Identification of the C-Terminal Activator Domain in Yeast Heat Shock Factor: Independent Control of Transient and Sustained Transcriptional Activity

The EMBO Journal vol.12 no.13 pp.5007-5018, 1993 Identification of the C-terminal activator domain in yeast heat shock factor: independent control of transient and sustained transcriptional activity

Yuqing Chen1, Nickolai A.Barlev2,3, Introduction Ole Westergaard and Bent K.Jakobsen4 In response to hyperthermia, and certain other forms of Department of Molecular Biology, University of Aarhus, C.F.Mollers stress, cells transiently increase transcription from a small Alle, Building 130, DK-8000 Aarhus C, Denmark group of genes that encode a characteristic set of proteins, lPresent address: Department of Zoology, University of Western the heat shock proteins. The heat shock proteins exercise Ontario, London, Canada protective functions in the cell during stress, for instance 2Present address: Laboratory of Structural Genome Organization, Institute of Cytology, Tihkoretsky Avenue 4, 194064 St Petersburg, by renaturing or solubilizing denatured proteins (for reviews Russia see Lindquist, 1986; Pelham, 1986; Bienz and Pelham, 3Present address: Wistar Institute, Pennsylvania University, 1987). 3601 Spruce Street, Philadelphia, PA 19104-4268, USA Heat shock promoters contain a universal sequence 4Present address: Institute of Molecular Medicine, University of element that is necessary and sufficient for their Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK transcriptional activation (the heat shock element, HSE). This Communicated by H.R.B.Pelham was originally identified as a 14 bp sequence by deletion analysis of the Drosophila hsp7O promoter (Bienz and In yeast, heat shock factor (HSF) is a trimer that binds Pelham, 1982; Pelham, 1982; Pelham and Bienz, 1982). DNA constitutively but only supports high levels of Careful examination of the HSE has demonstrated that it can transcription upon heat shock. The C-terminal regions be considered as tandem arrays of inverted 5 bp units with of HSF from Saccharomyces cerevisiae and Kluyveromyces the consensus sequence -GAA- (Amin et al., 1988; Xiao and lactis are unconserved yet both contain strong Lis, 1988). Natural promoters contain various numbers of transactivators which are correctly regulated when the 5 bp units in several different arrays (Amin et al., 1988; substituted for each other. We have performed high Xiao and Lis, 1989). HSEs are recognition sites for a nuclear resolution mapping of these activator domains which protein, the heat shock factor, HSF (Wu, 1985); the 5 bp shows that in K.lacts HSF (KIHSF) activity can be located sequence is the unit of interaction with HSF but stable to a confined short domain, while in S.cerevisiae HSF binding requires at least two inverted 5 bp motifs (Perisic (ScHSF) two separate regions are required for full et al., 1989; Xiao and Lis, 1989; Lis et al., 1990; Xiao activity. Alignment of the activator domains reveals etal., 1991). similarity, as both overlap potential leucine zipper motifs In Drosophila melanogaster and mammalian cells, HSF (zipper C) with a distribution of hydrophobic residues binds DNA only weakly under normal conditions but its sinilar to two highly conserved N-terminal domains affinity is increased markedly by heat shock (Kingston et al., which mediate HSF trimerization (zippers A and B). In 1987; Sorger et al., 1987; Zimarino and Wu, 1987). In these higher eukaryotes a C-terminal leucine zipper is required cell types the crucial modification to HSF may trigger a to maintain HSF in a monomeric and non DNA-binding conformational change that allows oligomerization and state under normal conditions and we therefore address thereby increases DNA affinity (Larson et al., 1988; the regulatory roles of the three leucine zipper motifs in Zimarino and Wu, 1990; Rabindran et al., 1993; Sarge KIHSF. Whilst the longest and most N-terminal of the et al., 1993). In the yeasts S. cerevisiae and K. lactis, by trimer region zippers, A, is dispensable for regulation, contrast, HSF binds DNA constitutively at all physiological mutation of a single leucine in zipper B makes HSF temperatures (Sorger et al., 1987; Jakobsen and Pelham, constitutively active. In contrast to the situation in higher 1988, 1991). Transcription in yeast must therefore be eukaryotes disruption of zipper C has no observable regulated through modification of prebound HSF. regulatory effect and therefore, although an intra- HSF genes have been isolated from a number of organisms molecular contact between zippers B and C cannot be including S. cerevisiae (Sorger and Pelham, 1988; ruled out, such contact is not required for restraining Wiederrecht et al., 1988), K. lactis (Jakobsen and Pelham, the C-terminal activator domain. We furthermore fimd 1991), Drosophila (Clos et al., 1990), human (Rabindran that deletions which abolish activator potential of the et al., 1991), mouse (Sarge et al., 1991) and tomato (Scharf C-terminus render the host strain temperature sensitive. et al., 1990). In yeast, HSF is a single copy essential gene However, deletion of a double proline-glycine motif in whilst in man and mouse two alleles have been identified. the activator, whilst leaving HSF unable to respond to All HSFs contain two conserved domains: a basic DNA heat shock, does not cause temperature sensitivity. This binding surface (Nieto-Sotelo et al., 1990) and a helical result demonstrates that independent mechanisms control domain which has been shown in S. cerevisiae and the transient and sustained activities of HSF. Drosophila HSF to mediate trimerization (Perisic et al., Key words: activators/heat shock proteins/leucine zipper/ 1989; Sorger and Nelson, 1989; Clos et al., 1990; Rabindran transcription regulation/yeast et al., 1993). In addition, the yeast genes share two short homologous stretches, termed CE1 and CE2.

Mutational analyses of the yeast genes have illustrated the formation by forming heat shock sensitive interactions with fundamental arrangement of these proteins. Both contain the coiled-coil structures in the N-terminal part of the protein C-termini with high transactivator potentials (the C-terminal (Nakai and Morimoto, 1993; Rabindran et al., 1993). activator, CTA) which are constitutive when assayed by Although the yeast HSFs form trimeric complexes at all fusion to the heterologous DNA-binding proteins lexA or temperatures, the possible association of the activator domain API (Nieto-Sotelo et al., 1990; Sorger, 1990; Jakobsen and with a conserved interaction surface nevertheless suggests Pelham, 1991). Deletion of the CTA reduces the activity that the leucine zippers may form similar structures in all of HSF 3- to 4-fold and causes temperature sensitivity for eukaryotic HSFs. We therefore address the importance of the host strain. The truncated HSF must contain another the three leucine zipper domains for yeast HSF regulation. activity domain though it is likely that this is much less potent Of the N-terminal leucine zippers, the longest, A, is than the C-terminal activator (CTA) since it has not been dispensable for normal activity regulation while the whole possible to identify such a domain by fusion of N-terminal of zipper B is required to restrain both the N- and C-terminal fragments of HSF to lex (Sorger, 1990; Jakobsen and activators in HSF. Thus, control of HSF activity does not Pelham, 1991). require trimerizationper se but is a specific function ofzipper Mutation or deletion of three regions of HSF results in B. In the C-terminus, the activator domain is essential for deregulation. Surprisingly, deletion of the trimer region is normal HSF activity and for the host strain's ability to grow not lethal but leads to HSF having high constitutive activity at high temperature. Regulation is not affected by the (Nieto-Sotelo et al., 1990; Jakobsen and Pelham, 1991). integrity of zipper C and therefore a zipper B -C contact Deregulation, with HSF having somewhat lower activity, is not required for repression of HSF activity in yeast. One is also the result of deletion or mutation of CE2 in the yeast mutation in the activator region, however, locks HSF in a HSFs. It has been suggested that this element is involved virtually inactive state, but despite being unable to respond in an intramolecular contact with another conserved domain to heat shock the host strain grows at all normal and that this structure is required for restraining the CTA temperatures. This result demonstrates that the C-terminal (Jakobsen and Pelham, 1991; for review see Sorger, 1991). activator domain is regulated in two independent ways to Recent results indicate that this element influences the supply heat shock induced and sustained activities. sustained activity mediated by yeast HSF rather than being Furthermore this observation shows that structure in the C- involved in the heat shock response (A.kHoj and B.K.Jakobsen, terminus can influence unmasking of both the C- and N- submitted). Finally, a mutation in the DNA-binding domain terminal activators in HSF which could indicate that close causes constitutive activity, albeit in a fusion protein where physical contact exists between N-terminal and C-terminal the CTA had been exchanged with a heterologous activator domains. We have therefore taken initial steps to look for (Bonner et al., 1992). contact potential between these regions of HSF. In an in vivo From these analyses it is clear that complex intramolecular transcription assay we find that oligomerization can take contacts in HSF are required to restrain activity of the CTA. place between A-B and A-CTA peptides but not between How then is the activator unmasked in response to heat A and A-CTA. Thus ability to form interactions may exist shock? Gel retardation experiments have shown that between zipper B and structures in the C-terminus. HSF-DNA complexes formed with HSF from heat shocked cells migrate more slowly than when formed with HSF from Results unshocked cells (Sorger et al., 1987; Sorger and Pelham, 1988). This difference can be virtually eliminated by treating Mapping of the C-terminal activator in K.lactis HSF HSF with phosphatase. Thus, phosphorylation may be It was previously reported that the C-terminus of K1HSF, involved in activation of HSF, either directly or by inducing comprised of residues 457 -677, mediates strong a conformational change. Alternatively, phosphorylation may transcriptional activity when fused to the DNA-binding be a consequence of activation and possibly involved in feed- domain of the bacterial repressor lexA (Jakobsen and back regulation of HSF. Mammalian HSF1, although Pelham, 1991). In a DNA fragment coding for this region undergoing phosphorylation upon heat shock, can be of KIHSF, deletions were made from either end and, having activated by other treatments that apparently induce no established the extent of deletion points by sequencing, the changes in phosphorylation (Sarge et al., 1993). Considering new shorter fragments were fused in the correct reading the existence of a masked, constitutive activation domain in frame to lex (see Materials and methods). Transcriptional HSF it seems most likely that a conformational change is activities of the fusion proteins were assayed in yeast required for activation. The role phosphorylation plays in employing a LEX-lacZ reporter plasmid (Figure 1). HSF regulation has still to be clarified. Deletions from the C-terminus to amino acid 623 have no Here, we describe detailed mapping of the transactivators significant effects on activity, though some variation is from the C-termini of K1HSF and ScHSF. In K1HSF, activity present. Because some shorter proteins are more active than is contained in one short domain, while in ScHSF the longer ones we assume that these variations may be due to activator appears to be divided into two domains of which differential protein stability, solubility or other indirect one can function autonomously but with reduced activity. effects. Deletion of one more amino acid from the C- Though very divergent in sequence, comparison reveals that terminus to position 622 (klCA6), however, causes a 10-fold both activators overlap potential leucine zippers. This finding reduction in activity. Proteins further deleted from the C- is intriguing because two recent reports show that mutations terminus to positions 618, 617 and 615 (klCA7-9) maintain in a similarly located leucine zipper in Drosophila HSF and this lower level of activity, whereas all shorter proteins, the chicken HSF3 lead to constitutive trimer formation and high longest ending at position 610, do not produce activity above DNA binding affinity. It has been speculated that a possible background level (klCA 10- 18). role for the downstream zipper is to suppress trimer Likewise, deletions from the N-terminal side to residue 5008 Activator domain in yeast HSF

-4 . . .-, , I **_,- - _ CiVItyt

-cc [EXL * 37 - mqr'a ? ti9oc q;p@, LEX 1-[7 - mgra,ia 8700

-CS?= LEX 1'97 - rTl(aJa ± . j_ .. . .. 148000 - -'CS? -1[L X rI.qr-a.77 - 9500 -S _~~~~~ .- .- ' A _--_,i.. _4 [-EX 1-81 - rvaia 5300 X ;~~~3 .?__,>..a.K^-,. -- ^Rew <-C ILEX -87 - mrala 8100 -C? FX 1-87 rnmcwaJa 1200 _ ~~~-3 _-2: 9- 1.;;'-i -C:' LEX r/-nrqrai'a 660

-CS LEX 1S rrcfaJa Z - 500

'CS- 1$.53LLEX m-rnala= 670

L EX 187 ITWI-IIJ - 25

-1-.'- ~LEX -87- rara [X -R7 m-'aia 25

_ .FX 997 rnxji* - >9W

'-FC?[XLEX 1-87 rTr;199 rala-ai 20

F X ryy - aiazi 28 54 +? 31 .2 LEX !-1' LEX -8 7-

e 7 LEX uy"aai./-p." 23

LEX 43 / T r t = =

LEX '-14.' - A1c. 6500

LX -TIC

LEX -8,? - IrTY ; s' 79Qo

;z I.., " --i I -P7 -rrjxj r- s1- -' - 7' 89-00 *; LEX 49P.7 rm -V.;s . " 11500

LEX r-n ; 1060O

' I.[ X --. Z _ ; - 1rlC '- - L

7EX r r '-;s1 3 1 -)

LEX - - rT11 :912st 1260

I LEX - -'- rns 2/27

-, LE-X 8-' - FTKi[Ist - :, eggam 24

LELX '-17

FX_FX-' rirxr .ENZEM

LEX -8' rnxr--:' Ia

I , ;) ZOWIM

Fig. 1. Deletion analysis assay of the K1HSF C-terminal activator. Schematic presentations of fusions between the DNA binding domain of lexA (amino acids 1-87) and deletion products from the K1HSF C-terminus are shown. The fragment of KIHSF included in each construct is indicated partly as shaded boxes and partly, where in the authors opinion the sequence is of particular importance, in one-letter code. Numbers above boxes or sequence indicate endpoints and correspond to positions in the K1HSF sequence; where no number is indicated the endpoint is identical to the previous construct. K1HSF sequence is shown in capital letters, amino acids introduced in junctions or the C-terminus are shown in lower case letters. Activity was assayed from cells grown at 30'C by expression of ,B-galactosidase from an indicator plasmid containing a lexA binding site. Average results from three to six experiments were standardized to the kICAO construct; activity of the lexA construct without fusion of any C- terminal sequence is -20 units. 5009 Y.Chen et al.

592 do not significantly affect activity (klNA1-7), but significant level of activity (584 -783) and this was reduced further removal of three, six or seven amino acids to - 6-fold in comparison with the original fragment positions 595, 598 and 599 (klNA8-10) respectively, (584-833). We used an approach similar to the one significantly decreases activity without completely abolishing described above for deletion analysis of a fragment encoding it. Proteins with K1HSF sequences starting at position 602 amino acids 482-833 of ScHSF. In our experiment or further downstream are all inactive (klNA11-16). (Figure 2), deletion of C-terminal residues to position 783 In conclusion, this experiment indicates that amino acids does not significantly affect activity (scCA1), but removal 592 -623 are essential for the transcriptional activity of the of two more amino acids (to 781, scCA2) causes a 6-fold CTA from KIHSF. Below we address the question of reduction. In deletion mutants with C-termini between 781 whether this domain alone is necessary and sufficient for and 705 (ScCA2 -9) no more loss of activity is observed the full activity of the C-terminus. but a further 5- to 10-fold reduction is seen in proteins with C-termini between 691 and 650 (scCA10 - 13). Finally, with Mapping of the C-terminal activator in S.cerevisiae the C-terminus truncated to positions 635 or 596 no activity HSF above background level is detectable. Sorger (1990) showed that the C-terminal of ScHSF (amino With N-terminal truncations to position 595 full activity acids 584-833) is a strong constitutive activator when fused is retained (scNAI1-5) but a 60-fold reduction is the result to lex, and tested four fusion proteins expressing of deletion to positions 662, 665 or 667 (scNA6 - 8). More subfragments of this region. Of these only one retained a extensive deletions to position 688 or further downstream

lp, ActiWty 482 627 786 833 pCAQ C53=^ 11800 783 KM LEX-87mgrvcLrgpxUWDNVHRNIDEO)ARLONLENMVH7LSPGYPNKSFNNKTSSTNTNSNMESAVNVNSPGFNLODYLTGESNSPNSVHSVP=.I * 3 3 TRVSPDIKFSATENTKVSDNLPS 9400 781 2 LEX1 -87 - mgrvirgp MLV LFWDNVHRNIDEODARLONLENMVHILSPGYPNKSFNNKTSSTNTNSNMESAVNVNSPGFNLQDYLTGESNSPNSVHSVP E = TRVSPDIKFSATENTKVSDNL vse 1540 776 3 LEX1-87 - mgrvdu!gpx xLxFx WDNVHRNIDE0DARLLONLNMVHILSPGYPNKSFNNKTSSTNTNSNMESAVNVNSPGFNLODYLTGESNSPNSVHSVP rTRVSPDIKFSATENTK 1500 770 si LEX1-87 - mgrvdkrgp * ULFWDNVHRNIDEODARLONLENMVHILSPGYPNKSFNNKTSSTNTNSNMESAVNVNSPGFNLODYLTGESNSPNSVHSVPxx x Coo=xTRVSPDIKFS wse 1330 756 LEXI1-J7 mgvupxxxUDVRIEDROUNVISGPKFNKSTTSMSVVSGNOYTENPSHV x 1170 . scC= LEX1-87 -mgrvdergp \\\\3M\C=. WDNVHRNIDEODARLONLENMVHNLSPGYPNKSFNNKTSSTNTNSNMESAVNVNSPGFNLODYLTGESNSPNSVHSVP c731 r EQ4 LEX1-87ULFWDNVHRNIDEODARLONLENMVHILSPGYPNKSFNNKTSSTNTNSNMESAVNVNSPGFNLODYLTGESNSPNSVHSVPmgrvdrgp x a 1260 724 g2WZ LEX1-87 -mgrvckrgpES CE3 LFWDNVHRNIDEODARLONLENMVFULSPGYPNKSFNNKTSSTNTNSNMESAVNVNSPGFNLQDYLTGESNSPNSVHSVPts ge 1470 713 1840 gQW LEX1-87 -mgrv5rgp\\\3LF \\\,WDNVHRNIDEODARLONLENMVHILSPGYPNKSFNNK TSSTNTNSNMESAVNVNSPGFNLODYLTGESNSPNSVHISvse 1630 691 wCA&I LEX1-87 mgrvcdrgp. SwDv = LFWDNVHRNIDEODRLONLENMVHLSPGYPNKSFN NKTSSTNTNSNMESAVNVNSPGFNLOD 365 6cU 370 EQAIZ 370

WCA1 LEX1-87 - mgrvdWrgp = WDNVHRODEODARLONLENM 154 CCAl1 34 mmal 59 wCAI LEX1-87 - mgrvdcrgp 27 &CCS1 498 LEXI 47WDNVHRNIDEODARLONLENMVLSPGYPNKSFNNKTSSTNTNSN47ESAVNVNSPGFNLODYLTGESNSPNSVHSVPmgrg = x C x - TRVSPDIKFSATENTKVSDNPSF N 15000 2C21 542 LEX1-87 - mgrg sxxU WDNVHRNIDEODARLONLENMVHILSPGYPNKSFNNKTSSTNTNSNMESAVNVNSPGFNLQDYLTGESNSPNSVHSVP^ x TRVSPDKFSATENTKVSDNPSF N 11300

LEX1-87 - mgrg m L WD WVHRNIDEOD ARLONENNVHILSPGYPNKSFNNKTSSTNTNSNMESAVNVNSPGFNLODYLTGESNSPNSVHSVP E33=TRVSP DIKFSATENTKVSDNLPSFN C:, 12300 eW4 LEX147 - mgnrpg 10500 595 0 LEX1-87 - mgvdara 12100 662 MM LEX 1-87 - mgrg SF NNKTSSTNTNSNMESAVNVNSPGFNLODYLTGESNSPNSVHSVP TRVSPADIKFSATENTKVSDNLPSF N 235 665 EMZ LEX 1-87 - mgrvdard NKTSSTNTNSNMESAVNVNSPGFNLODYLTGESNSPNSVHSVP *0=T RVSPDIKFSATENTKVSDNLPSF N VZ\ 230 667 E2 LEX 1-87 - mgrvcara 235 688 KgMQ LEX 1-87 -mgrrpg NLODYLTGESNSPNSVHSVP a xTRVSPDIKFSATENTKVSDNLPSFN = 78

715 Aid10 LEX 1-87 mgrg TVSPDIKVSPDKFSATENTKVSDNLPSFN 75 729 6A1dI LEX1-87 - mgrg TRVSPDIKFSATENTKVSDNLPSFN 68 748 wAfdI LEX1-87 - mgrg m TRVSPDIKFSATENTKVSDNLPSFN 63 762 131 LEX147- mgrg RVSPDIKFSATENTKVSDNLPSFN 0, 75

773 wA4i! LEX 1-87 - mgrg ENTKVSDNLPSFN 45 779 wAl8 LEX 1-87 - mrrpg DNLPSFN 60 788 wNdI LEX1-87 - mgrrpg 52

Fig. 2. Deletion analysis assay of the ScHSF C-terminal activator. Presentation and assay are similar to Figure 1. It should be noted that constructs scCAO and klCAO have similar activity levels but vary -3-fold with cell density; the correlation between different deletion constructs is not affected. Figures and 2 show the activity from two typical experiments performed at similar cell densities.

5010 Activator domain in yeast HSF

result in fusion proteins with activity only two to three times fusion carrying only the minimal full activity region as higher than background. defined by deletions, i.e. residues 592-623, is inactive In conclusion, this analysis suggests that two regions are (construct 3). Thus, the activator domain may not be entirely necessary for full activity of the ScHSF CTA. In one region contained within this region but requires additional elements activity is contained between amino acids 595 and 713; this on at least one of either the N- or C-terminal sides. region is in itself capable of supporting transcription in lex Alternatively, its function may be affected by adjacent fusions (see scCA2 - scCA9) but is approximately six times sequences (thejunction sequences in construct 3 are different more active when linked to an element located further from the N- and C-terminal junctions in k1NA7 and 1dCA5, towards the C-terminus. This element which has a C-terminal respectively, see Figures 1 and 3). To distinguish these border around amino acid 783 is not able to function as an possibilities we tested the effect of inserting the activator activator when fused to lex. Rather, the most C-terminal domain in a heterologous protein fragment from the N- element seems to have a supporting role for the activator terminus of K1HSF. This N-terminal fragment, spanning domain residing between residues 595 and 713. It has not, residues 14-155, is virtually inactive when fused to lex with the approach used here, been possible to determine the (construct 5) but replacement of residues 118-132 with N-terminal border of the C-terminally located element 592 -623 makes it an extremely potent activator (construct because it does not in itself support transcription. 4). We conclude that residues 592 -623 constitute a transcriptional activator domain containing the full activity Residues 592- 623 of KIHSF constitute a potential of the K1HSF C-terminus. transcriptional activator domain To investigate whether the results obtained by deletion analysis have identified the boundaries of autonomously The KIHSF and ScHSF activator domains overlap functioning activator domains we examined more closely the potential leucine zippers region of the KIHSF C-terminus within which full activity Although computer comparisons have previously identified was observed (Figure 3). An internal deletion of residues no homologies in the C-termini of K1HSF and ScHSF 560-653 abolishes activity from the lex-KICTA fusion (Jakobsen and Pelham, 1991) an alignment of the regions protein (construct 1) and, in accordance with this, residues which have been identified as activators by deletion analyses 559-654 retain full (construct 2). However, a lex shows that these domains may nevertheless be associated activity with a conserved structure (Figure 4). Residues 581-608 of K1HSF and 628-655 of ScHSF, which overlap their respective activator domains, share limited but striking homology (Figure 4) in that both regions contain two arrays of conserved heptad repeats of hydrophobic residues 3 _X 1 -87 rTgrraie ------(indicated by open or solid triangles in Figure 4). 2~LE~ ~ .4:e Furthermore, both sequences have high scores for a-helix potential (see Figure 4 legend). These features are 2 s 1-8-7 r-igrrgi .,-;a ------S characteristics of 'leucine zipper' type domains which oligomerize several transcription factors (Landschultz et al., 1988). In such structures heptad repeats of, most frequendy, 3 X 1r-8' ggrrip------leucine, isoleucine or valine form a hydrophobic side on a coiled-coil which constitutes the main contact surface to the coiled-coil of another subunit. The possibility that the activator domain in HSF forms a coiled-coil is particularly noticeable because HSF is trimerized through interactions in an N-terminal region consisting of two coiled-coil domains, called helices A and B (zipper A and B; Sorger and Nelson, 1989; see Figures 5 and 6). Furthermore, a Fig. 3. Residues 592-623 of K1HSF can confer transcriptional C-terminal leucine zipper has also been noticed in the HSFs activity. The same conventions are used for presenting constructs as in man et Figure 1 except that the region within borders of full activity, residues of mouse, Drosophila, chicken and (Clos al., 1990; 592-623, is presented as a box with different shading. Activity is Sarge et al., 1991; Nakai and Morimoto, 1993). In scaled to that of construct kICAO in Figure 1. Drosophila HSF, mutations that disrupt a-helix structure in

580 590 600 610 620 l V7 K.lactis SPDPAIFQDLQNNN K EESIEQEIQDWITKLNPGPGEDGNTPIFPELNM w IV 'v v 'w S.cerevisiae NESDL?WDNVHRg; E DARLQNLENMVHILS.P YPNKSFNNKTSSTNT- 668-763 -PDIKFSATENTKVSDNLPSFN

630 640 650 660 670 770 780 ( 595) A ffl Fig. 4. Comparison of the activator domains in the C-termini of KIHSF and ScHSF. Amino acid sequences were aligned according to two heptad repeats of hydrophobic residues; these are indicated above the sequences by open and closed triangles, respectively. Predicted ca-helix potentials according to Chou and Fasman (1974) are in the range 0.92-1.25 for residues 582-607 of K1HSF, 0.90-1.21 for residues 628-654, and 0.92-1.14 for residues 766-779 of ScHSF. Amino acids which are identical in the two sequences are darkly shaded, homologous amino acids lightly shaded. Pairs of arrows summarize the results of each deletion series experiment; long arrows indicate the borders of full activity, short arrows the following deletion point from which full activity had been lost. 5011 Y.Chen et al.

Activity DNA binding 0 \A i r irerizarior; 1a1 Ivator- C:u HS activity bind2ing A35-7B -4 wt EEII 20 200 + ±f 1! 94-. - qPi 356-375 451 -4f66 580-623 67/ Il -3,3 .:

HFAA L__ :: I I ..I 20 180

HFAB3 i - s- lii| _ 210 200

- KXX Fr<'Al r-IFB, .__ . IL .:-. ] 180 170

l3ow p 370 -` HF-B .A( _I _ 55 60 nt

Fig. 5. Zipper B controls transcriptional activity of HSF during normal temperatures. Schematic presentation of mutations introduced in the conserved trimer region of K1HSF. DNA binding was assessed by gel retardation experiments (Sorger et al., 1987). Activities of HSF mutants were determined after growth at 30°C either by harvesting cells directly (C) or after exposure to 39°C for 45 min followed by recovery at 22°C for 45 min (HS). the C-tenninal leucine zipper confer constitutive trimerization the ability of HSF to bind DNA we exchanged leucine370, and DNA binding to this factor. On this basis it has been which is at the C-terminal end of zipper B, to a proline suggested that the C-terminal leucine zipper inhibits HSF (HFBL-P). The effect of this point mutation with respect trimerization during normal temperatures by forming a heat to activity regulation is similar to deleting the entire zipper sensitive interaction with the leucine zippers in the N-terminal B, but HSF's ability to bind DNA is only slightly affected. part of the protein (Rabindran et al., 1993). It is intriguing, Finally, it should be noted that mutations in helix B appear therefore, to consider the existence of a third helical contact to unmask the C-terminal as well as the N-terminal activator surface in the constitutively binding yeast HSFs and its domains in HSF, since a C-terminally truncated HSF also possible regulatory role with respect to the overlapping becomes constitutive, with a lower level of activity, by the activator domain. Hence, we investigated the earlier Leu370-Pro mutation (HFBL-PAC). observation that deletion of the N-terminal leucine zippers These results show that the restraining role of the deregulates HSF and tested the effect of mutating the N-terminal zippers in K1HSF can be ascribed specifically C-terminal activator or its putative overlapping leucine to helix B which is functional even when the global structure zipper. of HSF is altered such that no DNA binding activity can be observed in a standard gel retardation assay. It is also Zipper B but not zipper A is required for restraining remarkable that the restraining effect by zipper B can be activity of HSF during non-heat shock conditions abolished by a point mutation (HFBL.P) which does not A previous investigation showed that deletion of the bipartite severely affect DNA binding ability. This suggests that not coiled-coil domain in the N-termini of the yeast HSFs leads all of helix B is required for correct trimerization and that to high level constitutive activity (Jakobsen and Pelham, zipper B has a specific role in repression of activity. 1991). Prompted by this finding, the possible existence of Prompted by these observations we next addressed the a leucine zipper in the C-terminus, and the observations on importance of the activator domain and its association with Drosophila HSF and chicken HSF 3 where deletion of the a putative leucine zipper structure for HSF function, and in distal C-terminal zipper confers constitutive binding of these particular whether zipper C could be a possible contact HSFs (Nakai and Morimoto, 1993; Rabindran et al., 1993), surface for interactions with zipper B. we investigated in more detail the restrainment of the N-terminal zippers on KIHSF activity (Figure 5). The activator domain of KIHSF is required for normal Surprisingly deletion of zipper A, while severely disabling heat shock response and growth at high temperature the DNA-binding ability of HSF in in vitro experiments, has In both ScHSF and K1HSF removal of the C-terminus no detectable effect on regulation of activity in vivo (HFAA, reduces transcriptional activity after heat shock - 3- to 4-fold Figure 5). In contrast, deletion of zipper B deregulates HSF and the resulting yeast strains are growth sensitive to which also assumes activity around normal heat shock levels temperatures over 34°C (Nieto-Sotelo et al., 1990; Sorger, at the control temperature (HFAB). Deletion of zipper B also 1990; Jakobsen and Pelham, 1991). To investigate whether abolishes high-affinity DNA binding. To see whether this is a result of losing the activator domain, a helical contact deregulation could be achieved without severely affecting surface, or indeed both, we introduced a number of less 5012 Activator domain in yeast HSF

]MDR ._= .___f _ .. . _.m ._ S~~~~~~~.L :

__a- __ z. - X Fig. 6. Assay of activity effects of mutations in the K1HSF C-terminus. On a linear representation of KIHSF indicating conserved and functional elements the helix C-activator region is highlighted as an enlarged bar with the sequence indicated in capital letters over it. Amino acids deleted (A) from this region are indicated over the respective bars except in cases where an entire stretch has been deleted; introduced junction sequences are indicated by minor letters in the gaps. Activities of HSF mutants were determined as described in the legend to Figure 5. Activity of lex fusions carrying mutated C-termini were determined after growth at 30°C. Average activities from four to six experiments are shown. Activities of HSF proteins are scaled to wt and of lex fusion proteins to construct kICAO. For growth assays an equal number of cells was spread on rich plates and grown at 30 or 37°C; observations of colony formation, '+ + +' being similar to wt and '-' meaning no colonies, was identical at 35 and 370C for all constructs except HFC4 (for HFC4 see text for details). Note that, in order to avoid GAL80 interference (Ma and Ptashne, 1987), activities of HFCgall and HFCgal2 were measured from cells grown with galactose as carbon source. They are therefore not directly comparable with activities of the other constructs, the hosts of which were grown with glucose. extensive mutations in the zipper C/activator region of loss of activity from HSF (HFC3) as well as from the KIHSF. The effects of these mutations on HSF activity and corresponding lex fusion (lexC3) and the resulting strain is ability to support growth of the host strain at 30, 35 and also rendered temperature sensitive. These results 37°C were determined and their influence on the potency demonstrate that disruption of the activator region in HSF of the activator domain was tested by fusing the mutant has identical consequences to removal of the entire C- C-termini to lex (Figure 6). terminus and also that the two suggested zipper C domains Deletion of the potential zipper C, residues 584-611, can functionally substitute for each other. This is remarkable causes a 4-fold reduction of HSF activity after heat shock because the exchanged sequences show little homology apart (construct HFC1), an -50-fold loss of activity from the from their heptad repeats of hydrophobic residues (see lex-C-terminus fusion (construct lexC1), and renders the Figure 4). host strain unable to grow at high temperature. Thus, this To see whether over-representation of negatively charged internal deletion, as predicted from the deletion analysis residues is an essential feature of the activator region we (Figure 1), disrupts the activator domain and has an effect also introduced mutations that alter the net charge from -5 similar to deleting the entire C-terminus. However, if the to +3. The effect of this is a 3-fold reduction of activity deleted region comprising helix C is replaced with the in HSF (HFC4) and a 6-fold reduction from the equivalent region from ScHSF-residues 633-657 (see corresponding lex fusion (lexC4) showing that whilst Figure 4) -normal activity is restored both to HSF negative charge may be of importance to the function of this (construct HFC2) and to a lex-CTA fusion (lexC2), and activator, it is not an essential feature. the resulting strain grows at all test temperatures. From this HSF construct (HFC2), containing the Mutations that disrupt zipper C heterologous helix C, we next deleted the downstream part We next tested whether mutations can be introduced to of the K1HSF activator domain, i.e. residues 612-623. The disrupt zipper C without disabling activator function. effect of this is similar to deleting the zipper C region with Deletion of all hydrophobic residues in the heptad repeats 5013 Y.Chen et al. of helix C was too crude for this purpose as this mutation Interactions between zipper B and the C-terminal reduced activity ofthe lex fusion - 10-fold (lexC5) and also activator domain -4-fold reduced the activity of HSF (HFC5); although some The results described above show that helix B is involved activity potential remains in the C-terminus, the host strain in controlling activity of HSF in a way that is independent is also in this case temperature sensitive. We therefore tried of maintenance of overall structure. An obvious way to two mutations designed to disrupt helix structure with the exercise control over the activator domains would be by minimal change of protein sequence. In one, the isoleucine forming interactions with those that mask the activity at position 597, which is at a central position in helix C, function. Though control over the CTA is clearly not was exchanged with a proline-glycine motif (HFC6/lexC6), achieved by a simple zipper B - C contact it cannot be ruled and in the other residues 599 and 600 were interchanged out that a contact on a different structural basis may exist and the leucine at position 607 was exchanged for a histidine between zipper B and the activator domain in the C-terminus. (HFC7/lexC7). Neither of these mutations have significant We therefore tested whether potential for forming contacts effects on regulation of HSF activity or on transactivity of between zippers A and B and activator could be demonstrated the lex fusion protein, and neither renders the host strain in an in vivo transcription assay (Fields and Song, 1989). temperature sensitive. Though it cannot be excluded that the In this assay the ability of a DNA-binding fusion protein to mutant C-termini in HFC6 and HFC7 can still form shorter form contacts with a 'free' activator peptide is tested through coiled-coils the introduced changes would be expected to the efficiency with which the DNA-binding protein can have changed the structure of the putative helix C region stimulate transcription by bringing the activator protein into dramatically and disrupted any helix forming potential (Pu an appropriate promoter context. From the results obtained and Struhl, 1991; Tzamarias et al., 1992; Rabindran et al., with this assay (Figure 7A) it appears that neither zipper A 1993). Thus it is clear that mutations which disrupt zipper (experiment 2) nor zipper B (not shown) are capable of C cannot produce an effect similar to that seen by disrupting self-association, while A and B in conjunction mediate zipper B and therefore formation of a direct contact between oligomerization (experiment 6). These observations are in these zippers is unlikely to constitute the means by which keeping with those of Sorger and Nelson (1989) who found the CTA of yeast HSF is restrained. that in vitro synthesized ScHSF fragments could not be crosslinked when deletions from the C-terminal side had The heat shock response can be suppressed by removed zipper B. The interesting observation comes from mutations in the activator domain comparing experiments 2 and 3 with experiment 5. Although In contrast to mutations that disrupt zipper C, a strong effect lex-A is not capable of forming contacts with A-CTA or is obtained by deletion of the predicted 'turn' which ends AB-CTA, lex-AB is capable of forming contact with zipper C, i.e. residues 609-612. Though this mutation A-CTA. This result could indicate that whilst the leucine reduces activator strength less than 2-fold (Figure 5, zippers in HSF are incapable of forming stable contacts construct lexC8) the ability of the mutant HSF to respond involving only one zipper, oligomerization by zipper A can to heat shock is virtually abolished (HFC8). This be stabilized if a supporting contact can be formed; in these demonstrates that structural features in the C-terminal experiments the additional contact can be supplied through activator region are required for accurate regulation of HSF B -B (experiment 6) or, possibly, B -CTA (experiment 5) activity and also that structure in this region can suppress interactions. Alternatively, the inability of lex-A or lex-B activation by the hitherto unidentified N-terminal activator. to associate with activator partners may be due to lack of To further approach the question of structural requirements ability to fold correctly or insolubility of these proteins. for correct regulation of a C-terminal activator in K1HSF However, if a trans contact between zipper B and CTA is we also exchanged the CTA with the heterologous activator the stabilizing factor in experiment 5 then it seems likely from GALA (HFCgall and HFCgal2). Hybrid constructs that a similar contact could also exist within the HSF carrying the GAL4 activator in place of the CTA are monomer. constitutively active although the GAL4 activator is significantly repressed in comparison with activity in the GAL4 protein (see Jakobsen and Pelham, 1988, for Zipper C oligomerization comparison of HSF and GAL4 activities). Furthermore, the We finally sought to clarify whether zipper C may in itself efficiency of repression depends on the presence of the CE2 possess oligomerization ability. As seen in Figure 7B, lex element as - 4-fold more activity is observed when this fusions carrying truncated and inactive K1HSF C-termini are is not present in the HSF-GAL4 fusion protein. These capable of supporting transcription when expressed in the observations indicate that HSF regulation is dependent upon presence of 'free' activator. This ability requires the entire activator structure and emphasize the significance of the zipper C domain on the lex fusion (experiments 7 and 8), compatibility between the two yeast CTAs. as activity is progressively lost when part (experiment 9) Finally, it is significant that HFC8, in which residues or the whole (experiment 10) of this has been deleted. 609-6 12 are deleted, lacks heat shock activity but this does However, the activating ability is not severely affected when not lead to a temperature sensitive phenotype. This result the free C-terminus is mutated so as to disrupt zipper C demonstrates that sensitivity to growth temperature is (experiments 11 and 12). We therefore tried to verify zipper independent of the heat shock response and therefore, as C oligomerization by cross-linking in vitro translated KIHSF suggested by Sorger (1990), that the C-terminal activator C-terminal fragments but were unable to obtain crosslinking is probably regulated by two mechanisms, which respond between these. However, we cannot rule out that a weak to transient and sustained elevation of temperature, oligomerization potential may reside in the activator domain respectively, only the latter being required for growth at high but that this requires support from the N-terminal zippers temperature. in order to be significant. 5014 Activator domain in yeast HSF

- Expeerr ..L. fL

-..

A 7x .-.11..I .7- AB MM- ...Io

-- .'

LEX

LFX

Yxp-rjTl.rim n ...... X v17W 'V X7 '_ 7_

L LA V7 _V

L[ .. V '7 wK f5-

LEX 1 (..I

::: .- -.- No. LEX IN,,.

12 1. -.. .. Fig. 7. Oligomerization potentials of the KIHSF leucine zippers. (A) Contact potentials involving zippers A and B. To avoid interactions with endogenous HSF the fusion proteins were expressed in a yeast strain expressing a K1HSF mutant from which zippers A and B had been deleted (Jakobsen and Pelham, 1991). (B) Zipper C mediated oligomerization. In all experiments PCR generated fragments encoding the respective leucine zipper regions (see Materials and methods for details) were fused to the DNA-binding domain of lex or to the TPI promoter for overexpression of non-DNA binding peptides, and cotransformed in respective yeast strains together with an indicator plasmid; virtually identical results were obtained when using a lex-A fusion in which a 15 amino acid C-terminus was included.

Discussion In K1HSF, where the activator has been most accurately The C-terminal activator domains of KIHSF and located, the sequence constituting this is not glutamine or ScHSF proline rich as is characteristic for certain activators from We have described high resolution mapping of the other organisms (Courey and Tjian, 1988; Mermod et al., transcriptional activators residing in the functionally 1989; Nicosia et al., 1990; Foulkes et al., 1992; Schindler interchangeable C-termini of HSF from the yeasts K. lactis et al., 1992). Indeed, the deletion analysis (Figure 1) and S. cerevisiae. In K1HSF, full activity potential is demonstrates that a region which is remarkably rich in contained within a domain of just 32 amino acids whereas glutamines (34% glutamines between positions 470 and 560) in ScHSF more than 180 amino acids are necessary. Within contains no activation potential in the yeast system. the latter region, however, resides an autonomously active Furthermore, the K1HSF activator is only slightly acidic domain, probably of similar size to that in K1HSF but full having a calculated net charge of -5 and may therefore be activity of the ScHSF activator requires support from a C- atypical for yeast activators in which acidity is a terminal element which does not possess transactivator characteristic, though probably not essential, feature (Giniger function of its own. and Ptashne, 1987; Hope et al., 1988; Ruden et al., 1991;

5015 Y.Chen et al.

Leuther et al., 1993). In an attempt to clarify the importance The C-terminal activator domain is required for the of the negative charge for this activator we introduced normal heat shock response and growth at high mutations which alter the charge to +3; this causes an temperature - 6-fold reduction, although not a complete loss, of activity. Through introduction of deletions or mutations in the Thus, acidity may be important for the KIHSF activator but activator domain of KIHSF, i.e. residues 592-623, we have is not essential. compared the effects on transactivation by the KIHSF Although in ScHSF the confines of the activator domain C-terminus when carried in its normal context or when fused were not determined so precisely, a scanning of this sequence with lex (see Figure 6). In all cases mutations that remove similarly shows no over-representation of proline, glutamine most or all activity, as measured from lex fusions, reduce or acidic residues. The mutations we have tested in the the heat shock response 3- to 4-fold and render the host strain KIHSF activator (Figure 5) show that the requirements for temperature sensitive for growth over 34°C. As these effects primary sequence are probably quite non-stringent but a are identical to those observed when deleting the entire careful examination of the importance of individual residues C-terminus, i.e. 470-677, we conclude that the activity in the activator domains will have to be performed to domain which has been identified is the domain which is elucidate this further. involved in mediating HSF activity upon heat shock as well as being necessary for growth at high physiological temperatures. Organization of the activator domain in a helical structure The C-terminal activator domain is independently Despite the difference in organization of the activators, that regulated for transient and sustained activities of KIHSF being short and contained within a single region In general there is a strict correlation between the effect a whilst that of ScHSF is much more extended and split into mutation in the activator domain has on transactivation by two cooperatively functioning parts, an alignment the lex fusion, K1HSF activity and the ability of the host nevertheless demonstrates that significant conservation may strain to grow at high temperature. One mutation, however, exist. Two arrays of heptad repeats of hydrophobic residues deletion of residues 609-612, shows a quite distinct overlap the KIHSF activator domain as well as the behaviour from this pattern. This deletion reduces the activity autonomously functioning part of the ScHSF activator and of the lex-CTA fusion <2-fold, but in K1HSF virtually the shorter downstream element which does not mediate abolishes the heat shock reponse. In this case, however, loss activity by itself. Furthermore, all three regions have high ofheat shock activity does not lead to a temperature sensitive scores for ce-helix potential and they may thus form helical phenotype. This result demonstrates that sensitivity to growth structures with a hydrophobic surface similar to leucine temperature is independent of the heat shock response and zippers. therefore, as suggested by Sorger (1990), that the C-terminal It has been suggested that the function of some activators activator must be controlled by two separate and may be associated with the ability to form an amphipathic independently regulated responses for heat shock and oa-helix. Giniger and Ptashne (1987) showed that an artificial sustained activation, only the latter of which is required for 15 amino acid peptide is an efficient activator when ordered growth at high temperature. It will be interesting in the future so that it can form an c-helix but not when the same amino to define which of the controlling elements in HSF are acids were ordered so that a helical structure could not be involved with heat shock and sustained activity regulation. formed. In the case of the yeast HSF activator domains, however, several results indicate that the ability to form an A coiled-coi contact surface overlapping the a-helical structure is not necessary for their transactivating activators function. First, the activators are located in a slightly Although the possible ability to form an amphipathic a-helix staggered position relative to the potential helix-forming appears to be unimportant for the transactivating function domain and the N-terminal part of this can be deleted without of the CTAs an alternative possibility would be a structural loss of function (see Figure 4). Second, insertion of helix role as a contact surface in intra- or intermolecular structures breaking mutations within the part of the helix which in the HSF trimer. Several considerations point to the C- overlaps the activator domain have no significant effects on terminal leucine zipper motifs in the yeast HSFs being of transactivator potential. An alternative interpretation of these significance. First, while leucine zipper candidates with some results is that the activator domain has a bipartite structure homology have been identified in the regions overlapping with a requirement for a short N-terminal helical region the activator domains of the yeast HSFs no such candidates followed by a non-helical element. This would account for can be identified in other parts of either CTA. Secondly, the ability of the helix C region of ScHSF to substitute the existence of similar leucine zippers with two hydrophobic functionally when inserted in conjunction with the C-terminal 'spokes' in the N-terminal part of HSF suggests that a general part of the KIHSF activator. structural design may exist in several regions of HSF; the Finally, neither of the activator domains have high scores significance of this is further supported by analogy to higher for forming $-sheets as has recently been suggested to be eukaryotes where the presence of a C-terminal leucine zipper a functional basis for the GCN4 and GAL4 activators is required for maintaining the non-DNA binding state at (Van Hoy et al., 1993). Thus, the HSF CTAs may represent control temperatures. Thirdly, the CTAs of K1HSF and a new class of activators in yeast but more analyses will be ScHSF can functionally substitute for each other yet HSF required to see which characteristics constitute the basis of is incapable of utilizing activity from the heterologous GCN4 their transactivating function. activator (Jakobsen and Pelham, 1991) or of regulating the

5016 Activator domain in yeast HSF

GAL4 activator in fusion proteins in which these have been this with interaction experiments (Figure 7A) which show exchanged with the CTA (Figure 6). that zipper A oligomerization possibly can be stabilized by Despite these indications it has not been possible here to contact between zipper B and the CTA. Considering the establish any regulatory effect of disrupting zipper C. restraining effect of zipper B on activity it seems likely that Mutation of zipper B, which according to size would be a this contact is mediated with the activator region and that suitable partner for zipper C, deregulates HSF whilst it may also be formed in the HSF subunit. mutation of zipper A does not (Figure 5). However, the Finally it is important to notice that mutation of zipper results presented in Figure 6, concerning mutations that B makes HSF constitutively active whether or not the CTA disrupt zipper C without affecting activation function, show is present and therefore zipper B presumably restrains the that disruption of zipper C cannot achieve the same effect. activity of both the C- and N-terminal activator domains. Therefore, a possible B - C contact cannot be the means by As both activator domains can supply a heat shock response, which yeast HSF restrains activity of the CTA. zipper B seems a likely candidate for a domain in HSF which is involved in triggering the heat shock response. In contrast, Zipper C as an oligomerization surface the short conserved element located between zipper B and An obvious structural role for the C-terminal helix would the C-terminal activator domain, in which mutations can also be to mediate protein -protein contacts between subunits of deregulate HSF (Jakobsen and Pelham, 1991), is more likely HSF. The results presented in Figure 6B suggest that a to be involved in regulation of sustained activity as mutations potential for mediating oligomerization may indeed exist in in this only affect the CTA. the zipper C region but surprisingly the contact does not appear to be hindered by mutations that disrupt helical structure. Though zipper C oligomerization could not be Materials and methods verified by cross-linking experiments, it cannot be ruled out Plasmids that it constitutes an efficient contact surface between the Deletion fragments of the parts of the K1HSF and ScHSF genes encoding the C-termini were generated with exonuclease IH and mung bean nuclease HSF subunits which have already trimerized with the zippers in plasmids containing linkers with termination codons in three reading in the N-terminus. Cooperative trimerization has previously frames. The truncated fragments were then inserted in the SalI and NotI been observed in the reovirus cell attachment protein where sites of the CEN3/URA plasmid pYCA19N so that the HSF fragments were trimerization by an N-terminally located leucine zipper is encoded C-terminally of lex1_87. pYCA19N is derived from plasmid a prerequisite for trimerization by a C-terminal zipper (Leone YCP88-lexA-gcn4-A19 (see Hope et al., 1988) by insertion of a Notl linker in the unique EcoRI site. Fusion proteins involving helices A and B were et al., 1992). Considering the low conservation of helix C, constructed with PCR-generated fragments encoding these regions of the and that the trimer region (helix A +B) forms a stable trimer respective HSF proteins. The PCR linkers contains Sall and NotI sites N- without helix C, it seems likely that helix C in this case must and C-terminally respectively, of the HSF fragment, which allows in frame play a subsidiary role for the stability of the HSF complex. fusion to lexl-87 in pYCA 19N as well as to the helix C-activator construct. The helix C-activator expression plasmid was constructed by Alternatively, interactions in the C-terminus could be of insertion of a DNA fragment coding for the K1HSF C-terminus behind the a nature that do not favour any particular oligomerization triose phosphate isomerase promoter in plasmid pBY102. This is derived state. A possible role for such a contact potential could be from the CEN6-ARSH4/HIS3 plasmid pRS313 (Sikorski and Hieter, 1989> to allow higher order interactions between HSF trimers by insertion of a PstI-EcoRI TPI fragment into the EcoRV-EcoRI sites forming the hexameric or even larger complexes which have and by insertion of a synthetic linker, containing a start codon and Sall, MluI and BamHI sites, into the EcoRI and NotI sites. been observed experimentally (Sorger and Nelson, 1989). Plasmids carrying HSF mutant genes were constructed by site-directed The ability to establish protein -protein contacts through mutagenesis and the resulting C-termini were fused to eXI-87as with wild- activator domains is not without precedent; in Spl, one type HSF. activator domain mediates multimer formation required for Strains so-called superactivation (Pascal and Tjian, 1991). In HSF, CEN6-ARSH4/HIS3 plasmids encoding mutant K1HSF or ScHSF proteins the ability to form large complexes from trimers would likely expressed from the ScHSF promoter were transformed into a haploid strain play a role in assisting the highly cooperative binding which derived from W303 and carrying a HSF: :TRPI disruption and a wild-type is observed to DNA sites with more than three -GAA- ScHSF gene on the URA3 expressing plasmid YCp5O (see Sorger and elements (Xiao et al., 1991). Pelham, 1988). Cells were then cleared from the presence of the wild-type HSF plasmid by selecting against URA3 expression on plates containing 1 mg/ml 5-fluoro-orotic acid, FOA (Boeke et al., 1984). For gel retardation Repression of activity by zipper B experiments, a protease-deficient haploid strain, BJ5462 (ura3-52, trpl, In yeast HSF, trimerization is constitutive but integrity of leu2AJ, his3A200, pep4::HIS3), was transformed with a URA3 plasmid the trimer region is required for restraining activity. We have expressing wild-type K1HSF and the region encoding residues 41-657 of the endogenous HSF gene was replaced by a fragment encoding the TRP1 here examined this control more closely: whilst the long gene. This strain was co-transformed with CEN6-ARSH4/HIS3 plasmids zipper A is dispensable for correct regulation the shorter expressing mutant KIHSF proteins and plasmid pRS315 which carries the zipper B is essential for it (Figure 6). This is a spectacular selective marker LEU2. The URA3 plasmid encoding wild-type KIHSF result because it demonstrates that the restraining effect of was then eliminated by counterselection with FOA as just described. The the trimer region can be attributed to a particular region and co-transformed HIS3 plasmids expressing mutant K[HSF proteins were quite stable in the cells even without selection; no transformation colonies were does not require maintenance of global HSF structure; tested that did not contain the unselected HIS3 plasmid. overall structure is clearly seriously disrupted by deletion of zipper A since this has a dramatic effect on DNA binding Activity assays ability in vitro (HFAA, Figure 6). Strains cured on FOA to contain only one plasmid-borne HSF gene were transformed with an indicator plasmid canrying a HSF binding site upstream Thus the restraining effect of zipper B could be exercised of a CYCI-TATA box-lacZ fusion gene (Sorger, 1990). For assay of HSF through direct contacts with other parts of HSF, most likely mutants, individual colonies were grown in medium lacking uracil to select the activator domains. We have taken initial steps to clarify for the indicator plasmid; because HSF is essential for cell viability it was

5017 Y.Chen et al. not necessary to select for the plasmid expressing HSF. For assay of lex Mermod,N., O'Neill,E.A., Kelly,T.J. and Tjian,R. (1989) Cell, 58, fusions cells were grown in medium lacking uracil and leucine, and for 741 -753. interaction assays in medium lacking uracil, leucine and histidine to select Nakai,A. and Morimoto,R.I. (1993) Mol. Cell. Biol., 13, 1983-1997. for the respective plasmid markers used in these experiments. Nicosia,A., Monaci,P., Tomei,L., De Francesco,R., Nuzzo,M., Stunnenberg,H. and Cortese,R. (1990) Cell, 61, 1225-1236. Protein - protein interaction assay Nieto-Sotelo,J., Wiederrecht,G., Okuda,A. and Parker,C.S. (1990) Cell, Lex fusion proteins and 'free' KIHSF C-terminus were co-expressed from 62, 807-817. CEN/URA- and CEN/LEU plasmids respectively, in a strain where, to Pascal,E. and Tjian,R. (1991) Genes Dev., 5, 1646-1656. avoid interactions with HSF, the endogenous HSF gene had been disrupted Pelham,H.R.B. (1982) Cell, 30, 517-528. and replaced by expression from a CEN/HIS plasmid of a KIHSF gene Pelham,H.R.B. (1986) Cell, 46, 959-961. with deletion of residues 311-380 (the trimer-region, see Jakobsen and Pelham,H.R.B. and Bienz,M. (1982) EMBO J., 1, 1473-1477. Pelham, 1991). No difference could be observed in assays only involving Perisic,O., Xiao,H. and Lis,J.T. (1989) Cell, 59, 797-806. the C-terninus (zipper C) when performed either in a strain expressing wild- Pu,W.T. and Struhl,K. (1991) Proc. NatlAcad. Sci. USA, 88, 6901-6905. type KIHSF or truncated K1HSF (1-470) lacking zipper C. Rabindran,S.K., Giorgi,G., Clos,J. and Wu,C. (1991) Proc. Natl Acad. Protein fragments from KIHSF fused to lex or the CTA were: 304-356 Sci. USA, 88, 6906-6910. (zipper A), 356-389 (zipper B) or 304-389 (zipper A+B). Junction and Rabindran,S.K., Haroun,R.I., Clos,J., Wisniewski,J. and Wu,C. (1993) C-terminal sequences introduced in all these lex fusions were mgrr and gr, Science, 259, 230-234. respectively, and in helix C-activator fusions mprr and gr, respectively. Ruden,D.M., Ma,J., Wood,K. and Ptashne,M. (1991) Nature, 350, 250-252. Gel retardation experiments Sarge,K.D., Zimarino,V., Holm,K., Wu,C. and Morimoto,R.I. (1991) Extracts were prepared from protease deficient strains (see Strains) expressing Genes Dev., 5, 1902-1911. only plasmid-bome HSF genes; binding reactions and non-denaturing gel Sarge,K.D., Murphy,S.P. and Morimoto,R.I. (1993) Mo. Cell. Biol., 13, electrophoresis were performed essentially as described previously (Sorger 1392-1407. et al., 1987; Sorger and Pelham, 1988). Scharf,K.-D., Rose,S., Zott,W., Sch6ff,F. and Nover,L. (1990) EMBO J., 9, 4495-4501. Schindler,U., Terzhagi,W., Beckrnann,H., Kadesch,T. and Cashmore,A.R. Acknowledgements (1992) EMBO J., 11, 1275-1289. Sikorski,R.S. and Hieter,P. (1989) Genetics, 22, 19-27. Yuqing Chen and Nickolai A.Barlev made equal contributions to the work Sorger,P.K. (1990) Cell, 62, 793-805. presented in this paper. We are grateful to Hugh Pelham for advice and Sorger,P.K. (1991) Cell, 65, 363-366. suggestions, and also to Jan Alsner, Torben Andersen, and Jesper Svejstrup. Sorger,P.K. and Pelham,H.R.B. (1988) Cell, 54, 855-864. We would also like to thank Anette H0j for letting us use the protease Sorger,P.K. and Nelson,H.C.M. (1989) Cell, 59, 807-813. deficient HSF deletion strain, Kirsten Andersen for technical assistance, Sorger,P.K., Lewis,M.J. and Pelham,H.R.B. (1987) Nature, 329, 81-84. and Hugh Pelham, Peter Hjorth, Jesper Svejstrup and Martyn Bell for Tzamarias,D., Pu,W.T. and Struhl,K. (1992) Proc. NatlAcad. Sci. USA, comments on the manuscript. Y.C. and N.A.B. received one year stipends 89, 2007-2011. for visiting PhD students from the Danish Research Academy. B.K.J. is Van Hoy,M., Leuther,K.K., Kodadek,T. and Johnston,S.A. 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