA, 6 ml of glycerol, 0.37 g of KCl, 2 ^il of 2mercaptoethanol, 50 ^ll of NP-40 and 50 |J.l of 4% bovine serum albumin (BSA) in 50 ml. The amount of protein on the bead was estimated by SDS-PAGE and Coomassie Brilliant Blue staining. GST-VP16 (413-490) and GST-VP16 (A456-FP442) were kindly provided by Dr. S. G. E. Roberts (Roberts and Green, 1994) as positive and negative controls.

PAGE 70

59 BaSmEXbNcSaXhScH I EcoR I, Klenow I LIgase pGEX-NE I Hind III, KJenow. Sal I I Insert Bgl U linker Xba i Vector 4. pGEX-SB is a GST fusion protein vector for expression in E. coli. It was derived from pGEX-KG (Guan and Dixon, 1991)

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60 BESc SalHNotX CTAin pPC97 Sal I and Not I Recover PC4 insert \1/ Bgl Sp N X T7.tiig BamHI— Sad Sail CTA Spel Noll Xhol •GGT,CGGGAmGA,Ga,CCG,TCGAC AGWAC,TAG,TGCGGCCGCACTCGAGCA Vector 5. p24d-SB is a E. coli expression vector with the T7-epitope. It was derived from pET-24d(+) (Novagen)

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61 Transcription factors and derived protein fragments were translationally fused with the T7-epitope to allow detection by western blot. TV-tagged hTFIIB in pET5a (Novagen) was provided by Dr. S. G. E. Roberts. hTBP from Dr. R. G. Roeder was tagged by cloning into Sal I and Bgl II sites of vector p24d-SB (Vector 5) which was derived from pET24d (Novagen) by blocking Bgl 11 and inserting a stuffer DNA fragment with Sal I site upstream and Bgl 11 downstream. Three cDNA clones, PC4, hTAF32 and hTAF55, in addition of the CTAl-Plus fragment of hHSFl were cloned from the HeLa S3 matchmaker cDNA library (Clontech) by PGR via gene-specific primers and tagged with the T7-epitope in vector p24d-SB. All of these T7-tagged clones were expressed in E.coll strain BL-21(DE3) by 0.1 mM IPTG induction for 3 hr at room temperature. The bacterial pellets from a 50 ml culture (OD600=l-2) were suspended in 3 ml Buffer A(IOO) and sonicated on ice. The bacterial protein lysates were collected after a 5 min cent rif ugation (microcentrifuge) at 4C and stored at -20C. The binding assays were performed in 100 |Il of buffer A(100, 150 or 300), containing about 5 fig of GST-fusion proteins on beads and 5 |ll of T7-tagged protein lysate The binding incubation was 2 hr at 4C with gentle rocking. The beads were extensively washed with washing buffer which is a derivative of Buffer A (150) with 10-fold less BSA. The beads were boiled in 40 ^ll of IXSDS loading buffer, and the proteins were resolved by 10% SDS-PAGE Epitope-tagged proteins were visualized by western blot using a 1:10,000

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62 dilution of anti-T7-tag monoclonal antibody (Novagen) coupled with ECL detection system (Amersham) HeLa Cell Transfection and Heat Shock Treatment HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum at 37C and transfected by the calcium phosphate precipitation method (Ausubel et al., 1991c). 0.5 \ig of Gal4-DBD-hHSFl expression constructs in vector pNG-SB (Vector 6) (derived from pCI-neo vector from Promega Corporation), 0.5 |lg of human growth hormone reporter (pXGH5 from Nichols Institute Diagnostics) for transfection efficiency, and 2 [Ig of pG5Luc luciferase reporter DNA (Patel et al 1995) were used in each transfection. Cells were harvested after 48 hr, and transfection efficiency was estimated with the hGH-TGES lOOT Kit (Nichols Institute Diagnostics) Luciferase activity of the reporter was measured using a commercially supplied kit (Luciferase assay system with reporter lysis buffer, Promega Corporation) Since hs inhibits enzymatic activity of luciferase, allowing for the recovery of HeLa cells at 37C after hs was required in order to measure activity. Forty four hr after transfection, HeLa cells were incubated for 2 hr at either 42C (hs) or 37C (control), and allowed to recover for 2 hr at 37C before harvesting.

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63 CTAinpPC97 J^Hind III, Klenow Not I Recover the insert BglHSpel Sail GAUDBD Bgin Spel Not I (damaged during cloning) g,tcg,ac|I^I^Dag,atc,tac,tag,tgcggccgc Vector 6 pNG-SB and pNeo-NB are mammalian expression vectors. pNG-SB contains GAL4-DBD for the production of GAL4-DBD fusion proteins. pNeo-NB lacks the GAL4-DBD. Both vectors were derived from pCI-neo vector (Promega)

PAGE 75

64 HeLa Cell Nuclear Extract and Far-Western HeLa cell nuclear extracts were prepared according to the method of Dignam (Dignam et al 1983) using IX 10^ cells. 500 ^11 of nuclear extract and 100 |Il of GST-CTAl immobilized on Sepharose-4B beads was used for the interaction of CTAl with nuclear proteins in buffer A(IOO). Interaction was allowed to occur for 2 hr at 4C with gentle rocking. The beads were then washed twice with 2 ml of buffer A(IOO) each time and bound proteins eluted with buffer A(500) (buffer A with 500 mM KCl) The eluted proteins were precipitated by cold 12% TCA and separated by SDS-PAGE before transfer to Immobilon PVDF membrane (Millipore) The blotted membranes were incubated with T7-tagged CTAl in buffer A(IOO) overnight at 4C. Washed membranes were then crosslinked for 30min at 4C in buffer A (100) containing 0.5% glutaraldehyde The membranes were probed by monoclonal antibody against the T7-tag, then visualized using ECL (enhanced chemiluminescense) The Squelch-Rescue Assay for Analysis of in vivo Interactions An assay based on the squelching of activated transcription was utilized. As a test for in vivo proteinprotein interactions between the activation domain of hHSFl (combined ADl and AD2, designated as CTAl-Plus) and various proteins that comprise the PIC. In this assay, the activity

PAGE 76

65 of a GAL4-DBD fusion with the CTAl-Plus fragment (activation domain ADl and AD2) was squelched in transient assays by cotransfection with vectors incorporating dysfunctional constructs of the potentially interacting proteins. If the test factor bound the GAL4-DBD-CTA1-Plus protein, a dysfunctional complex would be formed resulting in a decrease, or squelching, of luciferase activity of the reporter which was driven by a promoter containing the four GAL4 DNA binding sites This squelching should be alleviated, or the luciferase activity of the reporter increased, if a second mutation was introduced into the dysfunctional factor that eliminated its interaction with the CTAl-Plus construct. This strategy of squelch and squelchrescue was previously employed in the characterization of hTFIIB interactions with a glutamine-rich activator (Colgan et al., 1995) and in a demonstration of interaction between p53 and TFIIB and TFIID (Liu and Berk, 1995) A series of transcriptionally dysfunctional mutants of transcription factors including hTBP, hTFIIB, hTAF32 and hPC4 were cloned into the Sal 1 and Bgl 11 sites of the modified mammalian expression vector pT7-NLS (Vector 7) (derived from pCI-neo vector) The pT7-NLS vector has two unique features for the squelch-rescue assay: a T7-tag for the detection of protein expression, and a nuclear localization signal (NLS) for transporting expressed proteins into the nucleus. This NLS contains 13 aa residues from human DNA ligase I (Montecucco et al., 1995). HeLa cells were co-

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66 TJ-tag BamHI-ML Sad Sail PC4 Bgin NLS Not I GGT,CGG,ATC,CGA,An,CGA,GCT,CCG,TCG,AC AG,ATC,T GCGGCCGC P K R R T A R K 0 L P K R MIC. 5'G,ATC,m,(XG,AAG,CGT,CGC,ACA,GCT,aJG,AAG,CAG,CTC,CCG,AAA,CGC,C3' 3'AAA,GGCTIlC,GCA,GCG,TGT,a;A,GCC,TO,GT(;GAG,GGC,m,GCC,GCC,GG5' Vector 7. pT7-NLS is a mammlian expression vector with TVtag and nuclear localization signal. It encodes both a TVtag and NLS (Montecucco, et al 1995) used in squelch-rescue assays. It was derived from the pCI-neo vector indirectly via pNTV-SB.

PAGE 78

67 transfected with four plasmids : 3 |J.g of the transcriptionally dysfunctional mutants in the pT7-NLS vector, 2 of reporter plasmid pG51uc containing GAL4 binding sites upstream of luciferase gene, 0.5 |lg of the effector construct containing GAL4-DBD-CTA1-P1US fusion in the pNG-SB vector, and 0.5 [Lq of pXGH5 for monitoring transfection efficiency. The final amount of DNA for each transfection was equalized by adding empty vector pNeo-NB DNA where appropriate. All transf ections were conducted in triplicate, and activities of the luciferase reporter gene were normalized for transfection efficiency by dividing units of luciferase activity by units of human growth hormone produced. Co-immunoprecipitation A second method used to detect in vivo associations between the activation domains of hHSFl and GTFs was based on their co-immunoprecipitation. CTAl, mutated CTAl and CTAlPlus constructs of hHSFl in the GAL4-DBD vector pNG-SB were each co-transfected with TV-tagged hTBP (also in vector pNTSB) into HeLa cells. CTAl and T7-hTBP were transfected individually into HeLa cells as negative controls. Forty eight hr after transfection, cells were harvested and lysed by three rounds of f reezing-and-thawing in buffer A(500) plus a cocktail of proteinase inhibitors (0.1 mM PMSF, 2 )ig/ml leupeptin, 3 |Ig/ml pepstatin A, 40 P-g/ml antipain) The lysates were cleared by centrif ugation for 5 min at 4C and collected. For co-immunoprecipitation, the lysates were

PAGE 79

68 diluted with buffer A(0) to the same final salt concentrations as in buffer A (150) GAL4-DBD fusions were co-immunoprecipitated from cellular lysate with ant i-GAL4-DBD monoclonal antibody (mAb) which was covalently crosslinked to protein A Sepharose beads by using DMP (dimethylpimelimidate) (Harlow and Lane, 1988) The beads were washed twice with buffer A(150) and boiled for 5 min in SDS loading buffer. Released proteins were resolved by 10% SDS-PAGE and blotted to Immobilon PVDF membrane (Millipore) The membrane was then probed with mAb (monoclonal ^ntitiody) against the T7epitope to detect the T7-tagged hTBP Pull-down Assay of hTBP Deletions Transiently Expressed in HeLa Cells hTBP and its deletions were cloned into Sal 1 and Bgl II sites of vectors pNT7-SB (Vector 8) (without NLS for wild type TBP) or pT7-NLS (with NLS for deletions of TBP) and transiently expressed in HeLa cells. Cells were harvested and lysed in buffer A(500) plus proteinase inhibitors by three rounds of quick freezing and thawing. The lysates were collected after a 10 min centrif ugation (microcentrifuge) at 4C. The lysate was incubated with rocking for 2 hr at 4C with 5 |lg of GST-CTAl or GST-CTAl-Plus fusion protein immobilized on Sepharose-4-B beads in buffer A (150) The beads were washed three times with buffer A(150) and boiled for 5 min in SDS loading buffer. Released proteins were analyzed by western blotting as described above.

PAGE 80

69 Bgin PC4 in p24dNB Xba I, Not I Recover the Insert Bi,Sc3l I Bgl n.Spe I Vector 8. pNT7-SB is a mammlian expression vector with the T7-tag. It was derived from the pCI-neo vector (Pr omega)

PAGE 81

• If 70 hTFIID Comp lex Pull-down by GST-fusions of hHSFl For detection of interactions of CTAl and CTAl-Plus with hTFIID complex, 180 ^11 of buffer A(IOO) plus 0.1% NP-40, 50 |Il of the HeLa nuclear extract and 20 |Il of immobilized GSTCTAl, or GST-CTAl-Plus, were incubated for 2 hr at 4C with gentle rocking. The final salt concentration was adjusted to 100 mM, 150 mM or 250 mM by adding 3 M KCl. The beads were washed twice with buffer A (150) and then eluted by buffer A (500) and visualized by western blotting. Endogenous hTBP and TAF250 in the TFIID complex were detected by anti-hTBP mAb from Promega and anti-TAF250 mAb provided by Dr. R. G. Roeder. For detection of interactions between NR and the TFIID complex, 500 |Il of buffer A(IOO) and 500 |Il HeLa nuclear extract were mixed and passed through GST-NR affinity column (150 ^ll of GST-NR) by gravity. Bound proteins were washed with 1.5 ml of buffer A(IOO), eluted with buffer A(500) and visualized by western blot using anti-hTBP antibody

PAGE 82

RESULTS The Function and Structure Studies of HSFs fro m Human and Soybean Mapping the Transcriptional Activation Domains of hHSFl and hHSF2 in Yeast and HeLa Cells In order to identify domains of hHSFl involved in transcriptional activation and maintenance of basal repression, a series of Nand C-terminal truncations were initially evaluated in yeast cells. Activity of hHSF mutants was monitored using P-gal activity generated by a GALl-lacZ reporter integrated into the chromosome of yeast strain PCY2 All hHSF constructs were fused with the GAL4-DBD at the Nterminus (plasmid pPC97, Vector 1) Since nuclear localization and DNA binding functions were provided by the GAL4-DBD, this system tolerated mutations which eliminated the corresponding regions of the hHSF. Mutations could also be placed in the OD since HSF trimerizat ion was presumably not needed for binding to the GAL4 DNA binding site. Expression of the full length hHSFl protein fused to the GAL4-DBD showed activity in yeast under hs conditions and no detectable activity under normal growth conditions indicating that basal repression and heat inducible activation of hHSFl were able to occur in this heterologous system (Fig. 1,

PAGE 83

72 A. %-CTA1 GAL4-DBD (AD2) (ADD CTA1 1 2 3 4 5 6 7 8 hHSF1 Yeast 25C 37 C 529 0 0 25 53 100 0 0 0 11 9 32 52 90 0 0 16 GAL4 Activation Domain HeLa 37 op 42 C 0 3.2 0.2 4.4 1.4 13.4 131 129 114 100 <0.1 0.3 <0.1 0.2 <0.1 1.4 hHSF2 CTA2 9 10 DBD OP H HR3 397 536 0 10 3 11 <0.1 0 1.7 0.4 Fig. 1. Mapping of human HSFl and HSF2 regions involved in transcriptional activation and basal repression in yeast and HeLa cells. The transcriptional activity of construct 5 under non-heat shock conditions was designated as 100% in both yeast and HeLa cells, and the activities of other constructs were expressed as relative activity of construct 5. CTAl = C-terminal transcriptional activation domain of hHSFl; CTA2 = C-terminal transcriptional activation domain of hHSF2; DBD = DNA binding domain; HR =heptad repeat; OD = oligomerization domain.

PAGE 84

73 construct 1) Removal of 119 aa residues (aa) from the Nterminus demonstrated that the hHSFl DBD was not involved in basal repression or transcriptional activation (construct 2) Further deletion of aa 120 to 174, which removed HR-A of the OD, resulted in both an increase in overall activity and a loss of basal repression (construct 3) Activity under both control and HS conditions nearly doubled when the remaining portion of the OD (HR-B) and the region N-terminal to HR-C was deleted in construct 4 despite low levels of protein expression (Fig. 2) Loss of transcriptional activity was not seen until a portion of the C-terminus was deleted in construct 6. These results indicate that, when assayed in yeast cells, the transcriptional activation domain of hHSFl is located in the 107 aa C-terminal to HR-C. The difference in P-gal activity between constructs 4 and 5 was most likely artifactual due to the extremely low amount of stable protein derived from construct 4 (Fig. 2) All other constructs showed similar levels of expression and correct protein size by western blot analysis (Fig. 2) The C-terminal activation domain identified here in yeast corresponds to the AD2 region characterized by Shi et al. (Shi et al., 1995) and Newton et al. (Newton et al., 1996) and does not include the adjacent region surrounding HR-C (ADl domain) (Green et al., 1995; Newton et al., 1996; Zuo et al 1995). The same experiment was repeated in HeLa cells with similar results except in the case of construct 3. Comparison of the activity of construct 3 in yeast and HeLa

PAGE 85

74 1 2 3 6 7 8 4 5 Yeast 9 10 HeLa 1342 5 10 67 89 Fig. 2. Western blot of human HSF constructs expressed in yeast and HeLa cells probed with anti-GAL4-DBD antibodies. The bar indicates expected size of expressed protein for construct 4. Numbers correspond to constructs depicted in Fig. 1.

PAGE 86

75 cells, under both hs and non-heat shock conditions, indicated that HR-A was required for the basal repression of hHSFl in yeast, but dispensable in HeLa cells (Fig. 1) Similar levels of protein expression in HeLa cells were observed for each of the constructs except constructs 2, 6 and 7 (Fig. 2) There was no correlation between the efficiency of protein expression and activity of the reporter genes. For example, constructs 6 and 7 showed high level of protein expression, but only residual activity. The low activity of construct 6 was not surprising since the CTAl domain had been deleted. A possible mechanism for basal repression may include masking of the DBD under non-heat shock conditions. To test this hypothesis, gel retardation assays were performed using whole cell extracts from the strain containing construct 2 to determine if basal repression was due to the inability to bind DNA under control conditions. Analysis of extracts from construct 2 grown under non-heat shock conditions indicated that the GAL4-DBD was able to bind DNA despite the lack of transcriptional activity (Fig. 3) These results indicate that the GAL4-DBD was not masked during basal repression under non-heat shock conditions, and imply that the DBD of the wild-type protein may also be accessible under basal conditions. The slight reduction in DNA binding activity upon hs may reflect a partial loss of function of the GAL4DBD. The presence of the GAL4-DBD-hHSFl protein in the DNA complexes was confirmed by the lack of complex formation by the vector less control strain PCY2, and by removal of

PAGE 87

76 B F 1 2 3 4 5 Fig. 3. Gel retardation assay of whole cell extract prepared from yeast containing construct 2. The GAL4-DNA binding site was used as a probe to test the ability of construct 2 to bind to DNA under control and heat shock conditions. Lane 1 is free probe. Strain PCY2 containing no vector plasmid was loaded in lanes 2 (non-heat shock) and 3 (heat shock) Whole cell extracts containing construct 2 was loaded in lanes 4 (non-heat shock) and 5 (heat shock) The presence of GAL4-DBD fusion proteins in the bound complexes was confirmed by supershift using monoclonal antibodies against the GAL4-DBD (not shown) F = free probe; B = bound.

PAGE 88

11 complexes by immunoprecipitat ion with antiGAL4-DBD antibody (not shown) The C-terminal location of the activation domain of hHSF2 (aa 397 to 536, designated as CTA2) is similar to the CTAl of hHSFl as demonstrated by constructs 9 and 10 (Fig, 1) Although activity of full length hHSF2 was only one fourth that of hHSFl in yeast, basal repression and heat inducibility were still evident. As with hHSFl, removal of all portions of the HSF N-terminal to HR-C resulted in a large increase (4-fold) in constitutive activity. The CTAl of hHSFl (AD2) appeared to be approximately eight times stronger than CTA2 of hHSF2 when assayed in isolation in yeast cells. HSF2 constructs 9 and 10 showed little activity in HeLa cells compared to yeast. Full length HSF2 had no significant activity under either basal or hs conditions. In addition, activity of CTA2 (construct 10) was less than 2% of CTAl, whereas in yeast this domain exhibited between 10 and 11% of CTAl activity. Experiments were conducted to evaluate potential interactions between hHSFl and hHSF2 activation domains since activation of the Hsp70 gene appears to be synergist ically induced by hHSFl and hHSF2 in cells (Sistonen et al 1994), GAL4-DBD fusion proteins containing either CTAl of hHSFl, or CTA2 of hHSF2 were co-expressed and activity monitored using the GAL4 DNA binding site reporter. As shown in Fig. 4, activity of CTAl (construct 5), was approximately 65-fold greater than CTA2 (construct 10) when each was expressed

PAGE 89

78 > o < > 10050 1 1 1 1 1 1 1 I i 1 IHI CTAl CTA2 CTAl + CTA2 GAL4DBD fusions Fig. 4. Co-expression of GAL4-DBD fusion proteins containing the CTAl or CTA2 of hHSFl GAL4-DBD fusion proteins were expressed in HeLa cells using vector pNG-SB. DNA used in transf ections : lane 1, CTAl (0.5 ^g, construct 12); lane 2, CTA2 (0.5 |Xg, construct 10); lane 3, CTAl (0.5 |Xg) plus CTA2 (0.5 |J,g) Each transfection contained 0.5 |lg of growth hormone vector (pXGH5) and 2 |ig of reporter plasmid (pG5Luc) The amount of DNA per transfection was kept constant by adjusting transf ections (lanes 1 and 2) with the addition of "empty" vector DNA( 0.5 |lg of pNeo-NB) Relative luciferase activity was normalized using growth hormone production. Error bars were derived from three replicate experiments.

PAGE 90

alone. Co-expression of CTAl and CTA2 resulted in activity levels that were intermediate between that of the two activation domains expressed separately indicating a lack of synergistic interaction. These results suggest that the synergistic response seen previously in hemin and heat induced K5 62 cells must involve events not directly related to the C-terminal activation domains of hHSFl and 2. Two interesting phenomena (Fig. 1) are evident in the comparison of activities between either the full length protein (construct 1) or the isolated CTAl domain (construct 5) and a C-terminal deletion of CTAl which leaves ADl/HR-C intact (construct 6) The first is the amazingly low activity level of the full length protein compared to the isolated CTAl in both HeLa and in yeast. This result indicates that CTAl is heavily repressed in the full length protein, even under hs conditions. A second point of interest is the total lack of activity of ADl/HR-C when CTAl has been removed, but the remainder of the protein is present (construct 6) Identification of Subdomains within the Transcriptional Activation Domai.n of hH.qpi Deletions within CTAl were conducted to more precisely define the aa motifs involved in transcriptional activation (Fig. 5). The conservation of CTAl subdomains is shown in Fig. 6. Three subdomains were identified that make substantial contributions to activity: subdomain I located between aa 422

PAGE 91

80 A. GAL4-DBD | g 422 443 464 5 11 12 13 14 15 16 17 18 19 20 21 B. b III 489 509 529 o 443 464 454 464 443 443 o 464 o 494 509 489 489 509 509 489 13 16 20 18 19 17 5 11 12 14 15 21 P-Gal activity NHS HS 203 182 40 43 31 36 0 0 89 117 72 80 5 6 11 9 0 0 16 18 18 17 0 0 Fig. 5. Subdomain mapping of CTAl in yeast. All constructs were derived from pPC97 and were expressed in yeast strain PCY2 A.) Major regions of activity are designated I, II, III with minor regions designated a and b. B.) Western blots of human HSFl constructs expressed in yeast using anti-GAL4-DBD antibody as probe. Numbers correspond to constructs depicted in panel A. NHS = non-heat shock; HS = heat shock.

PAGE 92

81 hHSFl mHSFl chHSFl hHSF2 mHSF2 chHSF2 hHSFl mHSFl chHSFl hHSF2 mHSF2 chHSF2 hHSFl mHSFl ChHSFl hHSF2 mHSF2 chHSF2 VTVPDMSLPDLDSSIASIQEI LSPQEPPRPPEAENSSPDSG SALLDIQEl LSPQEPPRPIEAENSNPDSG mtvtdmnlpdldqmnptdyinntkmlsgrqfsidpdllvdsenkgleatIkssvvqhvseegrkskskpd ptdh i pntkm etkg i ett fcsnagpaasqetqvskpks d SSLASIQDL LSSQEQQKPSEADAAAADTG SENKGLETT iCNNWQPVSEEGRKSKSKPD 462 436 424 439 420 469 C-1 II S glSBaSaSSSiieiE F-LLD PGSVDTGSNDLI|-VLFELGEGSYFSE — GDGFAE :J( pv^5Ff*SPl F-LLD PDAVDTGSSELE — VLFELGESSYFSE — GDDYTD KSBVHSETA^EF-LVD SSAVDVGSGDLP — IFFELGEGS YFTD— GDEYNE t iitl52*JS!Uf*t LAFLDGNPASSVEQASTTAE SEVLSSVDKPIEVDELLDSSLDP t gKCSyCAFPI LAFLDGNSASAIEQGSTTAS SEWPSVDKPIEVDELLDSSLDP 8 BUcSXXWFl LAFLDGNPGSTVESGSSAT^Tiiiif(#* m i ** ETPSSVDKPLEVDELLESSLDP C-2 OPT jstwiwssre p III PKAKD PTVS HKAKD PTVS PKPKD PTVS |Wff$^$6j6StttjS|PLTEAEASEATLFyLCELAPAPLDSDMPLLDS Kt^fSgKEiHREB PLTEAEASEATLFYLCELAPAPLDSDMPLLDS i evas^xti^m plteaeaseatlfylcelapapmdtdmffldn 529 503 491 536 517 564 508 482 470 493 474 521 Fig. 6. Comparison of the CTAl of hHSFl with C-terminal regions of vertebrate HSFs. Amino acid sequences were compared using the CLUSTAL W(1.4) program. Identical residues are designated by an asterisk and those with conserved biochemical character are indicated by a closed circle. Major subdomains of activity determined for hHSFl are represented by open boxes superimposed on sequences from all HSFs. Two regions of high conservation are identified in the shaded boxes C-1 and C-2. h = hioman; m = mouse; and ch = chicken.

PAGE 93

82 and 443, subdomain II located between aa 4 64 and 489, and subdomain III located between aa 509 to 529. Contribution from regions of less importance (subdomains a and b) were also seen. Subdomain I was identified by comparison of constructs 5 and 11 where a drastic loss of activity resulted from deletion of 21 aa normally located immediately adjacent to HR-C in the context of the full length HSF Subdomain II was revealed by the deletion of 25 aa in the central portion of the CTAl (compare constructs 12 and 13) Removal of the C-terminal 20 aa also resulted in a large drop in activity (compare constructs 5 and 14), and this region was designated as subdomain III. When assayed individually in isolation, the major subdomains showed either no, or little activity. This phenomenon is illustrated by construct 16 for subdomain I, construct 21 for subdomain II, and construct 13 for subdomain III. The contributions of the minor subdomains is more context sensitive. The influence of subdomain a is seen in the differences in activities between constructs 11 and 12, and between constructs 16 and 17. Although subdomain a contributed positively to subdomains II and III in combination, and to subdomain I in isolation, it seems to have had little effect on subdomain II in isolation (compare constructs 19 and 20) Subdomain b contributed positively to subdomains I and II in combination (compare constructs 14 and 15) and, unlike subdomain a, to subdomain II in isolation (compare constructs 20 and 21) These results indicate that the entire region located C-terminal to HR-C functions as a

PAGE 94

83 transcriptional activation domain (CTAl) with each of the subdomains making a synergistic contribution to the activity of the whole Point mutations were introduced into a portion of CTAl in an attempt to identify individual aa residues critical to transcriptional activity. Since CTAl seemed to be comprised of multiple elements, single aa changes were not likely to result in large changes in activity which would facilitate screening of mutants To reduce this problem, only the Cterminal portion of CTAl (construct 12) from aa residues 464 to 529 (CTA4g4_529) was used for these studies. Although this fragment showed only 15% of intact CTAl activity (Fig. 2), it is a highly conserved region with two major (II and III) and one minor (b) subdomains for transcriptional activity (Fig. 5 and 6) Analysis of point mutations within CTA4g4_529 indicated the importance of conserved aa residues present in HSFl and HSF2 families. Random mutations were introduced into CTA4g4_529 of construct 12 using low fidelity PCR (Kassenbrock et al 1993). Approximately 200 yeast transf ormants containing cloned DNA fragments derived from PCR mutagenesis were then assayed for (i-gal activity. Roughly 15% showed impairment of activity ranging from 50 to 100%. The results of DNA sequence analysis of some of the clones with impaired activity are shown in Table 1. Mutations were assigned to two groups based on activity compared to the nonmutated CTA454_529: Group A showing zero or slight residual activity, and group B exhibiting 30 to 50%

PAGE 95

84 mutant %-GaI %-Lux C-1 II b C-2 III /hGH Group A: MA-1 0 6.9 D477V; L515P P522K D482A MA-2 0 D477V; L515P P521S D482A MA-3 <3 6.1 A470V F500Y L514P MA-4 <3 P489L T516S Group B: MB-1 30-50 L491Q; E508G MB-2 30-50 S480P L515P K524temi MB-3 30-50 17.7 E508K MB-4 30-50 20 A470V Table 1. Analysis of PGR generated point mutations of CTA464529. Random mutations were placed in CTAl domains II, b and III, selected in yeast. Four representative mutants were assayed in HeLa cells. Percentage P-gal and lucif erase activities were determined relative to wild-type activity (construct 12) in yeast and HeLa cells respectively. C-1 and C-2 are regions of the CTAl showing conservation among animal HSFl and HSF2 families (Fig. 6) Mutations affecting amino acid residues showing conserved identity among HSFl and HSF2 families are in bold letters, those affecting residues with conserved similarity are underlined.

PAGE 96

85 activity. Mutations which resulted in partial loss of activity (Group B) were the most informative and revealed the importance of alanine 470 (mutant MB-4) and glutamic acid 508 (mutant MB-3) Alanine 470 shows identity among all members of the HSFl and HSF2 families and is located within conserved region C-1 (Fig. 6) Amino acid residue 508 is adjacent to conserved region C-2 This position is occupied by negatively charged residues in vertebrate HSFl. The deleterious effect of removal of the negative charge at position 508 by mutations E508G and E508K, and the conservation of negative charge at position 509, suggests that both residues 508 and 509 may be required for C-2 function within the HSFl family. Mutants MB-3 and MB-1 had roughly the same activity suggesting that mutation of L491 to Q (MB-1) had little effect. The A470V mutation within C-1 occurred twice demonstrating the critical role of alanine 470. The difference in activity between MB-4 and MA-3 indicates that either phenylalanine 500 or leucine 514 (within region C-2), or both, are also involved in transcriptional activation. In addition, the occurrence of the L515P mutation in three of the mutants suggests that this conserved aa residue of C-2 is also required for full activity. Assay of reporter activity of select mutations in HeLa cells showed similar results to the yeast expression studies with all mutant protein present at comparable levels (Fig. 7) It seems significant that in this limited

PAGE 97

86 MA-1 MA-3 MB-3 MB-4 WT Fig. 7. Western blot of point mutations of CTA464-529. WT = wild type; Mutants from MA-l to iyiB-4 were mutations selected from Table 1. All mutants were GAL4-DBD fusions and transiently expressed in HeLa cells. The expression of each mutants were measured by western blots using anti-GAL4-DBD antibody as probe.

PAGE 98

87 analysis most changes involved either hydrophobic or negatively charged aa Attempts at Domain Mapping of Soybean HSF34 and HSFS in Yeast A strategy of deletion analysis similar to that used with hHSFs was used with soybean HSFs (GmHSFs) in order to identify domains involved in transcriptional activation and basal repression. In contrast to human HSFs, the full length soybean HSFs failed to show any activity when monitored using the GAL-lacZ reporter system (Table 2) To test whether the lack of activity was due to a failure to release basal repression, a series of N-terminal and C-terminal deletions were analyzed. As with the hHSFs, all constructs contained the GAL4-DBD fused to the N-terminus. No deletion was found for either GmHSF5 or GmHSF34 that resulted detectable P-gal activity (Table 2) suggesting that the transcriptional activation domains were not functional in yeast, or that activation domains were not present on these particular HSFs. In the next series of experiments (Fig. 8), the acidic activation domain of GAL4 (aa 768 to 881) (Chevray and Nathans, 1992) was fused to the C-terminus of GmHSF34 to provide an activator known to be functional when fused to hHSFl (Fig. 1, construct 8) As before, most constructs showed little or no activity under either control or hs conditions. However, removal of aa 103 through 154 from the N-terminus caused GmHSF34 to show 3 6 units of constitutive activity suggesting that this portion of the HSF was

PAGE 99

88 construct HSF deleted amino acids domains intact activity (units) 22 GmHSF34 full length D, HR-A, HR-B, HR-C 0 23 GmHSF34 DN 1-40 HR-A, HR-B, HR-C 0 24 GmHSF34 DN 1-102 HR-A, HR-B, HR-C 0 25 GmHSF34 DN 1-154 HR-A, HR-B, HR-C 0 26 GmHSF34 DN 1-175 HR-B, HR-C 0 27 GmHSF34 DN 1-203 HR-C 0 28 GmHSF34 DN 1-248 HR-C 0 29 GmHSF34 DC 250-282 D, HR-A, HR-B 0 30 GmHSF34 DC 205-282 D, HR-A, HR-B 0 31 GmHSF34 DC 156-282 D 0 32 GmHSF5 full length D, HR-A, HR-B 0 33 GmHSF5 DC 349-370 D, HR-A, HR-B 0 34 GmHSF5 DC 329-370 D, HR-A, HR-B 0 35 GmHSF5 DC 309-370 D, HR-A, HR-B 0 36 GmHSF5 DN 1-255 0 37 GmHSF5 DN 1-255; DC 349-370 0 38 GmHSF5 DN 1-255; DC 329-370 0 39 GmHSF5 DN 1-255; DC 309-370 0 Table 2. Analysis of GmHSF-GAL4-DBD fusions in yeast cells. Fusion constructs of GmHSF34 and GmHSFS were tested in strain PCY2 for P-gal activity under both hs (370C) and control (250C) conditions for 1 hr. No activity at either temperature was obtained for any of the constructs tested. Nand C-terminal deletions are denoted as DN and DC, respectively. The GmHSF34 protein is predicted to have 282 amino acids and GmHSFS to have 370 amino acids. Domains: D, DNA binding domain; HR-A, hydrophobic repeat A of the OD; HRB, hydrophobic repeat B of the OD; and HR-C, C-terminal hydrophobic repeat C. GmHSFS has no identifiable HR-C. Fusion constructs 22 and 32 contained full length HSFs.

PAGE 100

89 GAL4-DBD r-^vjccoA GAL4 Activation R-Gal activitV GmHSF34 Domain \ n hs and hs HR-A HR-B 240 A240-258 47 48 49 50 I 164-166 KLK to GPG 186-188 LVA to PGG r-|— h 282 28r 225-227 LKL to GPG =n — 0 0 0 0 B. 44 45 48 49 50 47 40 41 42 43 46 Fig. 8. Expression of GinHSF34 -GAL4 -DBD fusion proteins in yeast. A.) P-gal activity did not vary significantly between control (nhs) and heat shock (hs) conditions. All constructs were cloned in vector pYDA and expressed in yeast strain PCY2 Point mutations in constructs 48 through 50 were as follows: 164GPG (KLK 164-166 changed to GPG); 186PGG (LVA 186-188 changed to PGG) ; and 225GPG (LKL 225-227 changed to GPG) B.) Western blot of soybean HSF constructs expressed in yeast using anti-GAL4-DBD antibodies. Numbers correspond to constructs depicted in panel A. nhs, non-heat shock; hs, heat shock.

PAGE 101

90 inhibiting activity of the GAL4 activation domain. When the C-terminal portion of HR-C was deleted, a low amount of constitutive activity was seen (construct 45) An even more dramatic increase in constitutive activity (396 units) was obtained by removal of 19 additional aa (aa 241 to 258) from the C-terminus suggesting that these 19 aa alone were responsible for the repression of activity. However, removal of these aa by internal deletion (construct 47) failed to activate transcription indicating that the entire HR-C must contribute to inhibition of the GAL4 activator. Substitution mutations within the OD and adjacent region were designed to disrupt the predicted coiled-coil structure by replacing hydrophobic residues and, perhaps, relieve basal repression. However, none of the three substitutions resulted in activity under either basal or heat shock conditions (constructs 47 through 50) Similar mutations in hHSF2 resulted in nuclear localization. The lack of effect of point mutations in the present study may have several interpretations and offers no obvious explanation for the lack of activity of the GmHSF34 constructs. Although these experiments did not identify domains involved in either basal repression or transcriptional activation, they did indicate that at least two regions of GmHSF34 are very inhibitory to the function of the acidic GAL4 activator domain: the linker region between the DBD (aa 103 to 154), and the HR-C region in the C-terminus (aa 249 to 282)

PAGE 102

91 Substitution of scHSF with HSFs from Human and Soybean Cells Human and soybean HSFs were substituted for the endogenous scHSF of yeast to assess the degree of conservation in the mechanisms of basal repression and transcriptional activation. Yeast strain YJB341 has a deletion in the chromosomal scHSF and is totally dependent for survival and normal growth on a copy of the scHSF present on a URA3 vector plasmid ( YEpHSF^SuRA) (Bonner et al., 1992). The heterologous HSFs were introduced into YJB341 using plasmids pYGAL2 and pYDBD22 (VP16 fusion to C-terminal of HSF) and then selected on 5-f luoroorot ic acid plates which eliminated the URA3 plasmid containing scHSF. Strain viability and HSF activity are shown in Fig. 9. HSF activity was monitored using the HSE-lacZ reporter gene present in yeast strain YJB341. The only nonviable substitution was with GmHSF34 (construct 54) This result was expected since no activity was seen when full length GmHSF34 was fused to the GAL4-DBD (Table 2, construct 22) Surprisingly, GmHSFS was able to substitute for scHSF even though no activity could be detected in the GAL4 fusion system (Table 2; construct 32) No viable colonies were obtained using a deletion of GmHSFS missing 42 aa on the C-terminus (construct 52) suggesting that the transcriptional activation domain may be located at the C-terminus. Viability of cells containing the C-terminal truncation was restored by the fusion of the VP16 acidic activator to the C-

PAGE 103

92 A. GmHSFS DBD 51 ^ p-Gal activity B. HR-A HR-B 1 21 115 191 253 370 NHS HS 1.4 2 52 53 — not viable 328 20 23 328 VP16 GmHSF34 DBD 54 .1 HR-A HR-B HR-C 1 6 103 155 204 249 282 55 -C 56 -C 240 240 VP16 not viable not viable 1200 1200 hHSF1 57 DBD ""-'^ ""-^ 1 16 120 137 212 hHSF2 HR-C 382422 529 13 58 i DBD 1 8 112 126 201 358 399 536 kDa Growth at 30 C C1 53 56 Pi 57 [ 1 Growth at 37 C 53 C2 56 51 C1 m 57 58 Fig. 9. Substitution of scHSF with HSFs from humans and soybean. A.) p-gal activity determined for all constructs in strain YJB341 after removal of scHSF by selection on media containing 5-FOA. Constructs 53 and 56 were derived from pYDBD22, all other were from pYGAL2 B.) Colonies after continuous growth on plates at indicated temperatures, Numbers indicate constructs shown in panel A. CI and C2 were positive control strains containing either the heat inducible scHSF (strain YJB341) or constitutive scHSFM232V {341/BS16M232V) repectively. C.) Western blot of soybean GmHSF34 constructs 56 and 54 expressed in yeast using antiGmHSF34 antibodies. Due to the nonviability of GmHSF34 substitution, cells containing construct 54 were assayed before 5-FOA selection. Control CI same as in panel B.

PAGE 104

93 terminus (construct 53) In a similar manner, the fusion of the VP16 activator to a C-terminal truncation of GmHSF34 also resulted in viable cells (construct 56) In the case of the GmHSF34 (Ac-terminus) -VP 16 fusion, p-gal activity was unusually high (1,200 units) compared to the analogous VP16 fusion with GmHSFS (AC-terminus ) suggesting that remaining portions of GmHSF5 may be inhibitory. It should be noted that neither of the plant HSFs showed significant heat inducibility One interpretation of these results is that the mechanism of basal repression for these two plant HSFs is not compatible with the yeast system. The lack of substitution in the case of GmHSF34 seems to indicate that the activation domain, if present, is also not compatible with yeast expression. An alternate interpretation is that these two soybean HSFs lack transcriptional activation domain and possess no inherent ability to regulate basal transcription. Both human HSFl and 2 were able to substitute for yeast HSF (constructs 57 and 58) and exhibited basal repression and heat inducibility. As was the case in the GAL4-DBD fusion constructs, hHSFl showed greater activity (Pgal) than hHSF2 All HSF-substituted strains were able to sustain growth at 15C, 30C and 37C, but all grew better at 30C than at 15C (not shown) or 37C (Fig. 9) All strains, with the exception of hHSF2, were drastically inhibited at 37C. The hHSF2-substituted strain grew almost as well as the wild-type yeast strain at this temperature. There was no correlation

PAGE 105

94 between the activity of the HSF monitored by the P-gal assay and the ability to maintain sustained growth at high temperature Activity of hHSFl and hHSF2 in Yeast under Sustained Heat The time course of activity for hHSFs in yeast lacking the endogenous HSF (scHSF) was determined by heat stressing cells at either 37C (normal heat shock) or 40C (severe heat stress) for up to 20 hr. The yeast control harbored the plasmid YEpHSF^^S URA3 (Bonner, 1991) which contained a Cterminal truncation of scHSF (at aa 583) and was heat inducible. Under heat shock temperatures the two hHSFs exhibited delayed activation detectable after approximately 30 min (Fig. 10) The yeast HSF showed a typical transient response (Sorger, 1990) with activity peaking from 1 to 2 hr after induction and declining to slightly less than one half of peak activity by 4 hr In contrast, the hHSFs showed a gradual reduction in activity after a peak at 2 hr After 20 hr at 37C, scHSF had a residual activity of approximately 1 unit, hHSF2 had roughly 4 units, and hHSFl still exhibited 10 units of activity. Yeast HSF was attenuated by 96%, whereas hHSF2 lost 64% of its activity, and hHSFl was only reduced by 41%. Under severe heat stress (40C) the transient nature of scHSF expression diminished. Peak values of scHSF activity after 2 hr were nearly identical to those at 37C, but attenuation during extended periods of stress was impaired.

PAGE 106

95 -O-scHSF -^hHSFI -o-hHSF2 37C (hr) 8 20 -O-scHSF ^hHSFI a-hHSF2 8 20 40C (hr) Fig. 10. Activity of hHSFl and 2 under hs conditions in yeast cells lacking scHSF. Human HSFs were introduced into strain YJB341 using pYGAL2 vector and substituted for the endogenous HSF by selection on medium containing 5-FOA. The control strain was YJB341. HSF activity was monitored by assaying ^-gal activity.

PAGE 107

96 The hHSFs also showed less decline in activity under severe heat stress. During prolonged heat stress, two noticeable differences were seen when hHSFs were substituted for the scHSF: a delayed activation, and the lack of a clearly defined transient response. The inability of the hHSFs to down modulate after 2 hr of heat shock may explain the poor growth at 37C of yeast cells substituted with plant and human HSFs However, this can not be the only reason since GmHSFS showed relatively low activity (1 to 2 units. Fig. 9) under both control and heat shock conditions. The Functional Targets of hHSFT in Transcription Strategies Used to Characterize Protein -Protein Interact inns in vitro and in vivo General assumption that underlies these experiments to characterize protein-protein interactions between HSFs and components of the PIC is that the predominant mechanism in transcriptional activation is based on recruitment mediated through direct protein-protein contacts. One strategy employed here to identify and map interactions in vitro entailed the binding of GTFs in solution to immobilized GST fusions consisting of various full length and deletion constructs of hHSFs (Fig. 11) The GST-HSF fusion proteins were bound to glutathione Sepharose beads forming an affinity matrix that was incubated with extracts containing

PAGE 108

97 T7-tag bead GST bead GST glutathione Binding; Washing Western blot Fig. 11. GST-pull-down assay. Protein X is T7 epitopetagged and protein Y is a GST fusion immobilized on glutathione Sepharose beads. After co-incubation, pelleting the beads will pull -down X if X and Y interact stably. Protein X released from the beads is visualized on western blot by probing with anti-TV tag antibody. If protein X is not tagged, as in the case of endogenous factors from a HeLa nuclear extract, protein X-specific antibodies must be used in the western blot to determine interaction with protein Y.

PAGE 109

98 recombinant peptides derived from GTFs expressed in E. coli or HeLa cells. The binding reactions were conducted at near physiological salt concentrations and pH to approximate conditions in vivo. Ligand proteins and their respective deletion constructs were T7-epitope-tagged to facilitate detection of bound proteins by western blotting. Negative controls included interactions with immobilized GST and nonbinding mutants of scTBP(l-82) and VP16 (A456-FP442 ) Wild type VP16 (413-490) was also used as a positive control for binding where appropriate The choice of factor proteins selected for testing was determined by availability and by previously demonstrated ability to interact with the acidic activator VP16. Two approaches were employed to demonstrate interactions between hHSFl and GTFs in vivo: co-immunoprecipitat ion of transiently expressed from HeLa whole cell extracts, and the squelch-rescue assay. In co-immunoprecipitation experiments a GAL4-DBD fusion construct containing either CTAl or CTAlPlus was co-expressed with a TV-tagged hTBP GAL4-DBD fusions with the HSF activation domain fragments were used in order to provide an epitope for antibody precipitation, and to furnish a nuclear localization sequence (NLS) The GAL4DBD fusion protein was immunoprecipitated on beads containing anti-GAL4-DBD antibody from whole cell extracts and the bound protein analyzed by western blots probed with ant i-T7-epitope antibodies. The squelch-rescue approach will be discussed later

PAGE 110

99 A. B. Lane hHSFl DBD HR-A HR-B 5 6 7 8 9 10 11 12 13 14 1-529 1-120 -[ 120-175 175-217 120-217 217-422 422-529 212-380 212-310 212-297 221-310 529 hTBP binding 150mM 300mM +++ -/+ -/+ ++ +++ +++ +++ -/+ -/+ 1234 56 789 10 it 9 11 12 13 14 Binding Conditions: 150mMKCl 300 mM KCl 1234 56 789 10 Fig. 12. Interactions between T7-hTBP and GST-hHSFl fusions. A.) Summary of results of GST-pull-down assay at 150 mM and 300 mM KCl with T7-tagged hTBP expressed in E. coll. B.) Western blot of the bound hTBP probed with antiTV-tag antibody. Lanes: 1 = GST (negative control); 2 = GST-VP16 (A456-FP442) (negative control); 3 = GST-VP16 (413 490) (positive control) All other lanes are GST-hHSFl deletions represented in panel A.

PAGE 111

100 In vitro and in vivo int eractions between hHSFl and hTBP The GST-pull-down assay (Fig. 11) was used to map in detail the interactions between hTBP and hHSFl in vitro (Fig, 12). Binding was conducted at moderate (150 mM KCl) and high (300 mM KCl) Strong binding at both salt concentrations was observed between full length hTBP (lane 4), the 217 to 422 construct (lane 9) and CTAl (aa 422 to 529, lane 10) The interaction of hTBP with the 217 to 422 fragment was due to the presence of ADl within HR-C, since removal of HR-C resulted in loss of binding (lanes 11 though 14) Weak binding was observed with protein fragments containing the Nterminus and DBD (aa 1 to 120, lane 5) and HR-A (lane 6) Moderate binding of hTBP occurred with a hHSFl fragment containing the intact OD (aa 120 to 217, lane 8) at 150 mM KCl, but this interaction was significantly reduced at 300 mM KCl. In general, there was a correlation between the affinity of a fragment of hHSFl for hTBP and its known involvement in transcriptional activation in that protein fragments containing either (ADl construct 9) or AD 2 (construct 10), exhibited the strongest binding. The interactions seen with the DBD and the OD were weaker, and its biological relevance is not clear. A similar series of experiments were conducted to map the region of hTBP that interacts with the transcriptional activation domains of hHSFl (ADl and AD2) present in the CTAl-Plus fragment (aa 382 to 529) In panel B of Fig. 13,

PAGE 112

101 A. ViTRP Binding core GST-CTAl-Plus Fig. 13. In vitro interactions of hTBP deletions with CTAlPlus A.) Summary of interactions between hTBP and CTAlPlus. Binding results are indicated for panels B and C. B.) Western blot of TV-tagged CTAl-Plus bound to GST-hTBP fusion proteins. TV-tagged CTAl-Plus from E. coli lysate was used in binding reactions with immobilized GST-hTBP fusion proteins. C.) Western blot of TV -tagged hTBP deletions bound to GST-CTAl-Plus. TV-tagged hTBP deletions were used to interact with GST-CTAl-Plus fusion protein and detected with anti-TV tag antibody. Equal amounts of TVtagged hTBP deletion proteins were added in each pull -down assay. The bars indicate the expected positions of bands representing deleted hTBP proteins. Lane numbers correspond to hTBP constructs in panel A.

PAGE 113

102 matrix bound GST-fusion proteins consisting of various of deletion fragments of hTBP were incubated with TV-tagged CTAl-Plus The region of hTBP responsible for the interaction corresponds to Repeat 1 of the core from aa 156 to 221. The reciprocal experiment using GST-CTAl-Plus as the matrix bound protein and T7-tagged deletion fragments of hTBP (expressed in HeLa cells) as the ligand gave identical results (Fig, 13, panel C) These results do not rule out the possibility that interactions between CTAl-Plus and hTBP may also involve core repeat 2 (C-terminal) since deletion of the N-terminal repeat may also disrupt conformation globally As a further test of the potential for interaction between the transcriptional activation domain of hHSFl and hTBP, a GST-pull-down assay and co-immunoprecipitation experiments were conducted using proteins expressed in HeLa cells. In panel A of Fig. 14, immobilized GST-CTAl was incubated with T7-tagged hTBP from whole cell HeLa extracts. CTAl (AD2) was able to bind HeLa-expressed hTBP with high efficiency (compare input, lane 1, with lane 3) In the coimmunoprecipitation experiment, interactions were demonstrated between co-expressed GAL4-DBD-CTA1 or GAL4-DBDCTAl-Plus and T7-hTBP (Fig. 14, panel B, lanes 4 and 5). A clear increase in binding was evident with the CTAl-Plus domain compared to CTAl. Significantly, the GAL4-DBD-CTA1 mutant (MA-1) was unable to bind hTBP. This lack of interaction is consistent with the drastically reduced

PAGE 114

103 A. mm -T7-hTBP B. Transfected DNAs T7-hTBP + + + + GAL4-DBD-CTA1 + + GAL4-DBD-CTA1-P1US + GAL4-DBD-MA-1 + Fig. 14. Interactions between hTBP and hHSFl demonstrated by co-immunoprecipitation. A.) GST-pull-down assay with whole cell extracts containing T7tagged hTBP expressed in HeLa cells. Lanes: 1 = input; 2 = GST (negative control); 3 = GST-CTAl. B.) Co-immunoprecipitation of either CTAl, MA-l or CTAl-Plus with TV-tagged hTBP co-expressed in HeLa. Plus sign (+) represents the presence of each plasmid in the transfection. Different combinations of plasmid DNAs were cotransf ected into HeLa cells. Whole cell extracts were made and used for co-immunoprecipitation by anti -GAL4 -DBD antibody covalently cross-linked to Protein-A-Sepharose beads. The coimmunoprecipitated T7-tagged hTBP was detected by probing the western blot with anti-T7-tag antibody. hTBP was TV-tagged in vector pNTV-SB; CTAl, CTAlPlus (CTAl plus HR-C) and MA-l (a mutant of CTA464-529) were fused with the GAL4-DBD in vector pNG-SB.

PAGE 115

104 transcriptional activity of the MA-1 mutant (Table 1) suggesting that CTAl-TBP interactions in vivo are essential for full activity. Since hTBP is tightly associated with hTAFs in the TFIID complex in vivo, it is important to demonstrate that the activation domains of hHSFs are capable of interacting with the endogenous TFIID complex in addition to isolated TBP In order to demonstrate that the GST-CTAl-Plus affinity matrix could bind TFIID in a HeLa nuclear extract, the pull-down assay was conducted and the membrane was probed with anti-TBP and then by anti-TAF250 (Fig. 15) GST-pull-down assays demonstrated that endogenous TBP in nuclear extracts was able to bind to the CTAl-Plus at salt concentration ranging from 100 mM to 250 mM KCl (Fig. 15A, lanes 3, 7 and 9) It was further demonstrated that the TBP bound to the GST-CTAl-Plus affinity matrix was incorporated in the TFIID complex since TAF250 was visualized upon reprobing the membrane with antiTAF250 (Fig. 15B, lane 3) It is also noteworthy that the CTAl domain was not able to bind the TFIID complex as efficiently as CTAl-Plus since ten-fold more extract and GSTCTAl matrix were needed in order to detect the interaction (data not shown) This large difference in binding affinities may indicate synergism in recruitment of TFIID by the two activation domains (ADl/HR-C and AD2/CTA1) ; however, a simple additive effect in binding by CTAl-Plus can not be ruled out since the binding efficiency of isolated ADl/HR-C was not tested.

PAGE 116

105 A. 100 mM KCl _^T7-hTBP ^ hTBP 12 3 4 150 mM KCl 250 mM KCl — hTBP 5 6 7 8 9 B. 1^ hTAF250 Fig. 15. hTFIID complex in HeLa nuclear extracts binds immobilized GST-CTAl-Plus in pull-down assay. A.) Western blot of hTBP in the hTFIID. 50 )a 1 of nuclear extract and 20 1^1 of GST-CTAl-Plus were used for hTFIID complex pull-down assay at various salt concentrations. hTBP was visualized by anti-hTBP antibody. Lanes: 1, 5 = GST; 2, 6, 8 = GSTCTAl; 3, 7, 9 = GST-CTAl-Plus ; 4 = T7-tagged hTBP expressed in E. coli as a size marker. B.) Western blot of hTAF250 in the hTFIID. The blot in panel A was reprobed with antihTAF25 0 antibody.

PAGE 117

106 Fig. 16. The squelch and rescue assay. Column A: Expression of GAL4-DBD fusion with activation domain results in reporter gene expression when the target factor is recruited to the PIC by interaction with the activation domain. Column B: Co -expression of the GAL4-DBD/ activator with a dysfunction mutant of the target factor results in formation of a dysfunctional complex which squelches transcriptional activity. Column C: Co-expression of the GAL4-DBD/ activator with a double mutant of the target factor has little, or no effect on transcription if the second mutation removes the site of activator binding.

PAGE 118

107 The squelch-rescue assay (Fig. 16) served as a final demonstration of protein-protein interactions in vivo between CTAl-Plus of hHSFl and hTBP In these experiments activity of the GAL4-DBD-CTA1-P1US activator protein was inhibited by co-expression of a hTBP mutant protein. A titration of effector vector conducted to insure that optimal expression levels of the activator (effector) were achieved that minimized the self-squelching effect of over-expression. As seen in Fig. 17, from 0.2 to 2 fig of the GAL4-DBD-CTA1-Plus plasmid DNA showed no significant self-squelching of activity. Transfection with 5 and 10 |Ig of the effector plasmid reduced activity by approximately 30 and 70%, respectively. These results indicated that transfection with 0.3 |lg of the effector plasmid would be in the range of optimum expression and acceptable for use in subsequent squelch-rescue assays. When 0.3 |Ig of GAL4-DBD-CTA1-Plus plasmid was co-transf ected with 3 |lg of the hTBP (aa 156-335) mutant DNA, transcriptional activity was reduced by approximately 65% (Fig. 18B) consistent with the squelching of CTAl-Plus activity due to interaction between it and the hTBP fragment. Squelching was not seen when the CTAl-Plus effector was co-expressed with the hTBP (aa 221-335) mutant as predicted, since this hTBP fragment was shown by the GSTpull-down assay not to bind CTAl-Plus (Fig. 13) The reversal of squelching by the 221 to 335 aa fragment was not due to poor expression or stability of this protein as demonstrated by western blot analysis (Fig. 18A)

PAGE 119

108 1 0.2 0.5 1 2 5 10 GAL4-DBD-CTA1-P1US (^g DNA) Fig. 17. Optimization of expression of the GAL4-DBD-CTA1Plus effector in HeLa cells. Each transfection contained 0.2 to 10 [ig of CTAl-Plus fusion in vector pNG-SB (GAL4-DBDCTAl-Plus) 0.5 |Xg of growth hormone vector (pGXHS) 2 ^ig of luc if erase reporter (pGSLuc) and appropriate amount of enpty vector (pNeo-NB) added upto total 12.5 [Lg of DNA. Error bars were derived from three replicate experiments.

PAGE 120

109 hTBP 1-155: hTBP156-335: hTBP22 1-335: hTBP221-300: B. > <: (U > 100-1 75 50 25 pNeo-NB 1-155 156-335 221-335 22 -300 hTBP mutants Fig. 18. In vivo interactions between CTAl-Plus and hTBP analyzed by the squelch-rescue assay. A.) Western blot of TV -tagged hTBP mutants transiently expressed in HeLa cells and detected with anti-TV-tag antibody. B.) Squelch-rescue assay of hTBP mutants demonstrating their effect on transcriptional activity of CTAl-Plus. The first effector was the CTAl-Plus fusion in vector pNG-SB (GAL4-DBD fusion vector) The second effector consisted of indicated hTBP deletions with the TV-tag and nuclear localization signal in vector pTV-NLS. The reporter was the lucif erase gene with GAL4 DNA binding sites upstream of the TATA-box (pGSluc) pNeo-NB is a mammalian cell expression vector with only polylinker downstream of the CMV promoter (empty vector) The following DNAs were cotransf ected into HeLa cells: 0.3 ^ig of GAL4-DBD-CTA1-P1US, 3 [ig of hTBP mutant, 2 i^g of pGSluc and 0.5 \ig of growth hormone reporter (pXGH5). Lucif erase activity of triplicate transf ect ions was normalized using human growth hormone production to give relative activities.

PAGE 121

110 Interact ions between hHSF and hTFIIB In vitro mapping of interactions between hHSFl and hTFIIB revealed two regions of hHSFl that show affinity for hTFIIB: the OD and a portion of the C-terminus (Fig. 19) As in the case with hTBP, interactions between hTFIIB and the OD were reproducibly strong, but difficult to interpret since this region can be deleted without affecting transcriptional activity. The other interaction at the C-terminus is potentially more significant due to the presence of activation domain ADl/HR-C (Fig. 19, lane 9) It appears that hTFIIB differs from hTBP by showing a strong preference for the ADl/HR-C, whereas hTBP interacted strongly with both ADl/HR-C and AD2/CTA1 (Fig. 12) It is also noteworthy that the region containing NR (aa 212 to 310) did not bind hTBP or hTFIIB in vitro. Although interacts with hHSF2 were not mapped in detail, it does have potential for hTBP interaction as demonstrated in Fig. 19, lane 2. Results from the squelch-rescue assay were consistent with the occurrence of in vivo interactions between CTAl-Plus and hTFIIB. Substantial squelching of approximately 70% occurred with the hTFIIBi-207 protein which includes the Nterminal domain and the first core repeat (Fig. 20) It is also significant that the hTFIIBi-207 deletion containing a mutation (R185E, R193E) in helix El of repeat 1 showed a similar degree of squelching as the corresponding wild-type

PAGE 122

14 >, 111 HR-A HR-B HR-C CTAl hTFIIB binding + + + 529 + + + + + + + -/+ — T7-hTFIIB 11 12 13 9 14 15 16 17 12345 6789 10 Fig. 19. Interactions between hTFIIB and hHSFl Lanes: 1 = GST-scTBP (1-82) (negative control); 2 = GST-hHSF2; 11 = GST (negative control); 13 = GST-VP16 (413-490) (positive control) ; Other lanes are GTS -hHSFl deletions as indicated.

PAGE 123

112 hTFIIB 124 200 218 294 316 3207 R193E R185E 1)207 D 108 109C 1207 pNeo-NB Fig. 20. Two fragments of hTFIIB squelch transcriptional activity of CTAl-Plus. A.) Schematic of hTFIIB and deletion mutants. The two astericks indicate two point mutations R185 to E185 and R193 to E193 in El helix of hTFIIB. B.) Western blot of T7 -tagged hTFIIB mutants transiently expressed in HeLa cells and detected with anti-TV tag antibody. C.) Squelching of CTAl-Plus activity by wild-type and mutated hTFIIB(i-207) The first effector was the GAL4 -DBD-CTAl -Plus fusion in vector pNG-SB, the second effector was either of two hTFIIB deletion fragments with TV -tag and nuclear localization signal in vector pTV-NLS. The reporter was 2 |J,g of the luciferase gene with GAL4 DNA binding sites upstream of TATA-box. pNeo-NB is a mammalian cell expression vector with only polylinker downstream of CMV promoter (empty vector) 0.3 [ig of GAL4DBD-CTA-Plus 3 ^g hTFIIB mutant, 2 [ig of luciferase reporter (pGSluc) and 0.5 |xg of growth hormone reporter (pXGHB) DNA were cotransf ected into HeLa cells. The luciferase activity of each transfection triplicates was normalized by human growth hormone expression.

PAGE 124

113 peptides. The failure of the helix El mutant to rescue the squelch of hTFIIBi-207 indicates that CTAl-Plus, unlike VP16 (Roberts et al 1993), does not require the El helix for binding Although squelching of CTAl-Plus activity relative to the growth hormone gene was clearly evident for hTFIIBi-207 and hTFIIBi-207 mutant, the conclusion that this attenuation was due to interactions between CTAl-Plus and hTFIIB was not as strong as in the case of hTBP since no reversal of squelching could be definitively demonstrated. Two constructs, hTFIIBi108 and hTFIIBi-207, showed no squelching, but protein expression levels were to low to conclude that either of these constructs failed to interact with CTAl-Plus. In vitro and in vivo interaction between hHSFI and hTAF32 55 and PC4 In addition to hTBP and hTFIIB, which have been shown to interact with multiple activation domains, three others less well characterized components of the PIC were screened for potential interaction with hHSFs The first of these was hTAF32 which is a target for VP16 (Klemm et al 1995) and therefore a potential target for ADl/HR-C (resemble an acidic activator) hTAF55 was chosen as a potential negative control since it does not bind VP16 (Chiang and Roeder, 1995), and PC4 was selected because it is required for efficient function of many types of transcriptional activators (Ge and Roeder, 1994) When immobilized protein

PAGE 125

114 --T7-hTAF32 12 3456789 10 — T7-hTAF55 1 2 3 4 5 6 7 8 9 10 binding: hTAF32 hTAFSS Lane hHSFl UoL) HR-A HR-B HR-C CTAl 4 1-529 H H 1 1 16 81 120 212 382 422 5 1-120 H I 6 120-175 CID175 7 175-217 IZZh^'^ 8 120-217 I I k ++ 9 217-422 10 422-529 3 VP 16(4 13-490) Fig. 21. Interactions of GST-hHSFl with hTAF32 and hTAF55 Lanes: 1 = GST; 2 GST-VP16 ( A456 -FP442 ) ; 3 = VP16(413490) ; Other lanes are GST-hHSFl fusion proteins indicated.

PAGE 126

115 A. — T7-PC4 1 2 3 4 5 6 7 8 Lanes GST-fusions PC4 binding 1 GST 2 GST-VP16(A456-FP442) 3 GST-VP 16(4 13-490) +++ 4 GST-hHSFl +/5 GST-hHSF2 + 6 GST-CTAl 7 GST-scTBP(l-82) 8 PC4 input input Fig. 22. Interactions of PC4 with GST-fusions. A.) Western blot of TV-tagged PC4 using anti-TV tag antibody. B.) Summary of interactions between PC4 and hHSFs Lanes 1, 2 and V, negative controls; lane 3, positive control. Other lanes are GSTfusion proteins indicated. i

PAGE 127

116 fragments of hHSFl were incubated with hTAF32 and hTAF55, no significant interactions were observed with the exception of hTAF55 binding to a fragment containing HR-A and HR-B (Fig. 21, lane 8) Again, it is hard to evaluate the significance of this interaction due to the lack of evidence that the OD contains a transcriptional activation motif. In these experiments GST-VP16 served as a positive control for binding to hTAF32 (Klemm et al 1995) and a negative control for binding to hTAF55 (Chiang and Roeder, 1995) In the case of PC4, weak interactions were observed (Fig. 22, lane 5). Interactions with CTAl were weak and interpreted as negative, since the faint band observed was no more intense than obtained using the VP16A456-FP442 (Fig. 18, lane 2), presumably a negative control. The weaker binding of CTAl (AD2) relative to full length hHSFl (Fig. 22, compare lanes 4 and 6) may indicate that AD2 is insufficient for optimum interactions and suggests that stronger binding may be possible using CTAl-Plus (ADl/HR-C plus AD2/CTA1) Other negative controls included GST alone (lane 1) and a fragment of yeast TBP, scTBPl-82. The VP16 protein positive control showed evidence of strong interaction indicating that the PC4 protein possessed binding activity under these conditions. The VP16A4 5 6-FP442 mutant was used as a negative control for nonspecific binding based on its inability to interact with hTFIIB (Roberts and Green, 1994) However, its ability to bind hTAF32 and PC4 has not been previously characterized. The lack of binding to hTAF32 (Fig. 21, lane 2) and the

PAGE 128

117 PC4 mutants B. 100>. 75 < 50 _> '-4— 25 21-127 41-127 pNeo-NB 21-127 41-127 60-364 135-264 PC4 mutants hTAF3 2 mutants Fig. 23. A.) Western blot of TV-tagged PC4 and hTAF32 mutants. The TV-tagged PC4 and hTAF32 mutants were transiently expressed in HeLa cells, and detected with antiTV tag antibody. The expression of hTAF3 2 mutants were undectable. B.) Squelching and reversal of squelching effects of PC4 and hTAF32 mutants on the transcriptional activity of CTAl-Plus. The first effector was GAL4-DBDCTAl-Plus fusion in vector pNG-SB, the second effector was PC4 or hTAF32 deletions with TV -tag and nuclear localization signal in vector pTV-NLS. The reporter was 2 i^g of luciferase gene with GAL4 DNA binding sites upstream of the TATA-box. pNeo-NB is a mammalian cell expression vector with only polylinker downstream of the CMV promoter. 0.3 |j.g of GAL4DBDCTAl-Plus, 3 |ag of PC4 or hTAF32 mutant 2 ^g of luciferase reporter (pGBluc) and 0.5 |j,g of growth hormone reporter (pXGH5) were cotransf ected into HeLa cells. The luciferase activity of each transfection was normalized by human growth hormone

PAGE 129

118 greatly reduced binding to PC4 (Fig. 22, lane 2) suggests that phenylalanine 442 of VP16 required for hTFIIB binding is also involved in interaction with hTAF32 and PC4 Squelch-rescue assays using GAL4-DBD-CTA1-Plus confirmed in vivo the in vitro results which indicated that little or no interactions occur between hHSFl and either hTAF32 or PC4 (Fig. 23B) A slight inhibition of CTAl-Plus activity was seen with the PC4 (21-12?) mutant, which was alleviated by removal of 20 aa at the N-terminus of this fragment. Western blot analysis (Fig. 23A) indicated (construct PC4 (41-12?)) that the apparent result of squelch exhibited by PC4 (21-12?) was not due to lower levels of PC4 (21-12?) expression. Taken alone, the slight degree of squelching exhibited by PC4 (21-12?) seems consistent with the weak indication of interaction with hHSFl and CTAl obtained from the GST-pull-down assay (Fig. 22, lane 4). However, the level of squelch observed with PC4 (21-12?) is very similar to that obtained with hTAF32 (Fig. 23) which showed no indication in vitro of interaction (Fig. 21) For this reason, the slight squelch observed with PC4 (21-12?) must be interpreted with caution regarding the potential involvement of PC4 in hHSFl activation. Use Of the Yeast Two-hvbrid Sy.ste m to Screen for Proteins Interac ting with hHSFl In order to gain more insight into hHSFl-mediated transcriptional activation, the yeast two-hybrid system was used to screen a cDNA library of HeLa S3 cells in an attempt

PAGE 130

119 to select out any relevant proteins interacting with hHSFl Since hHSFl has a transcriptional activation domain, a Cterminal truncation to aa 422 removing CTAl of hHSFl was engineered in order to use hHSFl as a bait for screening. This strategy was resulted in selection of proteins that interacted with regions of hHSFl other than CTAl. The residual activity of the bait vector alone (GAL4-DBD-hHSFl (i422)) was eliminated by addition of 20 mM 3-AT. Several clones were obtained including three independently selected clones encoding the ^-subunit precursor of pyruvate dehydrogenase El component (Koike et al 1990), single clones for phosphoprotein PI (Koike et al 1990) and ferritin (Dhar et al., 1993) Since there was no obvious links to hHSFl or heat shock regulation for any of these selected proteins, this experimental approach was abandoned. A Coupled GST-Pull-Down and FarWestern Analysis of Nuclear Proteins Capable of in vitro Interactions with the CTAl nf hHSFl A novel technique combining the GST-pull-down approach (Fig. 11) with Far-Western analysis was developed to identify other potential targets of the CTAl of hHSFl. In this procedure immobilized GST-CTAl was used to bind proteins present in HeLa nuclear extracts prepared from hs and nonheat shocked cells. As a negative control for nonspecific binding, a fusion protein containing the negative regulator (NR, aa 212 to 310) was used in parallel at the GST-pull-down step (Fig. 24, lanes 4 and 5) Bound proteins were then

PAGE 131

120 GST GST-CTAl GST-NR GST (aa 212-310) Fig. 24. Far-western blot of nuclear protein binding CTAl Nuclear extracts were prepared from hs and non-heat shock HeLa cells and passed through either GST, GST-CTAl, or GSTNR affinity columns. Bound proteins were eluted, resolved by SDS-PAGE, membrane-blotted, and incubated with TV-tagged CTAl protein. Protein-protein interactions were stabilized by chemical cross-linking and CTAl containing complexes visualized by ant iTV -tag antibody. Lanes: 1 and 6, GST; 2 and 3, GST-CTAl; 4 and 5, GST-NR. NR is the negative regulation domain of hHSFl (aa 212 to 310) The approximate molecular weight of proteins (a, b and c) bound to TV -CTAl are indicated. NE, nuclear extract; hs heat shock.

PAGE 132

121 eluted with buffer A with 500 mM KCl and membrane blotted. The membrane immobilized proteins were then probed with T7epitope-tagged CTAl and the complexes stabilized by chemical cross-linking. CTAl containing complexes were visualized by probing with anti-T7 epitope antibody (Fig. 24, lanes 2 and 3) Three major protein bands of approximately 250 kDa (a) 160 kDa {b) and 70 kDa (c) were detected in the GST-CTAl pull-down experiment using extracts from non-heat shock cells (lane 2) Bands a and c were also pulled down from extract prepared from hs HeLa cells. The specificity of the interactions with CTAl was demonstrated by the lack of a strong signal in the GST and GST-NR (negative regulator) (lanes 1 and 4-6) Although this experimental result suggests that proteins other than hTBP and hTFIIB potentially interact with CTAl, this interpretation was hard to verify due to a lack of reproducibility in subsequent experiments (two out of a total of four) A possible reason for variable results may have been due to inefficient renaturation of SDS denatured proteins immobilized on the membrane. These intriguing results were not followed through to a definitive conclusion due to time constraints and are included here to provide a complete experimental record. Interaction between TFTTD and Ne gative Regulation Domain The GST-pull-down assay was used to detect possible interactions between TFIID and the negative regulation domain (NR) of hHSFl. Nuclear extracts prepared from hs and non-hs

PAGE 133

122 HeLa nuclear extracts HS + + hTBP — ^mmm | | GST-NR GST Fig. 25. hTFIID complex in HeLa nuclear extracts binds immobilzed GST-NR in pull-down assay. 500 |J, 1 of extract and 150 1^1 of GST-NR were used for hTFIID pull-down assays in 100 mM KCl salt. hTBP visualized by anti-hTBP antibody. Legends: GST-NR, GST fusion of the negative regulator domain of hHSFl; GST, a negative control; HS, heat shock treatment.

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123 HeLa cells were incubated with either GST-NR or GST (as a negative control) at 100 mM KCl. Proteins bound to the affinity matrices were blotted and probed with anti-TBP antibody. The presence of TBP is evident in the pull-down assay using GST-NR, and the specificity of the interaction is demonstrated by the lacks of binding to GST. It is also significant that similar amounts of TBP exist from hs and non-hs nuclear extracts (Fig. 25). Because the inability of NR to interact with TBP directly (Fig. 12), TBP is assumed to interact indirectly through tight association with other proteins that contact the NR. The most likely candidate for these bridging proteins are the TAFs (one or more) Repressor Function of the Nega tive Regulation Domain Previous studies (Green et al., 1995; Newton et al 1996) indicated that the NR does not have transcriptional activity when fused with GAL4-DBD, but has inhibitory effects on activators when it is directly fused with these activators. In order to understand the mechanism of the NRmediated repression, the full NR and two deletions were fused with the GAL4-DBD and transiently co-expressed with GAL4-DBDCTAl-Plus effector in HeLa cells. CTAl-Plus activity was monitored using lucif erase/human growth hormone reporters. Equal amounts of GAL4-DBD-CTA1-Plus were co-transf ected with a second effector plasmid containing either full length or deleted NR constructs, or the empty vector pNeo-NB. Repression (active repression) in this assay is defined as a

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124 reduction in transcriptional activity greater than that predicted to occur from the presence of a neutral peptide fused to the GAL4-DBD. For example, if no repression occurred when equal amounts of GAL4-DBD-CTA1-Plus and GAL4DBD-NR were co-t ransf ected, the activity should have been around 50% compared with the empty vector control. This reduction in activity (passive repression) would result from formations of GAL4-DBD-NR homodimers (with no activity) and GAL4-DBD-NR/CTA1-P1US heterodimers (with half the activity of GAL4-DBD-CTA1-P1US homodimers) However, if the NR actively repressed transcription by blocking transcription in trans by poisoning the heterodimer and inhibiting neighboring GAL4DBD-CTAl-Plus homodimers, reporter activity would be significantly less that 50%. The results presented in Fig. 26 indicate that the full length NR (212-310) reduced CTAlPlus activity by more than 50% suggesting that the NR activity inhibited transcription in trans. Active repression was also exhibited by the NR (221-310) construct, but not by a C-terminal truncation of the NR (Fig. 26)

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125 212-310 221-310 212-297 pNeo-NB GAL4-DBD-NR Fig. 26. Negative efffects of NR on the activity of CTAlPlus. GAL4-DBD-CTA1-P1US and GAL4-DBD-NRS were coexpressed in HeLa cells using vector pNG-SB. Each transfection contained equal amounts of GAL4-DBD-CTA1-Plus DNA (0.5 |Xg) and either GAL4-DBD-NR constructs (0.5 ^ig) or pNeo-NB (0.5 \lg) as the empty vector control. 0.5 ^ig of growth hormone vector (pXGH5) and 2 |Xg of reporter plasmid (pG5Luc) were included in each transfection. Relative luciferase activities were normalized using growth hormone production. Error bars were derived from three replicate experiments

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126 DISCUSSION The human and plant HSFs evaluated in this study showed clear differences in activity when expressed in yeast. hHSFl and 2 were active, and hHSFl was heat inducible, and both were able to substitute for the endogenous yeast HSF In contrast, soybean GmHSF34 and 5 were inactive under all conditions tested in yeast with the possible exception of GmHSFS which was able to substitute for yeast HSF. Overall, the degree of conservation in the mechanisms of HSF function between yeast and human cells was adequate to support growth of yeast cells when yeast HSF was substituted by hHSFl or hHSF2 However, several aspects of HSFl regulation were different in yeast cells when compared to HeLa expression. Examples of these difference include the degree of involvement of HR-A in basal repression, activity of HSF2, and the rate of hHSF attenuation after a period of heat stress. Mechanism of Basal Repression The strong activity of the isolated C-terminal portion of hHSFl (ADl and AD2) under non-hs conditions is consistent with the constitutive activity shown by the isolated Cterminal transcriptional activators (CTAs) of S. cerevisiae and K. lactis HSFs when fused to heterologous DBDs (Jakobsen

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127 and Pelham, 1991; Nieto-Sotelo et al., 1990; Sorger, 1990). The inherent activity of the hHSFl C-terminal domain is illustrated by the progressive increase in activity under non-hs conditions as aa residues N-terminal to the activation domains were sequentially removed (Fig. 1, constructs 1-4). Activity under basal conditions suggests that no stress specific modifications of the activation domains are required during hs expression, and lends support to models of basal repression based on masking of the HSF These results do not, however, rule out the possibility that the activation domains must be modified posttranscript ionally ; only that potential modifications are not stress specific. Masking models of basal repression propose that activity of the various functional domains of HSFs are inhibited due to their inaccessibility during non-stress conditions (for review see (Sorger, 1991)). Repression is thought to result from the conformational state of the HSF itself, or by interactions of the HSF with other proteins. Hydrophobic repeats located within the OD and HR-C, and a region within AD2 have been shown to play a role in maintaining basal repression (Jakobsen and Pelham, 1991; Nieto-Sotelo et al., 1990; Rabindran et al 1993; Sheldon and Kingston, 1993). One model for basal repression involves folding of the HSF under non-stress conditions so that HR-C interacts with one, or more, hydrophobic repeats of the OD (Nakai and Morimoto, 1993; Rabindran et al., 1993; Sheldon and Kingston, 1993). Our experiments do not distinguish between interand

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128 intramolecular masking, but indicate that several regions of the hHSFl protein contribute to repression of activity under both basal and hs conditions. For example, removal of HR-A of the OD (aa residues 120 to 174) caused activity under hs conditions to increase by 3-fold, and resulted in a significant loss of basal repression in yeast and a slight loss of basal repression in HeLa cells (Fig. 1) The large differences in activity between the isolated activation domains (constructs 4 and 5) and the full length protein (construct 1) indicates that a substantial amount of inhibitory potential exists in the full length protein, even during hs The loss of basal repression resulting from the removal of HR-A is consistent with the study of Sheldon and Kingston (Sheldon and Kingston, 1993) where point mutations introduced into either HR-A or HR-B resulted in constitutive nuclear localization of hHSF2 The involvement of HR-A in the regulation of basal activity in hHSF 1 and 2 differs from K. lactls HSF where only HR-B participates in the repression of activity (Chen et al 1993) In Fig. 1, a correlation seems to exist between basal repression of hHSFl and the presence of an intact OD In these experiments DNA binding was conferred by the GAL4-DBD and is presumably not dependent on the oligomerizat ion state of the HSF as indicated by the binding of construct 2 to the GAL4 DNA binding site under basal conditions (Fig. 3) The clear accessibility of the GAL4-DBD to DNA under non-hs conditions suggests that the

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129 masking thought to be responsible for basal repression does not directly involve the DBD Since DNA binding by the native hHSFl can be inhibited indirectly by the prevention of trimerizat ion, there is no requirement to physically block DNA access under conditions of basal repression. Negative Regulation of Transcriptional Activation Domains The lack of ADl/HR-C function after removal of AD2/CTA1 by C-terminal deletion (Fig. 1, construct 6) was unexpected since similar experiments by Green et ai. (Green et al., 1995) and Newton et al. (Newton et al 1996) showed from 5to 11-fold heat induction. In those studies the OD was not present in the GAL4or LexA-DBD fusions; whereas, in our construct 6 the entire protein was present except for AD2/CTA1, normally at the C-terminus In the previous studies the 201 to 370 aa fragment exhibited heat inducible activity in HeLa and non-regulated, high activity in yeast (LexA-DBD fusion) The lack of ADl/HR-C activity in our experiments may indicate that negative regulation is in effect under all conditions, with a defect in mechanism for the release of negative control under hs conditions. This failure to release repression during heat stress may be related to the presence of the OD which has been associated with negative regulation in previous studies (Zuo et al 1995) and by the results shown in Fig. 1. Another consideration is the possibility that the presence of LexA

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130 and GAL4-DBDS may have differential effects on expression in the yeast system. HSF2 Expression GAL4-DBD fusions with hHSF2 were heat inducible in yeast, but showed little activity in HeLa cells (Fig. 1) In addition, the isolated C-terminus (aa 397 to 536) exhibited activities from 10 to 11% of the isolated HSFl AD2/CTA1 in yeast under both basal and hs conditions. In contrast, very little activity was obtained in HeLa with the full length HSF2 (construct 9) and the isolated C-terminal domain (construct 10) showed less than 2% of the activity of the isolated AD2/CTA1. Furthermore, a decrease in the residual activity of the C-terminus was observed after hs This striking difference in expression of HSF2 in yeast and HeLa cells seems to imply that different mechanisms of regulation are utilized in these two organisms suggesting the possibility that a developmentally-specif ic regulatory pathway may be involved in human cells that is absent in yeast. It is noteworthy that HSF2 is predominately involved in non-stress-induced Hsp gene expression during development and is active in hemin-induced differentiation of mouse embryonic carcinoma cells, in heart and brain cells, and during specific stages of spermatogenesis (Goodson et al 1995; Sarge et al., 1994; Sistonen et al 1992). The co-expression of GAL4-DBD fusions of CTAl and CTA2 resulted in non-synergistic activation of the reporter gene

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131 in HeLa (Fig. 4) This result suggests that the synergistic response in HspVO expression seen previously (Sistonen et al 1994) in human K562 cells that were induced with both hemin and heat stress was not due synergism between transcriptional activation domains of HSFl and HSF2 In our studies we have assumed that opportunities for synergistic interactions between activation domains of HSFl and HSF2 were provided by the formation of both homoand heterodimers of the GAL4-DBD fusion proteins and by the occupancy of mixtures of heterodimers and homodimers (two types) bound to the same promoter. In view of the lack of synergism between HSFl and HSF2 activation domains, it seems likely that co-expression of endogenous hHSFl and hHSF2 facilitates the binding of HSFl to hs promoters. It has been reported that during hs HSF binds to over 150 sites scattered over the genome of Drosophila that are non-hs genes (Westwood et al., 1991). It is also known that HSFl binding to HSE elements is much more cooperative than HSF2 binding (Kroeger and Morimoto, 1994) Perhaps the non-hs sites are preferentially occupied by HSF2 during conditions of co-expression making more HSFl available for synergistic binding to hs promoters. Act i vat inn Dnmains in HSF.c; The striking difference in activities of the human HSFs and soybean HSFs in yeast cells may be interpreted in two ways: either transcriptional activation domains of mammals and plant HSFs may be different in their compatibility with

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132 yeast, or the soybean HSFs tested in this study lack efficient activation domains Although a number of HSF activation domains have not been mapped in detail, it appears that most possess an overall negative charge and are enriched for serines. The Kluyveromyces lactis HSF (klHSF) activation domain (aa 592 to 623) is comprised of 22% negative aa residues, the activation domain of Saccharomyces cerevisiae HSF 16% (aa 628 to 671), and CTAl of hHSFl has 13% negative residues (Chen et al 1993; Rabindran et al 1991). Serines represent from 12 to 24% of the aa residues in scHSF and hHSFl, respectively, but are only 3% of the klHSF activation domain. The aa composition comparison between CTAl and proline-rich activation domains indicates that CTAl should be designated as a proline-rich activator (Table 3) (Artandi et al 1995). In higher plant HSFs, only the tomato transcriptional activation domains have been characterized (Treuter et al 1993) Although the three tomato HSFs contain a concentration of negative charge in the C-terminal portion of the proteins (Treuter et al 1993), several soybean HSFs do not. The C-terminal region of soybean GmHSFS is nearly neutral, whereas GmHSF34 is basic. A high degree of conservation is present in the primary aa sequence of the C-terminus of HSFl and 2 in humans and mouse cells, and HSFs 3a in chickens (Nakai and Morimoto, 1993; Rabindran et al 1991; Sarge et al 1991; Schuetz et al., 1991). A comparison of C-terminal aa between six

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133 TFE3 134aa CTFl 102aa Oct -2 146aa AP2 70aa CTAl 108aa Pro 13 19 13 21 18 Ser 16 13 14 9 24 Leu 13 10 8 9 13 Gly 9 10 10 7 8 Acidic 15 5 4 13 Table 3. Comparison of amino acid residue composition {%) of proline-rich activation domains (Artandi et al 1995) with CTAl domain of hHSFl

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134 vertebrate HSFs and AD2/CTA1 subdomains of hHSFl mapped in yeast reveals two blocks of conserved aa (C-1 and C-2, Fig. 6) which show a general correlation with the location of subdomains II and III. This is especially true for subdomain II where 50% of the aa are either identical or similar between HSFl and HSF2 classes. Even though searches of available protein data bases failed to show matches with the consensus for subdomain II in other proteins, there is little to suggest that this region may be exclusively specialized for HSF function since heterologous activator domains were able to replace AD2/CTA1 of hHSFl without impairing heat inducible activation of hHSF in yeast cells (Fig. 1) One of the best characterized acidic activation domains is from the herpes simplex virion protein VP16. It maps to the C-terminal 78 aa residues and can be divided into two subdomains consisting of hydrophobic residues embedded within regions of negative charge. The N-terminal subdomain contains phenylalanine residue 442 (Phe-442) at its core and the C-terminal region includes Phe-473 and Phe-475. Although AD2/CTA1 resembles a proline-rich activator in term of amino acid composition (see detailed discussion in the section. Attributes of Activation Domain, Literature Review) it also has regions that are similar to acidic activators. AD2/CTA1 contains four phenylalanines, most of which are located near negatively charged or bulky hydrophobic residues in configurations that are roughly similar to that found in VP16.

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135 Plant HSFs contain a distinct activation motif designated as the TRP element which in tomato HSFs closely resembles the N-subdomain of VP16. Like VP16, the TRP element contains an aromatic residue (tryptophan instead of phenylalanine) located in a short region of negative charge (Treuter et al 1993) and bulky hydrophobic residues. All three of the tomato HSFs and Arabidopsis HSFl (atHSFl) contain at least one copy of this motif, but some soybean HSFs do not have recognizable TRP elements. The two soybean HSFs analyzed in this study contain one putative TRP element each. It is not known if the low activity observed is inherent to these particular HSFs, or simply reflects the inefficient function of this domain in yeast. The TRP elements in tomato HSFs appear to be responsible for basal as well as heat inducible activity (Treuter et al 1993) Transient assays in tobacco protoplasts indicate that all three tomato HSFs possess considerable amounts of basal activity. For full length tomato HSFs, hs causes transcription to increase from 1 6 to 4.4-fold. Removal of the C-terminal portion (76 aa residues) of tomato HSFS deletes one of the two TRP elements present and results in a sharp reduction in basal activity, but has little effect on heat inducible activity. Further deletion removing the second TRP element causes in a loss of heat inducibility as well. The activation domains of tomato HSF30 and HSF24 are also C-terminal, but regions responsible for basal and heat inducible activity more closely coincide.

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136 In each case, transcriptional activity (basal or heat inducible) is closely correlated with the presence of one or more TRP elements. In the case of tomato HSF8 and HSF30, one of the TRP elements is present within HR-C. Although both human and tomato HSFs appear to rely on acidic activation motifs that are similar to the transcriptional activation domains of VP16, soybean HSF34 and HSFS activation motifs have not been identified in this study. Recent experiments by Dr. Eva Czarnecka-Verner (unpublished) indicated that soybean HSF34 and HSFS have no activity when examined by transient expression assays in a variety of plants and plant tissues. These results suggest that the lack of transcriptional activity exhibited by the soybean HSFs in yeast cells was not due to an incompatibility in the function of activation motifs between plants HSFs and yeast, but most likely reflects the absence of activation domains in GmHSF34 and GmHSFS. A potential caveat is the ability of GmHSFS to substitute for yeast HSF which may be due to a cryptic activation domain of very low activity present in GmHSFS that resulted in activity that was below detection levels in liquid assays of p-gal, but sufficient for growth of yeast cells under normal condition. Mechanism of Transcriptional Ant lvation of hHSFI In Drosophila, transcription factors including dHSF, TFIID and pol II are pre-bound to Hsp70 gene promoters and transcription has been initiated waiting for productive

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137 elongation before hs Pol II was shown to be paused at a position 21-35 nucleotides downstream of the transcription start site by nuclear run-on analysis and in vivo footprinting in Drosophila Hsp70 genes (Giardina et al 1992; Rasmussen and Lis, 1995) Pausing of pol II was also observed in nucleosome-packed human Hsp70 gene in vitro. Activators such as isolated activation domain of hHSFl as well as SWI/SNF can inhibit the pausing and stimulate the readthrough of pol II in nucleosome-containing promoter of human Hsp70 gene when assayed with in vitro transcription system (Brown et al 1996). It is suggested that activators can decrease the nucleosome-mediated inhibitory effect on transcriptional elongation resulting in the suppression of the pausing (Brown et al., 1996) In the absence of the GAGA factor, the Drosophila Hsp70 promoter is not sufficiently accessible in chromatin, for dTFIID to bind to TATA-box (Shopland et al., 1995). The GAGA factor is a constitut ively expressed transcription factor in Drosophila, and binds to GA/CT rich elements called GAGA elements. The binding of the GAGA factor to DNA opens the chromatin and makes DNA promoter accessible for HSFs, dTFIID, presumably other transcription factors. The GAGA factor, along with TATA box and initiator element, can stimulate the pausing and the level of non-productive transcription, and dHSF has no effect on transcriptional elongation and pausing in the same context when GAGA factor is absent (Lee et al

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138 1992) However, the presence of both factors stimulates the high level of productive transcription (Lee et al 1992). HSF may have direct effect on processivity of transcriptional elongation. Alternative explanations are that HSF-mediated recruitment of GTFs, under hs, overrides the pausing and pushes pol II through pausing sites to generate non-abortive transcripts, or the GAGA factorfacilitated pausing is heat labile. Regardless the exact mechanism for HSF-f acilitated stimulation of transcriptional elongation, the simplest explanation is that the phenomenon observed in Drosophila is limited to a small number of genes with unique promoter structure such as having GAGA elements, pausing sites, etc. This explanation is supported by the observation that the region of upstream of the Drosophila Hsp7 0 TATA box, including GAGA elements and HSEs, can program the formation of a paused pol II on a non-hs gene promoter with no detectable pausing normally (Lee et al 1992), It is also reinforced by mutation analysis of GAGA element and HSEs: mutation of GAGA element reduces the pausing, and mutations of HSEs have no effects on pausing (Lee et al., 1992) Therefore, the GAGA-mediated pausing and HSFfacilitated stimulation of transcriptional elongation are not HSF-specif ic, nor hs-response specific; they are GAGA factor and GAGA-element specific. In other words, the main stream of HSF-mediated transcriptional activation is initiation instead of elongation or pausing. Moreover, the pausing under non-hs and quick release of paused pol II under hs in

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139 Drosophila HspVO genes only account for the first round of transcription, thereafter, the transcriptional initiation of these genes will become the key step of regulation by HSFs The Role of the Interaction betwe en hTBP and hHSFI TBP contains determinants for protein-protein interactions with pol II, some of the TAFs and discrete sets of activators and repressors (Tang et al 1996). TFIID or TBP binding to chromatin templates is rate limiting for gene expression, and the kinetics of this process is subject to regulation by activators in vivo (Klages and Strubin, 1995; Klein and Struhl, 1994) Two recently published papers (Moqtaderi et al 1996; Walker et al., 1996) indicated that in yeast activated transcription in vivo generally does not require TAFs. TBP and TFIIB can be the direct target of transcription activators. Results from the in vitro and in vivo studies described here strongly suggested that TBP and/or TFIID is one of the direct target of HSF which leads to transcriptional activation of hs genes. hHSFl can function at least through facilitation of hTBP or TFIID binding to the TATA box and thereby facilitate formation of the PIC. There is strong indication that TFIIB is also involved the pathway of HSFl activation of transcription. These studies also indicate that hTAF32 and hTAF55, as well as PC4, are not the direct target for transcriptional activation by hHSFl.

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140 In addition to clear evidence for TBP and TFIIB having important roles in HSFl-act ivated transcription, preliminary evidence from coupled GST-pull-down/Far-Western analysises (Fig. 24) suggested that other nuclear proteins may also be the direct targets of hHSFl and involved in transcription regulation of hs genes. Model for Heat Induc ible Regulation of HSF The results of binding studies indicate that two regions of hHSFl can form complexes with components of TFIID: activation domains (CTAl-Plus) with hTBP, and the NR with a protein (s) associated with hTBP, probably a TAF(s). The ability of an activator protein to bind a GTFs is generally thought to result in recruitment of the GTF to the PIC and result in transcriptional activation. This picture of activator function is consistent with the correlation in TBP/TFIID binding by CTAl and CTAl-Plus and their involvement in transcriptional activity. In contrast, the binding of the NR to TFIID under hs and non-hs conditions lacks any correlation between binding and transcriptional activity. These results and the strong trans repression of CTAl-Plus activity when CTAl-Plus and NR are co-expressed as GAL4-DBD fusions suggests that the NR/TFIID complex is dysfunctional with regard to transcription. A model for transcriptional regulation of HSFl is presented in Fig. 27. At normal temperatures, the activation domains are masked and inaccessible for the

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141 functional TFIID NR CTAI-Plus Fig. 27. Model for heat inducible regulation of hHSFl Legend: CTAl-Plus, activation domains (ADl and AD2) of hHSFl; NR, negative regulation domain; TFIID, a complex of TBP and TAFs PIC, pre-initiation corrplex. Under basal conditions affinity for TFIID is mediated by TAF interaction with the NR resulting in a dysfunctional complex. After hs the C-terminal activation domains are unmasked and accessible for interaction with TBP and TFIIB. TFIID is then in equilibrium between the NR and CTAl-Plus. Binding to CTAl-Plus facilitates formation of the PIC,

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142 formation of the functional TFIID complex on promoters; therefore, no activation occurs. The NR is capable of association with TFIID, presumably with one of the TAFs, forming a dysfunctional complex and leading to no-activation under non-hs conditions and inactivation of transcription during attenuation of the hs response, or upon removal of hs. According to the model, hs unmasks the activation domains resulting in the formation of functional TFIID complex and efficient recruitment of the TFIID to the promoter leading to transcriptional activation. The equilibrium between the dysfunctional TFIID (bound to the NR) and the functional TFIID complex (bound to the activators) is influenced by the conformational changes of HSFs under stress conditions. This model can explain the prompt and massive hs response by the rapid shifting of the equilibrium. This model is also consistent with the observation that the transcriptional activity of the activation domains in full length context can not be fully released even under hs conditions (Fig. 1) Only partial release of the activity under hs can also be observed in published studies (Green et al., 1995; Newton et al., 1996). For example, the levels of the released activities (hs conditions), range from 27% to 100%, and seem to depend on the activation domains located adjacent to the NR. When the activation domain is HR-C (aa 371-430), only 27% of the activity released under hs; when ADl (aa 401-420) present, 83% released; when VP16 (aa 413-456) fused, 59%

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143 released; when VP16 (aa 452-490) exists, full release obtained (Newton et al., 1996) Basis for Future Studies in Plants Information regarding hHSF targets of contact and the model of negative regulation of hHSFl will serve as a starting point in future studies to characterize the modes of transcriptional regulation employed by plant HSFs For example, since human HSFs have been shown to function in tobacco protoplasts (Treuter et al 1993), it is reasonable to expect that plant HSFs may also target TBP and TFIIB for activation. The question of negative regulation of plant HSFs is still very much open, since no heat-inducible intrapeptide negative regulation domain have been identified. However, the C-terminal domains of GmHSF3 4 and GmHSFS were able to repress transcriptional activation domains, GAL4 and VP16 (Fig. 8 and 9) These results indicate that plant HSFs, especially those lacking transcriptional activation domains, may form dysfunctional complexes in a manner analogous to the NR/TFIID association postulated for hHSFl. Such complexes may, or may not involve TAFs, since no TAFs have been cloned from plants and their role in activated transcription is unknown in plants.

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144 SUMMARY AND CONCLUSIONS Transcriptional activation domains were mapped in yeast to the C-termini of hHSFl and hHSF2 Detailed mutagenesis of CTAl identified multiple subdomains which make positive and synergistic contributions to activity. Analysis of point mutations in CTAl revealed the importance of hydrophobic and charged amino acid residues for the transcriptional activity. Although co-induction of hHSFl and hHSF2 in vivo has been reported to result in a synergistic increase in transcription of Hsp70 gene (Sistonen et al., 1994), the isolated CTAl and CTA2 domains were not able to synergistically activate transcription in trans when co-expressed as GAL4-DBD fusions in HeLa cells For hHSFl, removal of HR-A caused a loss of basal repression when assayed in yeast, but not in HeLa indicating a lack of complete compatibility in mechanism between these two organsims. Although yeast was able to survive when its endogenous HSF was functionally substituted with either hHSFl or hHSF2, differences in the hs response between yeast and HeLa cells were evident in the 30min delay in HSF activation after hs and the reduced ability to attenuate HSF activity after a transient hs GAL4-DBD fusions of hHSFl and hHSF2 were transcriptionally activated in yeast cells in response to

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145 heat stress. In contrast, neither of the soybean GAL4-DBDHSF fusions (GmHSF34 and GmHSFS) elicited any reporter gene activity in yeast suggesting that their transcriptional activation domains were either not present, not folded properly, or incompatible with the yeast system. The Cterminal 42 aa residues of GmHSF34 showed a strong negative effect on transcriptional activity of the GAL4 activation domain when both were adjacent in the same protein. As a whole, the functional analysis of human and soybean HSFs in yeast cells indicated that these types of heterologous expression studies have limited usefulness which may be partly attributed to differences in regulation between yeast, plant and human cells. For hHSFl, these differences were more apparent when studying the mechanism of basal repression and negative control by the NR domain. However, the yeast expression system was useful in special situations, such as the mapping of transcriptional activation domains and in the screening of point mutations with CTAl In the case of soybean HSFs, very little was learned from the yeast expression studies because too little was known regarding their function in plants. It is clear that in order to understand the mechanisms of HSF regulation, a more direct approach is potentially more informative. For this reason, protein-protein interaction studies involving transcriptional regulatory domains of hHSFl and GTFs were conducted both in vitro and in vivo in order to identify potential targets of contact in the PIC.

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146 The results of protein interaction studies suggest that both hTBP and hTFIIB are major targets for hHSFl in the activation of transcription. The evidence supporting involvement of hTBP is based on in vitro mapping studies indicating that it has affinity for full length hHSFl, ADl/HR-C and CTA1/AD2 domains. These interactions were demonstrated using both E. coliand HeLa-expressed TV-tagged hTBP. The link to biological relevance was made by showing that a hTBP mutant was able to squelch activity of a GAL4DBD-CTAl-Plus effector in HeLa cells, and by showing that this inhibition did not occur when a second hTBP mutant that was lacking binding site for CTAl-Plus (the N-terminal core repeat) was co-expressed. An additional proof was obtained by the correlation between the ability of hTBP to bind wild type and mutated CTAl (MA-1) and transcriptional activities in HeLa cells (Fig. 14 and Table 1) The final confirmation was achieved by showing a strong interaction between CTAlPlus and the endogenous TFIID complex (Fig. 15) The evidence regarding the involvement of hTFIIB is not as extensive, but still indicates that hTFIIB is also a major target for hHSFl contact. In vitro hTFIIB has relatively strong affinity for HR-C/ADl and weak affinity for CTA1/AD2 (Fig. 19) Evidence demonstrating a potential for in vivo interactions between hHSFl and hTFIIB is derived from the strong squelch of GAL4-DBD-CTA1-Plus activity obtained when two dysfunctional mutants of hTFIIB (hTFIIBi-ios and hTFIIBi09207) are co-expressed in HeLa transient assays (Fig. 20)

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147 PC4, TAF32 and TAF55 seem not to be the direct functional targets of hHSFl (Fig. 21 to 23) In view of the high degree in conservation in TBPs and TFIIBs between divergent organisms, it is not surprising that the transcriptional activation domains of hHSFl can function as strong activators in a variety of heterologous systems The ability of the negative regulation domain (NR) of hHSFl to bind TFIID and repress CTAl-Plus-mediated reporter gene activity (Fig. 25 and 26) suggests that the interaction between the NR and TFIID results in the formation of a dysfunctional complex that is transcriptionally incompetent. A model incorporating the formation of a dysfunctional complex between the NR and TFIID under both hs and non-hs conditions attributes the interplay among the NR and activation domains of hHSFl with TFIID as the basis for control of the final step in heat induced activation of gene expression These studies in yeast, HeLa and in vitro have mapped the transcriptional activation domains of hHSFl and hHSF2 to their C-termini and have identified two GTFs that have the potential to be the major targets of interaction in the PIC. Contact with the PIC presumably represents the final step in the signal transduction pathway responsible for heat stressinduced expression of hs genes. One of the unresolved questions concerns the process whereby the C-terminal domains of hHSFl are activated upon heat stress so that contact is made with hTBP and hTFIIB. At present, masking models for

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148 basal repression of the C-terminus are favored, but the nature of this unmasking, whether by folding or by interaction with other proteins, remains a challenging area of research.

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BIOGRAPHICAL SKETCH Chao-Xing Yuan was born on July 2, 1962 in Sichuan Province, China. He graduated from the Number 2 High School of Fushun County, Sichuan, in July of 1978, and entered Southwestern Agricultural University in August of that year as one of the youngest students at the University. He received a Bachelor of Science degree in agronomy in July of 1982. After graduation, he worked as a teacher of plant physiology in the Ningxia Agricultural College for three years. In August of 1985, he enrolled in the Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, and earned the Master of Science degree in August of 1988. After graduation, he worked as a researcher in the National Laboratory of Plant Molecular Biology and Genetics, Shanghai Institute of Plant Physiology. In August of 1992, he entered the Plant Molecular and Cellular Biology Program, Department of Microbiology and Cell Science, University of Florida where he began his doctoral studies. 168

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Docter of Philosophy. William B. Gurley/ Chair Associate Professor of Plant Molecular and Cellullar Biology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Docter of Philosophy. Francis C. Davis Associate Professor of Microbiology and Cell Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Docter of Philosophy. Professor of Plant Molecular and Cellullar Biology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Docter of Philosophy. Chkrles L. Guy (y Professor of Plant Molecular and Cellullar Biology

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Docter of Philosophy. This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Docter of Philosophy. December 1996 Donald f.. McQartlyx Associate Professor of Plant Molecular and Cellullar Biology Dean, College of Agriculture Dean, Graduate School