Cell, Vol. 62, 807-917,

August

24, 1990, Copyright

0 1990 by Cell Press

The Yeast Heat Shock Transcription Factor Contains a Transcriptional Activation Domain Whose Activity Is Repressed under Nonshock Conditions Jorge Nieto-Sotelo,’ Akihiko Okuda, and Division of Chemistry, California Institute of Pasadena, California

Greg Wiederrecht,t Carl S. Parker 147-75 Technology 91125

Summary Transcription of heat shock genes is induced by exposure of cells to elevated temperatures or other stress conditions. In yeast, it is thought that induction of transcription is mediated by conversion of a DNAbound transcriptionally inactive form of the heat shock transcription factor (HSTF) to a DNA-bound transcriptionally active form. We have identified domains in HSTF involved in transcriptional activation and in repression of transcriptional activation at nonshock temperatures. We present evidence that a temperature-regulated transcriptional activation domain exists in HSTF and that this domain is essential for survival of yeast cells at heat shock temperatures. We propose a model for temperature-regulated transcriptional activation by a derepression mechanism. Introduction Cells subjected to heat shock or other physiological stress conditions dramatically increase the expression of a specific set of genes encoding the heat shock proteins (hsps; for reviews see Craig, 1985; Lindquist, 1986). The promoters of these genes all possess a &-acting heat shock control element (HSE) that consists of a highly conserved sequence (Pelham, 1982; Perisic et al., 1989). A heat shock transcription factor (HSTF), which binds specifically to the HSEs, has been identified in yeast, Drosophila, and human cells (Parker and Topol, 1984; Top01 et al., 1985; Wiederrecht et al., 1987; Sorger and Pelham, 1987; Wu et al., 1987; Kingston et al., 1987). The gene that encodes the Saccharomyces cerevisiae HSTF has been cloned and partially characterized (Wiederrecht et al., 1988; Sorger and Pelham, 1988). The DNA binding domain was localized between residues 167 and 284 of the 833 amino acids that constitute the full-length protein (Wiederrecht et al., 1988). More recently, it has been demonstrated that yeast HSTF can oligomerize in vitro. The region of the protein responsible for this oligomerization is found between amino acids 327 and 424 and contains a putative hydrophobic a-helical structural element. This structural element is characterized by a 3-4 repeat of hydrophobic residues consisting primarily of isoleucine and leucine residues (Sorger and Nelson, 1989).

l Present address: University of California, Berkeley/USDA Plant Gene Expression Center, 800 Buchanan Street, Albany, California 94710. t Present address: Merck Sharp 8. Dohme Research Laboratories, PO. Box 2000, Rahway, New Jersey 07065-0900.

In response to heat shock, the HSTF is specifically modified to an active form capable of stimulating transcriptional initiation. In Drosophila and HeLa cells activation is accompanied by an apparent change in the DNA binding properties of HSTF. Binding activity is increased in heat-shocked cells, and it is independent of new protein synthesis (Zimarino and Wu, 1987; Mosser et al., 1988). In yeast cells, however, there is considerable evidence that the HSTF is bound to the HSEs prior to heat shock, suggesting that binding to the HSE is not sufficient for transcriptional activation (Jakobsen and Pelham, 1988). In yeast the same amount of HSE binding activity is found before and after heat shock (Sorger et al., 1987), making possible the isolation of HSTF from both shocked and nonshocked cells (Wiederrecht et al., 1987; Sorger and Pelham, 1987). Together, these results indicate that an increase in the levels of the HSTF is not responsible for increased hsp transcription of heat shock genes. It is clear, then, that the HSTF is an inducible transcription factor, and one of our primary interests is to understand the mechanism by which modifications of the HSTF lead to its transcriptional activation. We would also like to bring to light the molecular events in the signal transduction pathway that leads to activation of the HSTF. To this end, we have identified the domains of the HSTF that are responsible for transcriptional activation upon heat shock. In this article we present evidence that HSTF contains a cryptic constitutive transcriptional activator whose activity is repressed in the absence of heat shock by adjacent regions of the protein. We propose a model in which the interactions between these domains may result in a temperature-regulated transcription factor. Results A Domain of HSTF Is Required for Viability at Heat Shock Temperatures but Not at Normal Growth Temperatures Our previous work has shown that disruption of the HSTF gene results in a lethal phenotype (Wiederrecht et al., 1988). To identify the domains of the protein that are essential for viability, we constructed a series of carboxyterminal deletions of HSTF. These deletion constructs are expressed under the control of the HSF7 promoter on CEN plasmids to generate levels of expression comparable to the normal chromosomal copy. We determined whether these HSF7 deletions could rescue a haploid strain of yeast containing a disrupted chromosomal copy of HSFl (GWYA3-II; hsfl). This hsfl strain contains an episomal copy of HSF7 whose transcription is controlled by the GAL7,Xl promoter. When this strain is grown in minimal medium with galactose as the carbon source, the cell is provided with sufficient amounts of HSTF for growth (Figure 1). A switch to glucose-containing medium shuts off HSTF expression from the GAL7,70 promoter and results in cell death. Figure 1 shows the results of the analysis in which the

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various HSFl deletions in the hsfl strain were grown on plates containing either galactose at 30°C or glucose at 22% 30°C or 37%. As expected, all strains grew in galactose-containing medium. In glucose-containing medium, growth at 22% and 30% was no longer observed when 437 or more amino acids were removed from the carboxyl terminus (amino acid endpoint 396). The growth pattern of deletion mutants at 37°C however, was very different. Under heat shock conditions we observed that no more than 210 amino acids could be removed from the carboxyl terminus without loss of viability (amino acid endpoint 587). These observations suggest that a region of the HSTF between residues 623 and 587 is required for growth under heat shock conditions but is not required for growth at 22% or 30%. Viability at 22% and 30% is lost when the carboxy-terminal endpoint approaches the isoleucine repeat (located between residues 347 and 375). These data suggest that the isoleucine repeat and adjacent sequences contain an important feature required for HSTF activity. Temperature-Regulated Transcription Can Be Transposed onto Another DNA Binding Domain HSF7 is essential for growth at all temperatures. Therefore, it is not possible to measure transcriptional activity from truncated forms of HSF7 with an HSE-dependent reporter gene because the endogenous gene would interfere with the activity measurements. To identify and quantitatively measure the activity of potential transcriptional

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Frgure 1. Viability of hsfl Cells Rescued with Wild-Type /-/SF1 or with HSF7 Encoding C-Terminal Deletion Derivatives In these experiments expression of HSFl was driven by its own promoter (1 kb of 5’ untranslated region). CEN plasmids containing wildtype HSFl or HSFl encoding C-terminal deletions were introduced into hsfl cells harboring a second centromere vector containing a GALl,lO/HSF7 fusron. Cells containing these two kinds of plasmids were grown in minimal medium containing galactose, raffinose, and glycerol as carbon sources. Viability was measured by transferring cells to minimal plates supplemented with either galactose or glucose and grown at 22%, 30°C, and 37% for 24-72 hr. Growth of colonies: very good, +; slow, b; no growth, The length of wrld-type HSTF and positions of the DNA binding domain (DBD; diagonal lines) and isoleucine repeat (ILR; horizontal lines) are represented at the top. The amino acid endpoints of each deletion derivative are indicated by the numbers at the right end of each construct.

activation domains of the HSTF, we fused various portions of the HSTF to the DNA binding domain of the yeast AP-1 transcription factor (YAP-1). Previous work from this laboratory has shown that the YAP-1 gene, YAPl, is not essential for growth (Moye-Rowley et al., 1989). A YAPFdisrupted yeast strain containing a UAS& CYCl//acZ reporter gene (strain SM9/10), in which transcription of the CYCl//acZfusion is under the control of an ARE (AP-1 recognition element), has negligible P-galactosidase activity (Figure 2A, line 1; Moye-Rowley et al., 1989). When strain SM9/10 harbors a full-length YAP7 gene on a 2km plasmid, ARE-dependent transcription of the reporter gene is constitutive at both nonshock and heat shock temperatures (Figure 2A, line 3). The YAP-1 transcriptional activation domain is localized in the carboxy-terminal region of the protein (Moye-Rowley and Parker, unpublished data), while the DNA binding domain of YAP-1 is found in the amino terminus between amino acids 63 and 155 (Moye-Rowley et al., 1989). When strain SM9/ 10 is transformed with a YAP7 deletion mutant (yap7) containing the promoter and amino acids 1-155 of YAP-1 (YAP1 ,-,ss), one observes only residual B-galactosidase activity (Figure 2A, line 2). These results show that there are no other transcription factors present in a yap7 yeast cell capable of significant ARE-dependent transcriptional activation. This allows the fusion of portions of the HSTF to the YAP-1 DNA binding domain in order to monitor the activity of potential transcriptional activation domains of the HSTF. Additionally, we can monitor the levels of temperature-regulated tran-

Transcriptional 809

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Figure 2. Carboxy-Terminal Deletion Mutants of HSTF~,Q-M~ Fused to the Carboxyl Terminus of YAP-1 ,-,55

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scription brought about by the various fusion protein constructs and identify any inducible transcriptional activation domains. The results of the viability studies described above demonstrated that a region carboxy-terminal to the DNA binding domain, possibly a transcriptional activation domain, was essential for activity at shocked temperatures. To test this possibility, various portions of the carboxy-terminal three fourths of the HSTF were fused to the carboxyl terminus of a construct encoding the YAP-1 DNA binding domain (YAP-1 ,-,s5). Figure 28 shows the results obtained when the carboxy-terminal deletions of HSTF219-64s are fused to the carboxy-terminal end of YAP-~,-,~~. This fusion protein is capable of temperature-regulated transcriptional activation. Deletion to amino acid 611 (yAP-11-155/ HSTF219-611) causes a 3-fold reduction in reporter gene activity at 23%, resulting in higher levels of inducible transcription. Progressive deletions from carboxyl terminus 611 to 254 show a significant decrease in the ratio of activity at 40% versus 23%. In addition, deletion to residue 587 causes a significant reduction in activity at 40°C, indicating that the carboxy-terminal endpoint of a temperature-regulated transcriptional activator resides between residues 611 and 587. Those constructs with a carboxy-terminal endpoint of 551 (YAP-1 1-155/HSTF219-551)and smaller have essentially no inducible transcriptional activation. It is noteworthy that the carboxy-terminal endpoint of the

(A) 2um plasmids encoding the depicted YAP1 1-155/HSTF,-y fusion proteins were introduced into yap1yeast cells containing an AREKKW laci’ reporter gene. The 8-galactosidase assays were carried out on cells grown in minimal medium supplemented with glucose and the required amino acids. Typically, cells were grown at 23OC to an ODW of 1.0. Half of the culture was harvested and extracted. The other half of the culture was shifted to 40% for 1 hr to induce heat shock. The cells were transferred back to 23% for 1 hr to allow translation of the heat-induced 6-galactosidase transcripts. The length of wild-type HSTF and positions of the DNA binding domain (DBD; diagonal lines) and isoleucine repeat (ILR; horizontal lines) are represented at the top. In yAP-11-155 and the full-length YAP-1 construct, stippling represents YAP1 sequences, triangle represents the YAP7 promoter (372 bp of 5’ untranslated sequence), small rectangle represents sequences encoding the 155 amino acids at the YAP-1 amino terminus that includes both the basic region and the leucine repeat, and large rectangle represents the remainder of the sequence that encodes a YAP-1 full-length protein. The endpoints of each deletion derivative are indicated by the numbers on top of each construct. (B) Legend is the same as in (A). Amino terminus of HSTF is at amino acid 219 (Dral site). Fusion between DNAs encoding ~AP-1r-r~~ and HSTFz,s-x was by blunt-end ligation and did not incorporate sequences encoding additional amino acids,

HSTF region required for viability at 37% (see Figure 1) maps between residues 623 and 587, corresponding with the carboxy-terminal endpoint of the temperature-regulated domain identified here. To be certain that the transitions in transcriptional activ-

Figure

3. Analysis

of YAP-VHSTF

Fusion

Protein

Expression

(A) Analysis of the carboxy-terminal deletions where alterations in transcriptional activity are observed. The numbers on top of the autoradiogram indicate the carboxy-terminal endpoint of the fusion protein. The numbers alongside are for molecular weight markers: phosphorylase B. bovine serum albumin, and carbonic anhydrase. (B) Analysis of amino-terminal deletions that result in alterations in transcriptional activity. The numbers on top of the autoradiogram indicate the amino terminus of the fusion protein. Molecular weight markers are as in (A).

Cell 810

Figure 4. Amino-Terminal Deletion Mutants of HSTF20s-648 Fused to the Carboxyl Terminus of YAP-1 ,-,ss Reveal a Repressor Domain

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Legends are the same as in Ftgure 2. The solid rectanalereoresents additional amino 1 acids encoded by the linkers used during construction of the fusion proteins. Fusion between DNAs encoding YAP-1 ,.,s5 and HSTF208..64s derivatives introduced 8 extra amino acids GDPLESSP

ity were due to losses of specific regions of the HSTF, the levels of fusion protein expression were monitored. Because the monoclonal antibodies that we prepared against the HSTF react with epitopes within the HSTF DNA binding domain, they were not suitable for analysis of the fusion proteins. Protein blots of samples from the indicated strains were probed with a radiolabeled DNA oligonucleotide corresponding to the YAP-1 recognition element (Harshman et al., 1988). The strains analyzed in Figure 3A correspond to carboxy-terminal deletions in which considerable alterations in transcriptional activity are observed. As observed in Figure 3A, uniformly high levels of fusion proteins are expressed with sizes corresponding to the predicted molecular mass. Note that no background bands corresponding to YAP-1 family members are observed, because the fusion proteins are expressed on 2ttm plasmids in a strain in which the gene encoding the 90 kd YAP-1 has been deleted.

tional activity at both 23% and 40% (compare YAP-1 ,+J HSTF4,~+s with YAP-1 1-155/HSTFS12-648 and with YAP1 1-155/HSTF584-648 in Figure 4). The analysis of the levels of the amino-terminal fusion proteins is shown in Figure 38. Only those strains in which alterations in transcriptional activity occurred were analyzed. In all cases very high levels of fusion protein were observed. These data strongly suggest that temperature-regulated activation is brought about by the repression at 23% of a constitutive activator element by adjacent protein sequence elements. The inducible activation domain seems to consist of two functional components: a constitutive transcriptional activation element whose activity is modulated by an adjacent repressor function, which in turn regulates the activity of the activation domain in a temperature-dependent fashion. These two domains function together to bring about temperature-regulated transcriptional activation.

Amino-Terminal Deletions of HSTF208-648 Fused to yAP-ll-lss Reveal a Domain That Represses HSTF Transcriptional Activity at Nonshock Temperatures To map the amino-terminal endpoints of the temperatureregulated domain, a series of amino-terminal deletions were constructed beginning at residue 208 and a carboxyterminal endpoint at 648. As amino-terminal sequences are progressively deleted from the temperature-regulated domain (HSTF20,-6,s), a stepwise increase in constitutive transcriptional activation is observed (Figure 4). The level of transcriptional activation at 40°C however, remains the same, resulting in a decrease in inducible transcription. The first increase in constitutive transcription occurs when a region of the DNA binding domain between residues 208 and 222 is removed. This deletion results in a lo-fold increase in transcriptional activity at 23%, with no loss in the total activity observed at 40%. The second increase in constitutive activation occurs when a portion of the isoleucine repeat is removed, resulting in a 5-fold increase in activity (compare YAP-1 1-155/HSTF311-648with YAP-1 1-155/ HSTFss4+s). Further deletion causes a drop in transcrip-

Two Additional Constitutive Transcriptional Activation Domains Are Present within the HSTF The previously described viability studies indicated that only the amino-terminal 413 amino acids were required for cell growth at 23%. The transcriptional activation domains mapped as described above are outside of this region. Furthermore, no transcriptional domain has been identified between residues 219 and 439 (see Figure 4). This suggests that the amino-terminal portion of the HSTF might harbor an activation domain that functions at 23% at levels sufficient to allow low level expression of the essential hsp genes. To test this hypothesis, carboxy-terminal deletions were constructed from a YAP-1 1-15s/HSTF1-172 hybrid protein in which the initiating methionine of HSTF was fused in frame to YAP-1 ,-,ss. As described previously, ARE-driven 6-galactosidase activity was measured under both nonshock and heat shock conditions to assess the level of transcriptional activation of the hybrid proteins (Figure 5A). The three hybrid proteins tested, YAP-1 l-15r,/HSTF7and YAP-1 1-155/HSTF,-132, all 172, YAP-~,-,~~/HSTF,-,~O,

Transcriptional 811

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Figure 5. Amino- and tion Mutants of HSTF Terminus of YAP-1 ,-rss ence of Constitutive ulated Transcriptional

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showed transcriptional activity. However, none of these activities were under temperature control. These findings suggest that there is at least one constitutive transcriptional activation domain present in the amino terminus of the HSTF. It is possible that this domain functions well enough in the nonshocked cell to allow viability. Fusion of HSTFato YAP~,-,~ (yAP-11-155/HSTF21-; Figure 58) produced a hybrid protein with the properties of a con8titutive transcriptional activator, whereas a fusion protein containing the region between residues 219 and 611 of HSTF has inducible transcriptional activity with very low constitutive activity. The fundamental difference between these two constructs is the very high level of 23% activity of the HSTF2,s-sss construct. Similarly, a fusion of the HSTF element from residues 589 to 833 (YAP1 1-155/HSTF589-833; Figure 58) 8hOWS almost 50-fold higher constitutive transcriptional activity than does the YAP1 1-lsr,/HSTF~g411 hybrid protein. These observations tentatively localize a constitutive transcriptional activation domain between residues 589 and 833. Fusion of the ammo termrnus (YAP-1 1-155/HSTF1-833; Figure 58) of the full-length HSTF to the YAP-1 DNA binding domain restores inducible transcription. This fusion gene has considerably less activity, however, than the wild-type factor or other fusion proteins. Apparently, the amino terminus of the HSTF may play some role in regulating the constitutive activity generated by the carboxy-terminal activation domain. We are currently testing the roles of all the domains identified by the fusion protein experiments in the context of the native HSTF for their effects on viability and heat shock gene regulation. Analysis of the Amlno Acid Sequences Present within the Temperature-Regulated Transcriptional Activation Domain and Other Domains Analysis of the amino acid sequence and composition of the carboxy-terminal half of the constitutive transcriptional activation element (All) present within the temperature-

Carboxy-Terminal DeleFused to the Carboxyl Demonstrate the Presand Temperature-RegActivation Domains

(A) Carboxy-terminal deletion mutants of HSTFl-ln fused to the carboxyl terminus of YAP-1 ,-rss reveal a transcriptional activation domain in the amino terminus of HSTF. Legends are the same as in Figure 2. Constructs encoding YAP-1 l-rss/HSTF,-ln hybrid protein and C-terminal deletion derivatives incorporated only 9 extra amino acids: GDPLESSPA. (6) In the YAP-1 ,-&HSTF,~~ fusion the solid rectangle represents additional amino acids encoded by the linkers used to construct the chimeric gene. This fusion incorporated sequences encoding 18 extra amino acids: GDPLESSPMANSGCIVGA. The remaining fusions did not incorporate sequences encoding additional amino acids.

regulated domain is shown in Figure 6. The carboxyterminal deletions indicated that maximal induced transcription occurred at residue 611. Deletion to residue 587 greatly reduced the induced level from 72 U to 9.9 U. The

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of the carboxyTwo strongly half of the doacid sequence endpoints at and the values figure shows a protein region

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DNA binding (DSD) (Wiederrecht et al., 1998) and isoleucine repeat (ILR) (Sorger and Nelson, 1999) domains were previously described. Amino acid endpoints for each region, as well as their proposed functions, are indicated. The constructs labeled “viability”show the minimum amino-terminal regions derived from carboxy-terminal deletions capable of growth at the given temperature. Constitutive activators I, II, and Ill (Al, All, and Alll, respectively) are defined as regions of HSTF that when attached to the carboxyl terminus of YAP-~,-~~~ activate transcription equally well at 23% and at 40%. The temperature-regulated domain when fused to YAP-1 , - 155 activates transcription only at 40%. Repressor domain is defined as a region of the temperature-regulated domain that, when deleted, allows transcription to occur at 23%. At right are shown the net charge and content of certain amino acids found in the indicated domains

amino acid composition of this portion of the All domain (amino acids 588-811) reveals a significant level of acidic residues (25% D and E) as well as serine and threonine residues (20.8%). One unusual feature of this element is the high level of asparagine residues (16.7% N). Together, these five amino acids comprise 62.5% of the subdomain. Adjacent to this element is a second portion of the activation domain that is responsible for -9 U of the induced level of transcription. This domain (amino acids 551-587) is extremely acidic, containing 32.4% glutamic acid and aspartic acid residues. This domain is also rich in serine and threonine residues (18.9% S and T). Taken together, these two regions have 29% acidic residues and 20% serine and threonine residues; thus nearly half of this portion of the All domain is made up of only four amino acids. This analysis can be extended to the other functional domains present within the HSTF, as shown in Figure 7. The amino-terminal constitutive activation domain (Al) contains a significant level of serine and threonine residues (24.4% Sand T) btit is not very rich in acidic residues (9.3% D and E). The carboxy-terminal constitutive activation domain (Alll) is also rich in serine and threonine (20.4% S and T) and reasonably rich in acidic residues (15.9% D and E). Discussion Yeast HSTF Contains a Temperature-Regulated Transcriptional Activation Domain Required for Viability at Heat Shock Temperatures The viability studies presented here have demonstrated

that amino acids 1 to 623 of the HSTF are necessary for growth under heat shock conditions. The gene fusion experiments corroborated the viability studies by demonstrating that a temperature-regulated transcriptional activation domain is contained within this region of the HSTF (between amino acids 208 and 648). These observations further demonstrate that the temperature-regulated domain is fully functional when fused to the YAP-1 DNA binding domain, producing a hybrid temperature-regulated transcription factor. Because the gene fusion data and the viability data were generated by completely independent lines of experiments, the evidence is compelling that the region between amino acids 208 and 648 constitutes the minimal element necessary for temperature-regulated transcriptional activation. Temperature-regulated transcription is brought about by the action of two regions within the temperature-regulated domain. One region consists of a constitutive transcriptional activation domain, All, localized between residues 410 and 648. Deletion analyses of the amino-terminal portion of the temperatureregulated domain revealed an element between residues 208 and 394 that represses the All domain under nonshock conditions. Our results indicate that at least two regions are involved in repression of the HSTF at nonshock temperatures. These regions include a portion of the DNA binding domain and the isoleucine repeat. It is conceivable that the intermolecular association of monomeric HSTF units through the isoleucine repeat plays an important role in temperature-regulated transcription. Deletion of the isoleucine repeat results in a significant increase in constitu-

Transcriptional 013

Activation

Domains

of HSTF

tive transcriptional activation. These observations suggest that disruption of inter- and/or intramolecular interactions of the factor leads to transcriptional activation mediated by the adjacent activation domain. It is possible that a higher order oligomeric state of the factor represses transcriptional activation. It will be interesting to determine if the temperature-regulated repressing domain is dominant to any constitutive transcriptional activation domain. We are currently constructing hybrid genes that will place Al, Alll, and other transcriptional activation domains directly adjacent to the repressor region (HSTF DNA binding domain and the isoleucine repeat) to determine if this region has a dominant temperature-regulable repressor function. We are now in the position to test these different models of derepression of HSTF. Taken together, our observations demonstrate the complexity of interactions that occur to bring about temperature-regulated expression. This study also shows the modularity of the different structural domains of the HSTF and indicates that regions involved in constitutive transcriptional activation or in repression are compatible with other DNA binding domains (i.e., YAP-1). HSTF has been shown to trimerize in vitro (Sorger and Nelson, 1989). Our earlier and present studies have shown that the trimerization domain plays a limited role in DNA binding (Wiederrecht et al., 1988). It is our current view that the HSTF can bind to DNA as a trimer but that this is not a prerequisite for DNA binding. Our earlier studies showed that deletion of the isoleucine repeat did not significantly reduce DNA binding when carboxyterminal deleted proteins were analyzed (Weiderrecht et al., 1988). We also observed that when the DNA binding domain alone was fused to 8-galactosidase it was capable of high affinity binding (Weiderrecht et al., 1988). These observations suggest that there is considerable flexibility in the oligomerization state for DNA binding by HSTF. One concern about our gene fusion experiments is that there may be considerable oligomerization of the endogenous HSTF with the fusion HSTF, resulting in transcriptional activation by association with the endogenous HSTF. If this were true, then we would map only the position of the oligomerization domain and not a discrete transcriptional activation domain in the fusion protein. None of the results we have obtained are consistent with this possibility. The data presented in this report suggest that the isoleucine repeat may play an important role in temperature-regulated transcriptional activation. Roles of the Other Transcriptional Activation Domains of the HSTF In addition to the temperature-regulated domain, we identified two regions of yeast HSTF that conferred constitutive transcriptional activation functions to the YAP-1 DNA binding domain (see Figure 5). These regions are separate from the previously identified DNA binding (Wiederrecht et al., 1988) and trimerization domains (Sorger and Nelson, 1989). Constitutive activator domain I (Al) is contained within the amino-terminal 172 residues (see YAP1 r-155/HSTF1-,72 in Figures 5A and 7). A constitutive acti-

vation domain was also identified in the carboxyl terminus of the protein between residues 589 and 833 (Alll). One concern with any gene fusion study is that one cannot know for certain whether an artificial transcription factor has been fortuitously constructed by fusing portions of a given gene to a DNA binding domain. In the case of the temperature-regulated transcriptional activation domain, we are confident that it is involved in transcription of hsp genes because of the correlation between the gene fusion studies and the viability studies. The amino-terminal activation domain (Al) may also be important for HSTF function because it is in a region required for viability at 22% and 30% (see Figure 1). As the deletion analyses revealed no activation domain between residues 208 and 413, it is likely that the Al domain (between amino acids 1 and 172) performs the necessary activation function at nonshock temperatures. We are currently testing whether deletions of this region alter viability in the native HSTF. The role of the carboxy-terminal domain (Alll) is less clear. The viability studies demonstrated that its presence is not essential under shock or nonshock growth conditions. It is curious that in the gene fusion studies, the level of transcription at nonshock temperatures drops (concomitant with the hybrid protein becoming a heat-inducible transcription factor) only when this region is deleted. The deletions used in the viability studies and in the gene fusion experiments identified only the minimum region required for proper function and heat-inducible transcription. Region Alll, which is a powerful transcriptional activator on its own, is certainly repressed in the native HSTF molecule at nonshock growth conditions. However, in our gene fusion experiments it was never repressed (fusions encoding C-terminal deletions of YAP-1 1..155/HSTF21r,-833; see Figure 3). We suspect that the reason for this is that in our fusion experiments, the amino-terminal 218 amino acids (including Al and half of the HSTF DNA binding domain) were deleted and that an element in that region is necessary for repression of Alll. Support for this idea comes from fusion of the full-length HSTF at its amino terminus with the YAP-1 DNA binding domain, resulting in a temperature-regulated fusion protein (Figure 58). Therefore, it is possible that a repression function, similar to that seen for All, could reside in the amino-terminal portion of the HSTF and repress Alll. Furthermore, the isoleucine repeat, which in addition to the DNA binding domain is implicated in the repression of the All element, does not appear to be able to repress Alll function in the absence of the amino terminus. With this in mind, it is possible that regions All and Alll together constitute one inducible transcriptional domain but function this way only in the context of the intact HSTF protein. One other possibility is that although the Alll domain is not required for viability, it may be required for transcriptional activation under physiological conditions distinct from heat shock. A Model of Temperature-Regulated Transcriptional Activity of Yeast HSTF Two simple interpretations of our experiments are presented in Figure 8. In these models we describe diagram-

Cell 814

Figure

G

‘sACTIVE

INACTIVE

INACTIVE

INACTIVE

matically the temperature-regulated transition between a transcriptionally inactive form to a transcriptionally active form of the HSTF. In yeast, HSTF is free to interact with DNA at both low and high temperatures (Sorger et al., 1987). It is possible that under nonshock conditions there is direct intramolecular contact between the repressing domain and the transcriptional activation domain (Figure 8A). We speculate that upon heat shock, regions within the repressing domain are modified such that its conformation is altered to allow the adjacent transcriptional activation domain to function. Alternatively, the repressing domain may interact under nonshock conditions with another HSTF molecule or an as yet unidentified protein that, in turn, is responsible for repressing the transcriptional activity (see Figure 88). Upon heat shock, the blocking protein would dissociate as the result of some modification either to itself or to the HSTF, allowing the HSTF’s transcriptional activation domain to function. The elimination of all these negative interactions may allow the constitutive activators to interact with some component(s) of the transcriptional machinery (i.e., TATA factor [TFIID], RNA polymerase II, and other general factors). There is precedent for intermolecular interaction between a transcription factor and another protein to influence the activity of that transcription factor, For example, formation of an hsp90-glucocorticoid receptor complex prevents the glucocorticoid receptor from entering the nu-

8. A Model for Activation

of Yeast HSTF

Schematic representation of how yeast HSTF transcriptional activation is thought to occur (A) Intramolecular repression model. (8) Intermolecular repression model. DED, DNA bindmg domain (Wiederrecht et al., 1988). ILR, isoleucine repeat (Sorger and Nelson, 1989). FIP, repressor protein, which could be another HSTF monomer or an hsp protein or some umdentified factor. X represents some member(s) of the transcriptional apparatus (I.e.. TATA factor, RNA polymerase II, bridging factor, etc.). HSE, heat shock element. HS mRNA. heat shock mRNA

ACTIVE

cleus (Catelli et al., 1985; Sanchez et al., 1985, 1987; Howard and Distelhorst, 1988). Deletion mutants of the glucocorticoid receptor that lack the hormone binding domain become constitutive activators of transcription (Godowski et al., 1987; Hollenberg et al., 1987). Mutations of the transcription factor GAL4 have been identified that make it a constitutive transcriptional activator in the absence of galactose (Douglas and Hawthorne, 1966; Matsumoto et al., 1980). It is now recognized that GAL4 interacts with GAL80 to form an inactive complex, through a region of GAL4 that is involved in transcriptional activation. Deletion of this region in GAL4 renders the factor constitutively active, and insensitive to the presence of GAL80 (Johnston et al., 1987; Ma and Ptashne, 1987). Although a candidate for the blocking activity other than the HSTF itself has not been identified, one possibility is that it could be an hsp, perhaps hsp70. Studies in Drosophila cells indicated that the levels of hsp70 regulate the level of the heat shock response (DiDomenico et al., 1982). Further evidence in yeast cells showed that two hsp70 genes, YGlOO and YG102, are required for growth at 37% as well as for control of the synthesis of hsp90 at nonshock temperatures (Craig and Jacobsen, 1984). All of these possibilities are now amenable to direct experimental analysis. The nature and the role of temperature-regulated posttranslational modifications that control HSTF activation remain unresolved questions. It is reasonable that the out-

Transcriptional 015

Activation

Domains

of HSTF

come of the modification is the derepression of the transcriptional activator domain(s) we have identified. It has been shown that upon heat shock the yeast HSTF is phosphorylated (Sorger et al., 1987; Sorger and Pelham, 1988). It remains to be determined whether phosphorylation or some other kind of modification causes the derepression of transcription during heat shock. Experlmental

Procedures

Bacterial Strains, Yeast Strains, and Their Growth E. coli strains MM294 (F- endA hsdR17(r~-mk+), supE44 thi-7 h Restricfion: @k-m,,+) mcrA(+) mcrB(+) (Hanahan, 1983), DH5a (F- 1p8Od, laci’AM75 endA racA1 hsdR17 (rk-. mkf) supE44 thi-1 d- gyrAg8, D(lacZYRargFj, U189), and XL&Blue (fac47 lac- endA gyrA98 thi hsdR77supE44 m/Al {F’pm4S lacP lacZAM75 TnlO)) were used for all cloning steps involving the pSEY18 or pSEYC88 shuttle vectors. Bacteria were grown on media described by Miller (1972). S. cerevisiae strain SEY6210/6211 (a/a leu2-3,712 we%52 his%200 trpl-901 @2-807/i adeBlOl/+ suc2-9 mar) was used for all of the genetic manipulations described in this paper. Yeast were grown on media (YPD: yeast extract, peptone, dextrose; SD: synthetic minimal) described by Sherman et al. (1979). Diploid strains were sporulated on a solid sporulation medium (McClary’s medium: 1% potassium acetate, 0.1% dextrose, 0.25% yeast extract) supplemented with 50 uglml of the required amino acids. A new HSTF-disrupted strain with more of the HSF7 coding sequences removed in order to minimize potential recombination with episomal copies of HSF7 was constructed. A plasmid was constructed such that all HSF7 promoter and protein coding sequences between the Hindlll sites at positions 385 and 3382 (see Wiederrecht et al., 1988) were removed and replaced with a BamHl fragment containing the HIS3 selectable marker. An EcoRl fragment from the resulting clone containing the HIS3 gene flanked by HSFl sequences was used to transform the hiWhis3 SEY6210/6211 diploid yeast strain to histidine prototrophy by fragment-mediated spheroplast transformation (Rothstein, 1983). When tetrads from the resulting HSFV+ strain, called GWyA2, were dissected, two spores from each tetrad were always inviable and two spores were viable. The viable spores were always histidine auxotrophs. The haploid strain containing the chromosomally disrupted copy of HSF7 was constructed as follows. GWyAP was transformed to uracil prototrophy with the complete HSF7 gene carried on a YCp50 plasmid, which carries the URA3 marker. The resulting strain was transformed to tryptophan prototrophy with a GAL7,7O/HSF7 fusion carried on a centromere vector derived from SEYC68 having the URA3 selectable marker replaced by the TRPl selectable marker. The resulting chromosomally disrupted strain containing two episomal copies of HSF7 was sporulated, and the tetrads were dissected. Spores were selected for histidine, uracil, and tryptophan prototrophy. The haploids were then grown on plates containing 3% galactose, 2% raffinose, 2% glycerol, 5-fluoro-erotic acid (Boeke et al., 1984), and 20 uglml uracil in order to cure the strain of the YCp50 plasmid. The resulting strain, called GWyAS-II, grows only in the presence of galactose and dies on glucose-containing media. The haploid YAP-l-disrupted strain, referred to in this paper as SM9, has been described previously (MoyeRowley et al., 1989). To determine the minimal HSF7 sequence required to rescue the disrupted strain, the HSF7 deletion constructs in the centromere vector SEYC88, described below, were transformed into GWyAJ-II using the lithium acetate procedure (Ito et al., 1983). Uracil prototrophs were selected on minimal plates containing galactose, raffinose, and glycerol as carbon sources. Transformants were transferred to minimal plates having glucose as a carbon source. HSF7 fused to the GAL7,70 promoter is no longer transcribed because of catabolite repression and because viability of the transformants depends upon the particular HSF7 deletion. Plssmids, Shuttle Yectors, and Constructlon of Deletions The two shuttle vectors used in this work, pSEY18 and pSEYC68, are described in Emr et al. (1988). The HSFl gene in YCp50 is described

in Wiederrecht et al. (1988), and a map of YCp50 can be found in Rose (1987). The YAP1 clone in pUC19, RS2.5, is described in Moye-Rowley et al. (1989). The HSF7 3’ deletions used to determine the minimal HSFI sequences necessary for viability as well as the carboxy-terminal endpoints of the transcriptional activation domains were derived from the & deletions described in Wiederrecht et al. (1988). In that paper, the deletions were constructed in the vector pOTSNcol2 (Shatzman and Rosenberg, 1986, 1987) in order to overexpress H.W. HSF7 in pOTSNcol2 lacks the wild-type promoter sequences and is fused to the heterologous h promoter, PL. To determine the minimum piece of the HSF7 gene necessary to rescue the disrupted strain, this set of deletions was fused to the wild-type HSF7 promoter as follows. A 1.2 kb EcoRI-BamHI fragment from HSF7 clone 12, spanning a kilobase of promoter as well as 200 nucleotides of protein coding sequence, was cloned between the EcoRl and BamHl sites of pSEYC68. A Sall-Xhol fragment containing the TRP5 transcriptional terminator (Miyajima et al., 1984) was cloned into the Sall site of the resulting plasmid. The HSF7 deletions in pDTSNcol2 were digested with BamHl and Xhol and cloned between the BamHl and Sal1 sites of the shuttle vectors having the HSF7 promoter and TRP5 terminator. The final constructs, having both promoter and terminator sequences, were transformed into GWyAS-II and selected as described above. The YAPVHSF7 deletion fusion genes used to identify the transcrip tional activation domains of HSF7 were cloned as follows. Cutting RS2.5 with Hpal and Smal removes all YAP7 sequences downstream of the YAP7 DNA binding domain. The Hpal site defines the 3’ end of the YAP7 DNA binding domain. The HSF7 Dral fragment spanning nucleotides 1671 to 2851 was fused in frame to YAPl’s DNA binding domain at the Hpal site. This plasmid was digested with Stul (which cuts at nucleotide 2763 in the HSF7 sequence) and Xbal. A Stul-Xbal fragment from HSF7 clone 12 was inserted. The resulting clone, called AHl, has all HSF7 sequences downstream of nucleotide 1671 fused to all YAP7 sequences upstream of the Hpal site. For cloning of all fusion constructs having endpoints downstream of the Stul site, AH1 was digested with Stul and Sall and the Stul-Xhol fragments from the HSF7 deletions in pOTSNcol2 described above were inserted. For cloning of all fusion constructs having endpoints upstream of the Stul site, RS2.5 was digested with Hpal and Sal1 and the Dral-Xhol fragments from the HSFl deletions in pDTSNcol2 were inserted. All of these fusion genes can be removed from pUC19 by digestion with EcoRl and Sphl. They were cloned into the 2pm shuttle vector pSEY18 as follows. The Sall-Xhol fragment containing the TRP5 transcriptional terminator (described above) was cloned between the Sal1 and Xhol sites of pSEY18. The resulting plasmid was digested with Smal, Sphl linkers were attached, and the plasmid was digested with EcoRI. The YAPI/HSFl EcoRI-Sphl fragments were then inserted. The correct clones were transformed into the yeast strain referred to in this paper as SMSMO (Moye-Rowley et al., 1989), which is disrupted for the nonessential YAP7 gene and carries an AP-1 response element (ARE) functioning as an upstream activating sequence (L/AS) in front of a CYClNacZ reporter gene. The amino-terminal deletions of the HSTF derivative YAP-I,& HSTFsMe were made as follows. Twenty-five micrograms of plasmid DNA of HSFl deletion derivative encoding YAP-1 1155/HSrF1-&(8, which contains sequences between the EcoRl site of 1GW8’ and amino acid 848 (Wiederrecht et al., 1988), was digested to completion with BamHI. After phenol-chloroform extraction, ethanol precipitation, and resuspension in TE buffer, the DNA was digested with 2.75 U of Bal31 nuclease at 37°C. Aliquots were withdrawn at 2.5 min intervals for 30 min, and EGTA was added to 25 mM. Enzyme activity was destroyed at 70% for 5 min. After phenol-chloroform extraction, ethanol precipitation, and resuspension in TE, the ends were filled in using 2.5 U of T4 DNA polymerase and 20 FM dNTPs at room temperature for 15 min and incubated further with 2.5 U of DNA polymerase (Klenow fragment) at room temperature for 15 min. Reactions were phenol-chloroform extracted, ethanol precipitated, and resuspended in TE, and the DNA was digested to completion with Xhol. The resulting DNA fragments were separated from the vector by electrophoresis on a 1.2% agarose gel, and DNA inserts were eluted from the gel as described by Top01 et al. (1985). The deleted fragments were inserted directionally into the Smal-Sal1 sites of a phosphatased pSEY18 plasmid containing the TRPStranscriptional terminator (described above). In-frame

Cell 816

fusions as well as amino-terminal endpoints were determined by DNA sequence analyses of the mutants. The correct clones were transformed into the yeast strain SM9110 (Moye-Rowley et al., 1989) (described above). Induction of the Heat Shock Response in Yeast Cells One milliliter of a saturated yeast culture was inoculated into 100 ml of minimal medium selecting for the appropriate markers. The yeast culture was incubated with vigorous shaking at 22’C until the Am reached 1.0. Fifty milliliters of the culture was withdrawn, and the yeast cells were pelleted by centrifugation, washed with sterile water, and resuspended in permeabilization buffer (100 mM Tris [pH 7.51, 0.05% Triton X-100) at a concentration of 50 Asoo U/ml. The cells were frozen at -80% immediately. The other half of the culture was transferred to a 40% water bath for 1 hr to induce heat shock. The cells were then transferred back to 22°C for 1 hr to allow translation of the heatinduced b-galactosidase transcripts. The absorbance was measured, and the cells were resuspended at 50 Asoo U/ml in permeabilization buffer and frozen at -80%. BGalactosldase Assays The P-galactosidase assay is derived from the assay described by Miller (1972). The reaction was started by mixing the yeast cells with a solution containing 900 ~1 of 2 buffer (60 mM sodium phosphate [pH 7.01, 10 mM KCI, 1 mM MgSO.,, and 50 mM b-mercaptoethanol) and 200 PI of ortho-nitrophenylgalactoside (ONPG; 4 mglml in 100 mM sodium phosphate [pH 7.01) such that the final volume was 1.2 ml. The reaction was terminated when yellow color became visible by the addition of 0.5 ml of 1 M Na$Os. Start and stop times were noted, the cells were spun down, and the &PO of the supernatant was determined spectrophotometrically. The volume of cells added depended upon the transcriptional activity of the HSf7 construct and was adjusted such that the A420 was between 0.3 and 0.8 after 5 to 15 min from the time of addition of cells. To measure protein concentration, 100 PI of cells was mixed with 900 ~1 of HZ0 and 0.5 ml of 3 N NaOH, and the suspension was incubated at 90°C for 10 min. The mixture was cooled on ice to room temperature, 0.5 ml of 2.5% CuSO4 was added, and the mixture was incubated at room temperature for at least 10 min. The precipitate was removed bycentrifugation, and the absorbance at 555 nm was determined. Specific activity was calculated according to the equation: specific activity = 1.2(A.&min)ml-1/(5.3 x 106)(mg/ml protein). One unit of activity is defined as 1 nmol of ONPG hydrolyzed per min per mg of protein. Plasmid Sequencing To determine the endpoints of all of the deletions used in this study, sequencing was performed on plasmid miniprep DNA. In brief, 1.5 ml of an E. coli overnight culture was subjected to the standard DNA miniprep procedure, and the DNA pellet was resuspended in 100 ~1 of TE, 8 11 of 2 N NaOH and 2 mM EDTA were added, and the suspension was incubated at 90% for 5 min. Twelve microliters of 3 M sodium acetate (pH 5), 28 PI of H20, and 320 ~1 of ethanol were added to precipitate the DNA. The pellet was washed with 70% ethanol, dried, and resuspended in 25 ~1 of TE. The 3’ deletions were originally cloned into pOTSNcol2 and were sequenced in that vector. The primer 5’-AGGCTCTCAAGGGATCG-3’ (2.5 ng) was annealed to the DNA and the deletion endpoint determined by dideoxy sequencing. To ensure that YAP-1IHSTF fusions were in frame, a primer was used that annealed 69 bases upstream of the Hpal site in YAP7 with the following sequence: 5’-AATATAGACCAGAGACAAGA-3’. DNA sequencing reactions were carried out according to Sequenase protocols. Protein Blot Analysis Fifty milliliter cultures of a given yeast strain harboring the YAP-1lHSTF fusion proteins were grown to an A Bw of 1.0 and quickly harvested by centrifugation at 5000 rpm. The cells were resuspended in 1 ml of 50 nM Tris (pH 8) and lysed as described in Maniatis et al. (1982). The ly sates were subjected to SDS-polyacrylamide gel electrophoresis and the protein was transferred to nitrocellulose. The immobilized proteins were probed with a =P-labeled YAP-1 recognition oligonucleotide as described in Harshman et al. (1988).

Acknowledgments J. Nieto-Sotelo and G. Wiederrecht made equal contributions to the work reported in this article. J. N.-S. is a recipient of a Proctor and Gamble Fellowship. G. W. is a recipient of acalifornia Division, American Cancer Society Senior Fellowship (S-72-87). This research was funded by grant NP-482 from the American Cancer Society to C. S. P. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adverfisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

June

16, 1990; revised

July 20, 1990

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