MOLECULAR AND CELLULAR BIOLOGY, July 1991, p. 3504-3514 0270-7306/91/073504-11$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 11, No. 7

Modular Recognition of 5-Base-Pair DNA Sequence Motifs by Human Heat Shock Transcription Factor NINA F. A. CUNNIFF, JOHN WAGNER, AND WILLIAM D. MORGAN* Department of Biology and Centre for Human Genetics, McGill University, Montreal, Quebec H3A IBI, Canada

Received 14 February 1991/Accepted 8 April 1991

We investigated the recognition of the conserved 5-bp repeated motif NGAAN, which occurs in heat shock promoters of Drosophila melanogaster and other eukaryotic organisms, by human heat shock transcription factor (HSF). Extended heat shock element mutants of the human HSP70 gene promoter, containing additional NGAAN blocks flanking the original element, showed significantly higher affinity than the wild-type promoter element for human HSF in vitro. Protein-DNA contact positions were identified by hydroxyl radical protection, diethyl pyrocarbonate interference, and DNase I footprinting. New contacts in the mutant HSE constructs corresponded to the locations of additional NGAAN motifs. The pattern of binding indicated the occurrence of multiple DNA binding modes for HSF with the various constructs and was consistent with an oligomeric, possibly trimeric, structure of the protein. In contrast to the improved binding, the extended heat shock element mutant constructs did not exhibit dramatically increased heat-inducible transcription in transient expression assays with HeLa cells.

gene

Heat shock genes provide a valuable model for studying inducible gene expression. They represent a group of several highly conserved multigene families that occur throughout the eukaryotic kingdom and provide opportunities for studies of gene regulation in diverse systems, including plants, insects, fungi, and mammals (5, 24). Exposure of a cell or organism to high temperature or other physiological stresses causes a rapid, strong increase in the synthesis of proteins encoded by these gene families. This involves several levels of regulation in various species, including mRNA translation and stability in some cases, but a significant increase in the rate of transcription initiation is the most common aspect of the response. Molecular genetic analysis has shown that the cis-acting DNA sequence responsible for the heat inducibility of these promoters, the heat shock element (HSE), is also highly conserved among eukaryotes. Heterologous promoters or their HSEs in promoter fusion constructs are generally able to mediate the appropriate stress response when transfected into cells from other species. A specific transcriptional activator protein, heat shock transcription factor (HSF, also referred to as HSTF or HAP), interacts with the HSE DNA sequence. The structure and function of HSF is being characterized in organisms such as yeasts, Drosophila melanogaster, and mammals (6, 12, 21, 34, 25, 41, 42, 44, 54, 58, 60, 61). This represents an excellent opportunity to study the evolution of a specific transcriptional regulatory protein whose physiological function has been conserved throughout the eukaryotic kingdom. At the same time, specific mechanistic differences in transcriptional regulation among these frequently studied organisms can also be identified. In the work presented here, we examined the structural and functional requirements for the HSE in a well-characterized human heat shock gene, HSP70. This gene, which has been mapped to the HLA III region of human chromosome 6 (11, 17, 39, 40), encodes a heat-inducible and also growth-regulated copy of the major 70-kDa heat shock protein. This gene is designated HSP70 (24) and is also referred to as hsp70-1 and hsx70 elsewhere. This promoter *

contains a single perfect Pelham consensus sequence centered 100 bp upstream of the transcription start site and a second, nonessential partial match further upstream at -180. The element in the -100 region is both necessary and sufficient for heat shock induction (57). Interactions of several constitutive transcription factors have been identified with multiple sites in both the essential proximal promoter domain downstream of -74 and in the nonessential upstream region, and these are summarized in the promoter map of Fig. 1. Using this HSE in its natural promoter context, we performed oligonucleotide-directed mutagenesis to create extended blocks of NGAAN and NTTCN both upstream and downstream of the original element. We first compared the efficiency of these constructs relative to the wild-type HSP70 promoter in binding human HSF in vitro. We also pursued a detailed investigation of protein-DNA contacts with partially purified HeLa HSF. This was directed toward an investigation of the molecular topography of interactions between human HSF and both the wild-type promoter and the mutant constructs. High-resolution hydroxyl radical protection and diethyl pyrocarbonate (DEPC) interference techniques were used in combination with DNase I footprinting. We then analyzed their in vivo heat-inducible transcriptional activity in HeLa cell transient expression assays. The increased binding affinity observed with our series of mutants confirms that the repeated NGAAN motif proposed by Xiao and Lis (59) and Amin et al. (2) is a valid description of the recognition sequence for mammalian systems as well as for D. melanogaster. High-resolution studies of DNA-protein contacts demonstrated the modular nature of interactions between human HSF and the repeated 5-bp motifs within the HSE and contributed toward an understanding of the basis for increased binding affinity of the mutant constructs. This modular organization, as well as the detailed contacts within each 5-bp motif, further illustrate the high degree of conservation of the DNA binding function within this transcriptional regulatory system among a wide variety of eukaryotic species. Analysis of different binding modes for HSF with the extended HSE constructs also gave information on the possible oligomeric structure of the human protein. How-

Corresponding author. 3504

HUMAN HEAT SHOCK FACTOR-DNA INTERACTIONS

VOL. 11, 1991 SPI -241

AP-2

-210

HSF

SPI

CTF

AP-2

HSF

-190

-167

-147

-130

-100

-85

HEAT SHOCK INDUCTION

0-0*

AP-2

CTF

SPI ATF

-65

-46 -37 -28 -16

3505

AP-2

+1

+16

BASAL ACTIVITY

N

-100

I

WID

-----G-

TYPE

BSE 114/97

Galrn

tCAG ;GAaCTttCAG FIG. 1. Summary of transcription factor binding sites on the human HSP70 promoter. Each solid box represents a specific binding site identified by DNase I protection with fractionated HeLa nuclear extracts and in some cases with affinity-purified proteins (CTF, Spl, AP-2) (30, 31, 33, 34). These sites were mapped with probes extending from the NcoI site at approximately -270 to the BamHI site at + 150. Some USE

104/87

USZ

114/87

__

ttczc

of these sites have also been observed by exonuclease III protection, electrophoretic mobility shift assays, and DMS interference (21, 35, 57). The distal half of the previously observed footprint from -110 to -78 in fractions of non-heat-shocked HeLa extracts over the HSE consensus (34) appears to correspond to HSF from heat-shocked nuclei. The proximal half of that footprint appears to result from a distinct protein, present in non-heat-shocked cells, that binds to a sequence at -85 (31). This protein has not yet been identified but appears not to be essential for expression in HeLa cells. CTF refers to the CTF/NF-1 protein described previously (34); the unrelated CCAAT box binding protein CP-1 has also been suggested to bind to the HSP70 CCAAT box region (46), and a recent report has also described a novel protein that interacts with this and other CCAAT sequences (28). Domains for in vivo basal and heat-inducible expression, indicated by bars below the map, were previously determined (55-57). In vitro basal expression depends mainly on the proximal CCAAT box and Spl binding sites (33, 34). The four AP-2 binding sites appear not to be essential for expression in HeLa cells (32, 46). Binding of TFIID to the HSP70 TATA box region has also been reported (open box) (36). Potential interaction of ATF with the sequence at -37 (open box) has also been suggested (19), but the significance of this possibility is not yet clear. The sequence of wild-type and mutant HSP70 promoter HSEs from -117 to -85 is presented beneath the map. Mutant constructs are described in detail in the text. Base substitutions are indicated by lowercase letters. ever, the increased in vitro binding affinity with extended NGAAN-block arrays is not necessarily matched by a proportional improvement in efficiency of heat shock-inducible transcription in vivo.

MATERIALS AND METHODS Construction of promoter mutants. Plasmids A-133 and AS-8 containing the human HSP70 promoter sequence from nucleotides -133 to +150 fused to the bacterial chloramphenicol acetyltransferase (CAT) gene were described previously (34). The pseudo-wild-type control template AS-8 is identical to A-133 except for a 9-base deletion within the CAT untranslated leader sequence. The 285-bp HindlIlSmaI fragment from A-133 containing the HSP70 promoter sequence was subcloned into pUC118. This construct, HS133, was used for the preparation of a single-stranded uracil-containing template in Escherichia coli CJ236 (dut ung) as described previously (22, 51, 62). Oligonucleotidedirected mutagenesis was performed as described by Zoller and Smith (62) to produce the extended HSE mutants shown in Fig. 1. Additional mutants not shown in the figure were produced by similar methods. Two mutants without functional HSEs were created by multiple substitutions that eliminated the entire 8-bp Pelham consensus at -100, resulting in the following sequences (mutations indicated by lowercase): -107/tTGaggTAcctCCa/-94 and -107/gTGctt TAaagCCc/-94. Two linker-scanning-type substitutions flanking the HSE created the following sequences: -116/ atct/-113 and -91/agatCt/-86. The mutant promoter sequences were subsequently cloned back into A-133 for expression assays. All mutations were confirmed by dideoxy

sequencing. Plasmid DNA from E. coli NK5772 (deficient in DNA cytosine methylase activity [dcm]) was prepared by alkaline hydrolysis as described by Maniatis et al. (29) and purified twice by CsCl gradient centrifugation. Restriction fragment binding probes used in these experiments were derived from the pHS133 and pHE series of plasmids. Plasmid pHE is a subclone of pHS133 in which the Sacl-Sacl fragment from -72 of the promoter to the Sacl site of the polylinker has been deleted. Probes for DEPC interference and hydroxyl radical protection analysis were prepared as follows. pHS133 cut with Hindlll (for topstrand-labeled probes) or pHE cut with EcoRI (for bottomstrand-labeled probes) was dephosphorylated, end labeled with T4 polynucleotide kinase and [.y-32P]ATP, cut with a second enzyme, and gel purified. The resulting series of probes denoted pHS133 HindIII* Sacl and pHE EcoRI* Hindlll (where * denotes the site of the label) were 63 and 67 bp long, respectively. Probes for DNase I analysis (pHS133 BamHI* EcoRI series) were prepared in a similar manner except that gel purification was omitted. Preparation of HeLa HSF. HeLa S3 cells were grown in suspension in Joklik modified minimal essential medium supplemented with 5% calf serum, 100 units of penicillin G per ml, and 100 ,ug of streptomycin sulfate per ml at a density of 5 x 105 cells per ml. Nuclear extract was prepared essentially as described previously (8, 9). Harvested cells were heat shocked at 43°C for 20 min while cells were swelling in 4 packed cell volumes of hypotonic buffer consisting of 10 mM Tris-HCI (pH 7.9), 10 mM KCI, 1.5 mM MgCl2, and 1 mM dithiothreitol just prior to homogenization. In some cases, nuclear pellets were frozen in liquid nitrogen and stored at -80°C before extraction. The ammonium

3506

CUNNIFF ET AL.

sulfate pellet was resuspended in 0.1 volume of high-speed supernatant volume in TM-0. 1 M KCl (TM is 50 mM Tris-HCl [pH 7.9], 1 mM EDTA, 12.5 mM MgCl2, 1 mM dithiothreitol, 20% [vol/vol] glycerol) and applied to a 650-ml Sephacryl S300 column equilibrated in the same buffer. Chromatography conditions were similar to those described previously (34), except that HSF fractions were detected by band shift assays. The HSF pool from the S300 column was diluted with an equal volume of TM buffer to a final concentration of 0.05 M KCl and applied to a 10-ml heparin agarose column. The column was eluted with a step gradient of TM buffer containing 0.1, 0.2, 0.4, and 1.0 M KCl. HSF activity eluted in the 0.2 M KCl fraction, and this was dialyzed against either TM-0.1 M KCl or a buffer without glycerol (50 mM Tris-HCl [pH 7.9], 1 mM EDTA, 1 mM dithiothreitol, 0.1 M KCl, 0.05% Nonidet P-40). Fractions in glycerol-free buffer were used for hydroxyl radical protection experiments. All fractions were aliquoted, frozen in liquid nitrogen, and stored at -80°C until further use. Band shift assays. Band shift assays with nonradioactive competitor plasmids bearing HSE sequences were performed as described previously (10, 50), with modifications. Buffer contained 5 mM Tris-HCl [pH 7.5], 25 mM KCl, 0.5 mM dithiothreitol, 0.05 mM EDTA, and 2.5% (vol/vol) glycerol. Reactions were performed in a 10-t1l volume with 800 ng of poly(dI-dC)-poly(dI-dC), 8 to 10 fmol of a 24-bp 32P-labeled double-stranded synthetic oligonucleotide probe (with the sequence from -112 to -89 of the HSP70 promoter), various amounts of nonradioactive competitor DNA as indicated in the figure legends, and S jig of HeLa nuclear extract (added last). Reaction mixtures were incubated on ice for 30 min before being electrophoresed on a nondenaturing 4% polyacrylamide gel (79:1 acrylamide-bisacrylamide) in 10 mM Tris-acetate [pH 7.5]-1 mM EDTA. Gels were run at 400 V for 90 min with buffer recirculation. Autoradiographs were quantified by densitometry. Similar results were obtained by prior addition of either probe or competitor, indicating that equilibrium conditions were established during the competition. Multicomponent band shift assays were performed as above, with the following modifications. One labeled probe consisted of a 24-bp synthetic oligonucleotide (HSE24) with the human HSP70 promoter sequence from -112 to -89. The second labeled probe in each lane was a HindIII-SacI restriction fragment with either the wild-type or mutant HSE sequence. Both probes (approximately 105 cpm of each) were incubated for 30 min on ice with 25 to 30 ,u1 of an HSF heparin agarose fraction (0.45 mg of total protein per ml) and 2.4 ,ug of poly(dI-dC)-poly(dI-dC) and then electrophoresed as described above. The protein-bound and free probe bands were electroeleuted onto an NA-45 membrane (Schleicher & Schuell), separated on denaturing polyacrylamide gels, autoradiographed, and quantified by laser densitometry. The relative distribution on each probe between free and bound states was used to determine the ratio of their equilibrium binding constants for HSF (25, 27) according to the following relation: K1/K2 = ([C1]/[D1])/([C2]/[D2]), where [CI] is the concentration of probe n in the bound complex, and [Dn] is the concentration of free probe n. To confirm that these reactions reached equilibrium, the HSF fraction was first incubated with one probe for 15 min; this was followed by the addition of the second probe and continued incubation for 15 min, before processing as described above. Order of addition had no effect on the relative distribution of free and bound probes. Hydroxyl radical protection. Hydroxyl radical footprinting

MOL. CELL. BIOL.

of gel-separated complexes was performed with solutions and concentrations as previously described (48, 49). The following components were assembled in a 1.5-ml microtube on ice: 300,000 cpm of 5'-end-labeled probe in TE (20 pul) (10 mM Tris-HCl [pH 7.5], 1 mM EDTA), 2.0 RI of poly(dI-dC)poly(dI-dC) at 10 A260 units/ml, and 25 pul of an HSF heparin agarose fraction (0.45 mg of total protein per ml), as described above, in a buffer containing no glycerol. Mixtures were allowed to incubate for 15 min and then were preincubated for 2 min at room temperature before the addition of hydroxyl radical-generating reagents. The composition of each aqueous solution was as follows: iron(II)-EDTA solution (made fresh by mixing equal volumes of 0.4 mM ferrous ammonium sulfate and 0.8 mM EDTA), sodium ascorbate (20 mM), and hydrogen peroxide (0.6% [vol/vol]). These reagents were obtained from Aldrich and prepared fresh before use. A 3-pI sample of each solution was added individually to the side of each tube and then combined with the protein-DNA mixture and allowed to incubate for 2 min at room temperature. The reaction was terminated with 0.1 volume of loading dye (50% [vol/vol] glycerol, 0.1% [wt/vol] xylene cyanol, 0.1% [wt/vol] bromophenol blue), and the contents were immediately electrophoresed on a native polyacrylamide gel. Subsequent DNA isolation procedures were done as described below for DEPC interference except that piperidine cleavage and its removal were omitted. DEPC interference analysis. DEPC interference experiments were performed as previously described (45) except that DEPC modification was performed after purification of probes. Gel-purified 32P-5'-end-labeled restriction fragment probes were modified as follows. Probe (300,000 cpm) in 10 pI of TE was denatured at 100°C for 3 min and then transferred to ice where 200 ,ul of DMS buffer (50 mM sodium cacodylate [pH 8.1], 1 mM EDTA) was added. DEPC (4 pA) (Sigma) was added, and the tubes were vortexed and incubated at 37°C for 20 min with a brief vortex halfway through the incubation. DNA was recovered after two successive ethanol precipitations in 0.3 M sodium acetate (pH 5.0) and a 70% ethanol wash. DNA was resuspended in 18 pA of TE with 0.1 M NaCl and renatured at room temperature for 30 min. Poly(dI-dC)-poly(dI-dC) (2 pA of 10 A260 units/ml) and 20 pA of HSF-containing heparin agarose fraction (0.45 mg of total protein per ml in TM-0. 1 M KCl) were added to the renatured probe on ice and incubated for 15 min. Loading dye (4 RI.a) (50% [vol/vol] glycerol, 0.1% [wt/vol] xylene cyanol, 0.1% [wt/vol] bromophenol blue) was added, and the mixture was electrophoresed on a 4% native polyacrylamide gel (79:1 acrylamide-N,N'-methylene bisacrylamide) as typically used for band shift assays with buffer circulation. The wet gel was autoradiographed overnight at 4°C, and bands were electroeluted onto an NA-45 membrane (10). After extraction and piperidine treatment, sample aliquots with equal Cerenkov counts were electrophoresed on 10% sequencing gels and autoradiographed with intensifying screens at -80°C. DNase I footprinting analysis. DNase I footprinting was performed essentially as previously described (9). The following components were assembled in microtubes on ice: 10 ,ul of 10% (wt/vol) polyvinyl alcohol, 0.5 fmol of 5'-endlabeled probe pHS133 BamHI* EcoRI (1.0 ,ul), 0.5 pI of poly(dI-dC)-poly(dI-dC) at 10 A260 units/ml, a total of 25 pul of a protein fraction and TM-0.1 M KCl buffer as indicated in the figure legends, and water to a final volume of 50 RI. Samples were preincubated for 15 min on ice, and DNase I reactions were performed at room temperature. Subsequent steps were performed as described previously (9).

VOL . 1 l, 1991

HUMAN HEAT SHOCK FACTOR-DNA INTERACTIONS

Transfections. HeLa cells for transfection were maintained monolayers in Dulbecco modified Eagle medium containing 5% calf serum at 37°C in 5% CO2. Dishes (100 mm) that were 50 to 60% confluent were calcium phosphate transfected with 10 ,ug of each plasmid using the Pharmacia Cellphect transfection kit according to the manufacturer's protocol. After 6 h, the medium was removed from each dish and 2 ml of 15% glycerol in isotonic HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (14) was added for 1 min. Dishes were rinsed once, and fresh medium was added. The cells were harvested 48 h after transfection, and either the cytoplasmic RNA was extracted (2) or CAT enzyme activity was assayed as described previously (13), with modifications (7). Primer extension assays with cytoplasmic RNA were performed as previously described (20, 34). HeLa cells were heat shocked at 42TC for 2 h before harvesting, as indicated.

3507

B F

as

RESULTS Band shift assays. Heat shock treatment of the HeLa cells as they were harvested strongly induced HSF binding to the HSE oligonucleotide probe. Binding affinity of wild-type and mutant HSP70 promoters for HSF in crude nuclear extracts of HeLa cells was first measured by competition in band shift assays. The properties of the observed HSF-HSE complex were in good agreement with the previous results of other groups (23, 35, 42). HSF binding to the HSE probe was readily abolished by competition with the unlabeled HSE oligonucleotide but not by a heterologous oligonucleotide (data not shown). This binding activity was also specifically abolished by the plasmid HS133 (wt) containing the wildtype HSP70 promoter sequence but not by the plasmid vector pUC118 nor by promoter mutants (sequences given in Materials and Methods) with the multiple base substitutions that eliminated all eight conserved positions of the 14-bp consensus CNNGAANNTTCNNG, as the HSE was originally described by Bienz and Pelham (5). This complex exhibited dimethyl sulfate (DMS) interference similar to that described in previous reports (23, 35) and also appears to contain an approximately 100-kDa protein by UV crosslinking (53), in agreement with other reports (12, 47). Two other kinds of complexes, which were nonspecific in one case and noninducible in the other case, were observed in addition to the heat-inducible specific HSF complex (35, 47). To determine whether the promoter mutants were able to bind to heat shock-induced HSF more effectively than the wild-type sequence, we performed competition band shift assays using HS133 plasmids containing either the wild-type or extended NGAAN-block sequences as competitor DNA (data not shown). All three mutants with extended HSEs were able to compete much more efficiently than the original element for the HSF, as determined by the relative concentration needed for 50% reduction of the bound complex signal. The relative affinity of wild-type and mutant sequences was then more precisely quantified by band shift assays with multiple probes (25, 27). In these multicomponent band shift assays, the relative distribution of one or more variant sequence probes between the free and bound states is simultaneously compared with a wild-type reference probe. The extended HSE variants, located on restriction fragments, were compared with the 24-bp oligonucleotide probe containing the wild-type sequence (Fig. 2). Since bound complexes of the restriction fragment and oligonucleotide probes were not completely resolved from each other by nondenaturing gels, the bound complexes containing both

15

HSF> %d

-_-

RF

)

>0 frag p0 1 C0

~

f (

results frommltpe probe bandshif FIG.roho2.e Sumryghtofn aeF las-B bAndsit lesheuhassayswt says.-bun Acsmplex) frcioaedrHe ferforme HwE24 oligonucaneotide probtyeiestinicatedn byagen0and the resutrito prbe fromnsioety feragmeantife 104/87esindicatedb RFereaine bohinsets The upper o ec labele complex, HE HSF2, conitsolamxureofthedtwo to HSF protein. Thesexobandswepresenute and proesnboud Mtheril eletadropoee (rvight-hand* winsertyp; laeX, frEe probe7 bands lanEB,497

protenboun complex).rAtleated toehree suchasayherespertveformed withbeach mutanateandwid-tpesaturicing foyaryagmen,adte reslts,n weequantified byatrdenitomerapy.VausidctTherelative dsrbuiond-f ingthraffntyo of eachibiu HinEin comparedtwith HtE24 calcla-tyeda in Materials and advratsqecsasdescribed Method.Teerrbr-ersn n pobes werei ecthproeiuted toethert with the.rEspectivhen ofre by atrdoaph.Terelative equanifibru aofred odisribuaddtiono

experiments (27). The results in Fig. 2 demonstrate that, in agreement with competition band shift assays, the three extended HSE mutant sequences have significantly greater affinity than the wild-type element for HSF. The six-NGAAN-block construct HSE 114/87 had the highest affinity, 12-fold higher relative to wild type, while HSE 114/97 and HSE 104/87 had intermediate levels at 6.5- and 5-fold higher, respectively. These increases reflect recognition of the new NGAAN motifs in the mutant elements, as confirmed by additional binding studies described below. The 63-bp restriction fragment containing the wild-type HSE had the same affinity as the 24-bp oligonucleotide probe, indicating that the sequence outside of -112 to -89 does not contribute to the primary HSF binding site in vitro. Hydroxyl radical protection. We investigated binding of partially purified HSF from heat-shocked HeLa nuclear extract fractions by hydroxyl radical footprinting (48, 49)

3508

CUNNIFF ET AL. PROBE HSF

MOL. CELL. BIOL. 114-97 104-87 114-87

WT -+-

I

-+E--l

1111 I

PROBE

WT 114-97104-87114-87

HSF

-85 1 15

-90-

-

.95

-110

-100

-1 05 -

-1 05

-1 00 -

-110 -

-95

-115

-90

-

--

_ - 85

-oi*

-0

._ h

-

,M

v

4UdO

56 8 B A 1 2 1 1 1 2 3 4 5 6 7 8 9 1 1 2 3 4 5 6 7 8 9 101112 FIG. 3. Hydroxyl radical protection of gel-separated HSF complexes from wild-type HSE and extended NGAAN-block mutants of the human HSP70 gene promoter. Probes are indicated by numbers which denote the 5' and 3' boundaries of NGAAN blocks upstream relative to the transcription start site. Autoradiogram represents electrophoretic separation of hydroxyl radical-generated cleavage products obtained from free probe (-) and HSF-bound complex (+) after native gel separation. The complete nucleotide sequence of this region is given in Fig. 7. (A) Analysis of top-strand protection of pHS133 HindIII* Sacl series of probes. (B) Analysis of bottom-strand protection of pHE EcoRI* HindlIl series of probes. Numbers on left show location relative to start site. 0

and other methods described below to detect protein-DNA contacts at high resolution. Both the wild-type and extended GAA-block promoter elements were examined by these methods. The hydroxyl radical technique, compared with DNase I footprinting, provides a more detailed picture of contacts because of the small size of the chemical probe and the relatively even cleavage at every base pair. This was particularly valuable in this case, since DNase I cleavage within the human HSP70 promoter HSE sequence is unusually infrequent (see below). Although some protection of the HSE was visible in direct hydroxyl radical footprinting experiments, the signal was enhanced by performing gel isolation of bound complexes versus free probe on nondenaturing gels. Comparison of the hydroxyl radical protection on both strands in bound versus free probe, for the wild type and three mutant constructs, is shown in Fig. 3. These experiments demonstrated strong protection of the perfect pair of 5-bp motifs, NGAANNTTCN, contained within the core of the wild-type HSE symmetrically on both strands. Similar strong protection of this central core sequence was also observed with the three extended GAA-block mutants. The protection characteristically reached a maximum of 85 to 95% relative protection, by densitometry, near the two thymidine residues of the TTC sequence on each strand (summarized in Fig. 7). This protection gradually decreased 5' to the TTC motif, but there was still significant protection of the two intervening base pairs and through the GAA sequence of the other half of the core. The virtually complete protection of the TTC side of the element against hydroxyl radical cleavage may indicate interaction of the protein with the minor groove of the helix at this point. This could represent surface contact, rather than base-specific recognition. Studies with a limited sample of proteins, such as prokaryotic repressors, suggest that hydroxyl radical attacks

primarily through the minor groove (48, 49). In contrast, CCAAT/enhancer-binding protein C/EBP, which is believed to interact almost exclusively with the DNA major groove, produces only a very weak protection signal against this reagent (52). The decreasing degree of protection of the GAA sequence may result because contacts here are primarily in the major groove, which is consistent with alkylation interference experiments described below, although intrinsically weaker binding to the minor groove here cannot be ruled out. The extended GAA-block mutants showed similar protection of the original HSE core centered at -100, but there were dramatic differences outside this region, at the locations of specific point mutations introduced in these constructs. Each of the extended four-GAA-block mutants exhibited an additional strongly protected site corresponding to one of the perfect TTC sequences created by site-specific mutagenesis (Fig. 3 and see Fig. 7). The additional protection was upstream for HSE 114/97 and downstream for HSE 104/87. There was a new peak region of hydroxyl radical protection on the strand containing the TTC motif, and in addition, there was also somewhat increased protection extending in the same direction on the opposite strand, although as a shoulder rather than as a separate peak. In these mutants, each with four contiguous GAA blocks, the inner new block, adjacent to the original core, was completely resistant to hydroxyl radical cleavage, while the outer block was not strongly protected. The presence of new contacts to an artificial NGAAN consensus motif in each of these mutants is consistent with the observed increase in binding affinity for HSF, detected by band shift assays (Fig. 2). This reinforces the idea that this motif represents a valid description of the modular recognition element for human HSF, as well as for other species. Hydroxyl radical footprinting of the extended six-GAA-

HUMAN HEAT SHOCK FACTOR-DNA INTERACTIONS

VOL . 1 l, 1991

A

r-.om" I

-

PROBE

v. 0

o o* _

%.

0t

o

3

PROBE

%.

Ct

Rt

I

_

FC FC FC FC

FREE/COMPLEX

%.

B

.

CT

3509

-

_

FC FC FC FC

FREE/COMPLEX

nnninir

-85 -89 -94

-

-100

-

-115 -110 -106

-

A

i. I

-

_

_

_

_~~~~t

-101

-105 -r -95 0-

a

I-

-1 14

-

*--

ma _mOm_g _

(a

ft

-90 -87 _

m

.7N

w _

-_ -t '14

1 2 34567 8

1 2 34 5678

FIG. 4. DEPC interference analysis of wild type (WT) and extended NGAAN-block mutant series of the human HSP70 gene promoter. Probes are indicated by numbers which denote the 5' and 3' boundaries of NGAAN blocks upstream relative to the transcription start site. Autoradiogram represents electrophoretic fractionation of piperidine cleavage products of probes modified by DEPC and then subsequently separated into free probe (F) and HSF-bound complex (C). Darker bands (within a lane) represent A residues, and lighter bands represent G residues. (A) Analysis of top-strand residues from pHS133 HindIII* Sacl series of probes. (B) Analysis of bottom-strand residues from pHE EcoRI* HindIll series of probes. Numbers on left show location relative to start site.

block construct HSE 114/87 showed a very interesting pattern (Fig. 3 and see Fig. 7). There was a clear increase in protection, relative to the wild type, of each mutant TTC sequence adjacent to the original pair within the core on both sides (i.e., of the second of six blocks [-109 to -107] and the fifth block [-94 to -92]). The maximum protection level for the second and fifth blocks in HSE 114-87 (45 to 70%o), however, was significantly less than the level seen at these positions in the four-NGAAN-block mutants (85 to 95%). This is rather surprising, since with the higher binding affinity of HSE 114/87, correspondingly stronger protection might be expected. However, this intermediate degree of protection can be explained by alternative modes of binding of HSF to the HSE 114/87 element at either of two different overlapping positions. Each of these forms would have three contact regions, consisting of two with the original pair of TTC motifs and an additional flanking one either upstream or downstream. Thus, the average signal of the two forms would show strong protection of the core and intermediate protection on both sides. Protection of the upstream motif of HSE 114/87 on the top strand is more clear than protection of the downstream motif on the bottom strand, and this binding mode may therefore be preferred to some extent. These distinct hydroxyl radical protection patterns were obtained with isolated complexes that migrated identically on preparative nondenaturing gels and presumably were of the same stoichiometry. This suggests that an HSF molecule of a particular oligomeric form is capable of binding with dif-

ferent contact patterns, depending on the number of NGAAN motifs that are present on the template. DEPC interference. Since DEPC is reactive both at adenine residues and, to a lesser extent, at guanine residues, contacts with both types within the GAA sequence (and at flanking positions) of the HSE motif could be determined. This reagent is therefore useful as a supplement or replacement for DMS modification experiments at G residues, especially for the adenine-rich HSE motif. DEPC interference was monitored by comparison of DEPC-modified probe from bound complexes with unbound probe. The complexes were isolated from nondenaturing gels similar to those used for hydroxyl radical footprinting. Interference data for the wild type and three extended GAA-block mutants are shown in Fig. 4 and summarized in the diagram shown in Fig. 7. The wild-type HSE was subject to strong interference by DEPC modification at residues in the GAA sequence of the core on each strand, as well as to some flanking adenine and guanine residues within 1 to 2 bp on either side. Since carbethoxylation of adenine and guanine residues occurs at the N-7 position (18), these data may indicate contacts to the NGAAN motifs through the major groove. This includes strong DEPC interference with the pair of G residues located at the first two positions of each 5-bp motif in the wild-type HSE, where DMS interference is also seen, suggesting major groove contact (21, 35, 53). There was very weak interference by modification of the flanking guanine residue at -94, which possibly indicates an additional protein contact. The

CUNNIFF ET AL.

3510

o

c0

uL

_.;

MOL. CELL. BIOL.

0o

Ln

T-

N

i- r-

HSF (

0

Q.

EP!Is

ew

r-

01) co co

)

LII

2

o

-

LU LU LU 0 n n u)

m3Z"l

*b' S _ m* t 1001

.j G=Template 0*-_ Internal control

173 207 111

FIG. 6. Primer extension analysis of RNA levels. Each dish of HeLa cells was transfected with 10 tg of the pseudo-wild-type plasmid, AS-8, and 10 jig of either the wild-type or mutant HSP70CAT plasmids as indicated above each lane. Cytoplasmic RNA was harvested after the cells had been heat shocked for 2 h at 42°C. In each lane, the activity of the mutant promoter relative to that of the pseudo-wild-type promoter (AS-8, set at 100%) is reported below the gel, as determined by the average of at least six assays. One standard deviation for HSE 114/97, HSE 114/87, and HSE 104/87 was 55, 52, and 54%, respectively. 3

2

1

4

5

6

footprinting of HSE 114/87.

FIG. 5. DNase I

The HSE 114/87

probe was eId labeled on the bottom strand at the BamHl site (+ 150). Footprinting reactions were performed as described in

increasing amounts of HSF heparin agarose fraction (in microliters) as indicated above the gel. Rectangles on the left and right sides indicate protected regions at low and high protein concentration, respectively. Materials and Methods with

various

mutant

exhibited

constructs

interference within the

core

similar pattern

a

sequence, from -104 to

of

-97,

although with somewhat less intensity at some residues there compared with HSE 104/87

in HSE 114/87 and HSE 114/97 and

wild

type.

Additional

protection

also

NGAAN motifs in the extended

new

The presence of residues

sensitive

to

occurred

at

the

mutants.

consensus

DEPC interference

within the extended GAA-block sequences of HSE 114/97 and HSE 104/87 is consistent with hydroxyl radical protection results with the constructs. HSE 114/87 again subject to interference resembling that with both HSE 114/97 and HSE 104/87 the upstream and downstream sides, was

same

on

respectively,

less

but with

intensity.

This

suggests that with

this extended six-GAA-block site, modification at position within the element does not completely inhibit binding, which may reflect the alternative binding modes observed in hydroxyl radical protection experiments. DNase I footprinting. While DNase I endonuclease protection has much lower resolution than the hydroxyl radical one

technique, due

infrequency of

to the

DNase I

cleavage-

sites

within the HSE, it was possible to obtain clear DNase I protection in direct assays without isolating bound plexes gels. The improved protection observed with com-

on

DNase

I

the

with hydroxyl radical appears First, in the protocols

compared

from two factors. the assays (9, 49), DNase

standard

the

I method.

to result

used

for

protein concentration was greater for Second, use of the hydroxyl radical

method necessitated removal of interfering glycerol (a free

radical

scavenger)

dialysis, resulting

some

The six-GAA-block very

protein fractions by extensive activity. construct HSE 114/87 exhibited a in DNase I experiments.

from the in

interesting patter

loss of

footprinting

observed in titration experiments with increasing HSF fraction concentration (Fig. 5). The HSE smaller 20-bp footprint region at 114/87 template showed lower protein concentration. Similar footprints of approxi-

This was

most

clearly

a

mately 20 bp in this region were also observed with HSE 114/97, HSE 104/87, and wild-type probes (data not shown; see Fig. 7 for summary). At a higher protein concentration with HSE 114/87, there was an extended footprint extending almost as far as -80 in the downstream direction, with partial protection against cleavage at the position near - 120. This 35- to 40-bp footprint appeared to be approximately twice the size of the one observed under more dilute conditions and may therefore result from a different binding mode. The shorter form of the DNase I footprint with HSE 114/87 probably corresponds to the complexes analyzed by hydroxyl radical and DEPC experiments, since the latter were also performed under more dilute conditions. Since the transition between the two DNase I footprint patterns depends on protein concentration, the extended 35-bp form probably represents a higher stoichiometry. In vivo transcription activity. Both CAT assays and primer extension analysis (data not shown) were used to study basal transcription activity. It had previously been shown that the domain containing the HSE could be deleted without reducing basal levels of transcription in HeLa cells (16, 57). None of the three mutants with extended blocks of the NGAAN element had significantly altered basal levels of transcription. Primer extension assays were used to analyze cytoplasmic RNA from heat-shocked HeLa cells (Fig. 6). Both HSE 104/87 and HSE 114/97 contain four contiguous NGAAN elements in alternating orientation, yet HSE 104/87 did not appear significantly more active than wild type, while HSE 114/97 showed a slight increase. The double mutant HSE 114/87, with six contiguous NGAAN blocks, was clearly somewhat more active than wild type, with 20% relative activity, although this improvement was not directly proportional to its 12-fold-higher binding affinity. While this report was in preparation, an independent study examined transcriptional activation with an extended six-GAA-block construct similar to HSE 114/87 (15), although binding affinity was not investigated there. In that study, no improvement in heat-inducible transcription was seen with the extended HSE at the normal -100 location. The small discrepancy between this result and the twofold improvement in the data presented here may be due to slight differences in experimental protocols, such as heat shock conditions (42°C for 2 h here, versus 43°C for 4 h in the other study).

HUMAN HEAT SHOCK FACTOR-DNA INTERACTIONS

VOL. 11, 1991

N

WT

-85

-100

-115

3511

~~AAAA A ooooooo^soooooo

I

CGRRRCCCCTGGRRTRTTCCCGRCCTGGCRG GCTTTGGGGRCCTTRTRRGGGCTGGRCCGTC a---oooo A AAAAA

114-97

CGRRRCTTCTGGRRTRTTCCCGRCCTGGCRG GCTGRGCCT RGGGCTGGRCCGTC oo^oooooo*oomooooooo A AAAA AA AAAA A

10447

A

CGRRRCCCCTGGRRTRTTCCCGRRCTTTCRG * ooooomooooooommooo * GCTTTGGGGRCCTTRTRRGGGCTTGRRRGTC A AAA AA AA*A A ooooooooooo---oDD

AA

ooo

CGRRRCTTCTGGRRTRTTCCCGRRCTTTCRG 114-87 * * * * GCTTTGRRGRCCTTRTRRGGGCTTGRRRGTC oooooooooo-DE ooo ooo AA

A AAAAA *AA

FIG. 7. Summary of protection and interference results with NGAAN-block mutants. Solid squares next to the sequences show maximal hydroxyl radical protection (typically 85 to 95% by densitometry); open squares show positions of weaker protection (greater than 40%). Solid and open triangles show strong and weak DEPC interference, respectively. Arrows below the sequences indicate the DNase I footprints (approximate boundaries are between the ends of thick line segments and the arrowheads) determined only for the bottom strand; short and long arrows for HSE 114/87 represent footprints observed at low and high protein concentration, respectively, as in Fig. 5. Solid rectangles between the complementary strands show the location of perfect NGAAN-block motifs. WT, wild type.

DISCUSSION

The results described here indicated that all three extended GAA-block mutants had substantially increased binding affinity for HSF from heat-shocked HeLa cells. High-resolution protection and interference studies permitted analysis of a variety of different HSF binding modes to the original wild-type element and the extended site constructs. The primary interactions with the wild-type HSE are centered on the pair of perfect NGAAN motifs (in inverted relative orientation) at -100. These contacts are symmetric, reflecting the dyad character of the sequence. When the number of perfect NGAAN blocks is increased from two to four, additional contacts are formed within the new motifs. These increased contacts are accompanied by a significant increase in binding affinity but appear to occur without changing the stoichiometry of the protein-DNA complex. Human HSF may occur as a trimer, as suggested for HSF in yeasts and D. melanogaster (38, 43). The exact nature of oligomeric Drosophila HSF is still under investigation, and hexameric and larger forms have also been proposed (6). In both studies, however, the Drosophila HSF oligomer had apparent threefold symmetry and seemed to interact with three 5-bp motifs. The oligomeric state has not been established for the human protein, although the apparent molecular weight of the polypeptide, from UV cross-linking, and

its migration on gel filtration chromatography and nondenaturing gel electrophoresis are consistent with a large oligomeric form (11, 53). The higher-affinity complexes with extended consensus mutants appear to represent three strong contact regions between the protein and three perfect 5-bp motifs, as seen most clearly in the hydroxyl radical protection maxima (Fig. 7). If different contacts occur without changing the oligomeric form of the protein, a conformational change or flexibility of relative position of the promoters may be required. These results appear to be consistent with results of functional binding studies of Drosophila HSF and with the trimeric (or hexameric) model of HSF structure. However, the interpretation is somewhat speculative, since connections between different protein contact regions to the 5-bp motifs are not directly established but indirectly inferred. Binding to the wild-type human HSP70 HSE may involve tight contact with only two protomers, represented by the strongest protection and interference positions within the two perfect 5-bp motifs at -100. Potential contacts of other binding sites on the oligomer with adjacent imperfect repeats do not have equivalent protection and interference patterns, although the GAC sequence at -94 to -92 in particular may contribute to binding. The affinity of the 24-bp oligonucleotide with the sequence from -112 to -89 is identical to that of the complete wild-type

3512

CUNNIFF ET AL.

region on a restriction fragment, indicating that the primary high-affinity HSF binding site occurs within these limits. At high protein concentration, the six-NGAAN-block element of HSE 114/87 showed a dramatically different binding pattern in DNase I protection experiments. In this case, an extended 35- to 40-bp footprint was observed, which may represent a higher stoichiometry, such as two HSF oligomers. Since this was seen with the six-block construct but not with four-block elements, it is consistent with binding of a pair of trimers, requiring six NGAAN motifs, and is similar to observations with Drosophila HSF (38). The shorter form of the DNase I footprint on HSE 114/87 at lower protein concentration was similar to that observed with the four-block constructs and may result from binding of a single oligomer. Band shift assays, performed at lower protein concentration for technical reasons, showed predominant formation of a single complex, assumed to contain one HSF oligomer, with the wild type and all the extended mutants. There is a smaller amount of a slowermigrating band, especially with HSE 114/87, which may correspond to a higher-order complex (data not shown). This has not been analyzed for high-resolution contacts because of the low signal intensity, although it may be the form represented by the extended DNase I footprint and is expected to contain additional contacts. It is possible that the wild type and other constructs would also be able to form complexes at higher protein concentration with multiple oligomers that would contain lower-affinity contacts to imperfect 5-bp motifs. It is valuable to compare the above results with a recent report of in vivo genomic footprinting of the HSP70 promoter by DMS protection (1). This study demonstrated that the strongest protection of guanine residues in vivo during heat shock corresponds to the two perfect 5-bp motifs at -104 to -97, in good agreement with our in vitro results. Additional, weaker interactions were seen with the imperfect flanking repeats at -107 and -94. The model proposed for these interactions describes alternative binding modes of HSF oligomers to groups of three perfect and imperfect NGAAN blocks. This is similar to our independent conclusions based on in vitro interactions described above. Less clear protection data suggest a possible interaction with the -114 position as well. This could represent another minor binding mode for a single oligomer. Alternatively, the HSE region in a fraction of cases may be occupied simultaneously by two oligomers. This resembles the extended complex observed in vitro with the higher-affinity mutant HSE 114/87, although not with the wild-type element. A high concentration of activated HSF in vivo might allow formation of complexes with two oligomers on the endogenous promoter. Multicomponent band shift assays show that HSE 114/87 has an additional modest increase in apparent binding affinity relative to the four-NGAAN-block elements. If these HSE 114/87 complexes also represent binding of a single oligomer, as suggested by lack of change in the electrophoretic migration, the increased affinity may be due to the presence of multiple binding sites on the six-block construct. In the simplest case, if two independent, nonoverlapping sites were present on HSE 114/87 and the extent of complex formation is such that the concentration of complexes with two oligomers bound is negligible (which appears to be the case in these experiments), the apparent affinity in multicomponent band shift assays would be the sum of the equilibrium association constants for the individual sites. However, since protection experiments indicate more than one overlapping binding mode for this construct, and since the

MOL. CELL. BIOL.

difference in sequence context of individual motifs may have an influence, it is difficult to interpret the magnitude of the increase in terms of a unique model of interactions with individual 5-bp motifs. Predominant binding of a single oligomer under some conditions appears to indicate absence of strong cooperativity between adjacent HSF oligomers. Alternatively, a higher intrinsic affinity for the sites in the central core of the element, based on flanking sequence context, may counteract the increased affinity resulting from cooperativity. It is possible that a different degree of cooperativity between two HSF oligomers could be observed if the spacing or orientation between them was varied. More definitive determination of possible cooperativity between adjacent HSF oligomers would require measurement of the intrinsic affinity of individual subsites and further quantification of the binding isotherms. However, there is no clear indication with the present constructs, containing tandem arrays of NGAAN blocks with inverted orientation relative to their neighbors, that multiple blocks necessarily result in strongly increased overall binding affinity due to cooperativity. The above observations indicated that the extended GAAblock constructs have higher binding affinity as well as more extensive protein contacts in vitro (and, potentially, in vivo during transfection experiments, although this could not be measured directly). What are the corresponding heat-inducible transcription activities? Basal transcription was unaffected by either improvement or destruction of the HSE. This is consistent with previous reports that the HSE is not necessary for this (16, 57), although in other organisms such as yeasts the HSE does appear to influence basal transcription (37). The six-GAA-block promoter HSE114/87 showed a modest but significant twofold increase in heat-inducible transcription relative to the wild-type promoter. This indicates that improved HSF binding is capable of leading to increased functional activity, although the magnitude of the increase is not directly proportional to the binding affinity change. There may also be a difference between HSE 114/97, which was 1.7-fold better than wild type, and HSE 104/87, which showed no clear improvement. Since both constructs have four contiguous GAA blocks, this apparent difference could reflect the position relative to the start site and other promoter elements or the context of flanking sequences. It is interesting that the topography of contacts suggests that a trimer bound to HSE 114/97 compared with HSE 104/87 is not only 5 bp further upstream but is also inverted with respect to orientation on the helix axis. While the conditions for the transfected template in transient expression assays might be different than for the endogenous promoter, the overall degree of induction (approximately 24-fold) seen with both was similar (data not shown). Perhaps the most interesting result is that the extended six-block template, with greatly improved in vitro binding affinity, showed only a modest twofold increment compared with this high degree of inducibility given by the wild-type promoter. This suggests that HSF binding is not the limiting factor for heat inducibility of this promoter or that the differential binding observed in vitro between the constructs is not as great in vivo. One important factor may be distance to the transcription initiation site, since additional Drosophila HSE repeats were required when located at a distance of 800 bp upstream of the TATA box (4). More recently, an extended HSE mutation similar to HSE 114/87 was found to have a greater effect when placed more than 500 bp upstream of the transcription start site than when located at the natural -100 position (15). It is useful to compare the topography of high-resolution

VOL . 1l, 1991

HUMAN HEAT SHOCK FACTOR-DNA INTERACTIONS

protein-DNA contacts between human and Drosophila HSFs and the 5-bp motif. The experimental approaches used in the two cases were somewhat different, with hydroxyl radical protection and DEPC interference employed here, and DNase I, methidiumpropyl-EDTA-Fe(II), and DMS protection and interference used for the Drosophila work (26, 38). Only for DMS interference are similar data available (26, 35, 38, 53). This indicates a very similar strong effect of modification of purine residues in the first two positions of the 5-bp motif. This appears to be an important conserved major groove recognition contact, although there is a possible species-specific preference, since this is usually AMiAAN in D. melanogaster but GGAAN for the wild-type human HSP70 element (5). Despite the technical differences, the symmetry and extent of protection and interference observed here with hydroxyl radical and DEPC are in excellent agreement with data obtained with other reagents for the Drosophila protein. This is most clearly seen by comparing the wild-type human HSP70 element with the similar 10-bp sequence, consisting of two blocks in head-to-head orientation, of Perisic et al. (38). Thus, in addition to the similar modular nature of recognition of NGAAN repeats by HSFs from the two species, the topography of protein-DNA contacts within the motif is also consistent with a high degree of conservation in this area as well. This can be further investigated by applying the same methods to each protein. It will also be valuable to extend this work by a combination of structural investigations of the HSF protein and continued genetic analysis of the recognition sequence to understand its DNA binding properties in greater molecular detail. ACKNOWLEDGMENTS This work was supported by a grant from the Medical Research Council of Canada. We are grateful to J. Lis for comments on the manuscript. REFERENCES 1. Abravaya, K., B. Phillips, and R. I. Morimoto. 1991. Heat shock-induced interactions of heat shock transcription factor and the human hsp70 promoter examined by in vivo footprinting. Mol. Cell. Biol. 11:586-592. 2. Amin, J., J. Ananthan, and R. Voelimy. 1988. Key features of heat shock regulatory elements. Mol. Cell. Biol. 8:3761-3769. 3. Berk, A. J., and P. A. Sharp. 1977. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonucleasedigested hybrids. Cell 12:721-732. 4. Bienz, M., and H. R. B. Pelham. 1986. Heat shock regulatory elements function as an inducible enhancer in the Xenopus hsp70 gene and when linked to a heterologous promoter. Cell 45:753-760. 5. Bienz, M., and H. R. B. Pelham. 1987. Mechanisms of heatshock gene activation in higher eukaryotes. Adv. Genet. 24:3172. 6. Clos, J., J. T. Westwood, P. B. Becker, S. Wilson, K. Lambert, and C. Wu. 1990. Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation. Cell 63:1085-1097. 7. Crabb, D. W., and J. E. Dixon. 1987. A method for increasing the sensitivity of CAT assays in extracts of transfected cultured cells. Anal. Biochem. 163:93-99. 8. Dignam, J. D., R. M. Lebowitz, and R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475-1489. 9. Dynan, W. S. 1987. DNase I footprinting as an assay for mammalian gene regulatory proteins, p. 75-87. In J. K. Setlow (ed.), Genetic engineering principles and methods, vol. 9. Plenum Press, New York. 10. Gilman, M. Z., R. N. Wilson, and R. A. Weinberg. 1986.

11.

12.

13.

14. 15.

16.

17.

18.

19. 20. 21. 22. 23.

24. 25.

26.

27. 28.

29. 30.

31.

3513

Multiple protein binding sites in the 5'-flanking region regulate c-fos expression. Mol. Cell. Biol. 6:4305-4316. Goate, A. M., D. N. Cooper, C. Hall, T. K. C. Leung, E. Solomon, and L. Lim. 1987. Localization of a human heat shock hsp70 gene sequence to chromosome 6 and detection of two other loci by somatic-cell hybrid and restriction fragment length polymorphism analysis. Hum. Genet. 75:123-128. Goldenberg, C. J., Y. Luo, M. Fenna, R. Baler, R. Weinmann, and R. Voellmy. 1988. Purified human factor activates heat shock promoter in a HeLa cell-free transcription system. J. Biol. Chem. 263:19734-19739. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051. Graham, F. L., and A. J. Van der Eb. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456-467. Greene, J. M., and R. E. Kingston. 1990. TATA-dependent and TATA-independent function of the basal and heat shock elements of a human HSP70 promoter. Mol. Cell. Biol. 10:13191328. Greene, J. M., Z. Larin, I. C. A. Taylor, H. Prentice, K. A. Gwinn, and R. E. Kingston. 1987. Multiple basal elements of a human HSP70 promoter function differently in human and rodent cell lines. Mol. Cell. Biol. 7:3646-3655. Harrison, G. S., H. A. Drabkin, F.-T. Kao, J. Hartz, I. M. Hart, E. H. Y. Chu, B. J. Wu, and R. I. Morimoto. 1987. Chromosomal location of human genes encoding major heat-shock protein HSP70. Somatic Cell Mol. Genet. 13:119-130. Herr, W. 1985. Diethyl pyrocarbonate: a chemical probe for secondary structure in negatively supercoiled DNA. Proc. Natl. Acad. Sci. USA 82:8009-8013. Hurst, H. C., and N. C. Jones. 1987. Identification of factors that interact with the ElA-inducible adenovirus E3 promoter. Genes Dev. 1:1132-1146. Jones, K. A., K. R. Yamamoto, and R. Tjian. 1985. Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell 42:559-572. Kingston, R. E., T. J. Schuetz, and Z. Larin. 1987. Heatinducible human factor that binds to a human HSP70 promoter. Mol. Cell. Biol. 7:1530-1534. Kunkel, T. A., J. D. Roberts, and R. A. Zakour. 1987. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154:367-382. Larson, J. S., T. J. Schuetz, and R. E. Kingston. 1988. Activation in vitro of sequence-specific DNA binding by a human regulatory factor. Nature (London) 335:372-375. (Erratum, 336: 184.) Lindquist, S., and E. A. Craig. 1988. The heat-shock proteins. Annu. Rev. Genet. 22:631-677. Li, R., J. Knight, G. Bream, A. Stenlund, and M. Botchan. 1989. Specific recognition nucleotides and their DNA context determine the affinity of E2 protein for 17 binding sites in the BPV-1 genome. Genes Dev. 3:510-526. Lis, J. T., H. Xiao, and 0. Perisic. 1990. Modular units of heat shock regulatory regions: structure and function. p. 411-428. In R. Morimoto, A. Tissieres, and G. Georgopoulos (ed.), Stress proteins in biology and medicine. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Liu-Johnson, H. N., M. R. Gartenberg, and D. M. Crothers. 1986. The DNA binding domain and bending angle of E. coli cap protein. Cell 47:995-1005. Lum, L. S. Y., L. A. Sultzman, R. J. Kaufman, D. I. H. Linzer, and B. J. Wu. 1990. A cloned human CCAAT-box-binding factor stimulates transcription from the human hsp70 promoter. Mol. Cell. Biol. 10:6709-6717. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Mitchell, P. J., and R. Tjian. 1989. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245:371-378. Morgan, W. Unpublished data.

3514

CUNNIFF ET AL.

32. Morgan, W., and J. Wu. Unpublished data. 33. Morgan, W. D. 1989. Transcription factor Spl binds to and activates a human HSP70 gene promoter. Mol. Cell. Biol. 9:4099-4104. 34. Morgan, W. D., G. T. Williams, R. I. Morimoto, J. Greene, R. E. Kingston, and R. Tjian. 1987. Two transcriptional activators, CCAAT-box-binding transcription factor and heat shock transcription factor, interact with a human HSP70 gene promoter. Mol. Cell. Biol. 7:1129-1138. 35. Mosser, D. D., N. G. Theodorakis, and R. I. Morimoto. 1988. Coordinate changes in heat shock element-binding activity and HSP70 gene transcription rates in human cells. Mol. Cell. Biol. 8:4736-4744. 36. Nakajima, N., M. Horikoshi, and R. G. Roeder. 1988. Factors involved in specific transcription by mammalian RNA polymerase II: purification, genetic specificity, and TATA box-promoter interactions of TFIID. Mol. Cell. Biol. 8:4028-4040. 37. Park, H. O., and E. Craig. 1989. Positive and negative regulation of basal expression of a yeast hsp70 gene. Mol. Cell. Biol.

9:2025-2033. 38. Perisic, O., H. Xiao, and J. T. Lis. 1989. Stable binding of Drosophila heat shock factor to head-to-head and tail-to-tail repeats of a conserved 5bp recognition unit. Cell 59:797-806. 39. Sargent, C. A., I. Dunham, and R. D. Campbell. 1989. Identification of multiple HTF-island associated genes in the human major histocompatibility complex class III region. EMBO J. 8:2305-2312. 40. Sargent, C. A., I. Dunham, J. Trowsdale, and R. D. Campbell. 1989. Human major histocompatibility complex contains genes for the major heat shock protein HSP70. Proc. Natl. Acad. Sci. USA 86:1968-1972. 41. Shuey, D. J., and C. S. Parker. 1986. Binding of Drosophila heat-shock gene transcription factor to the hsp70 promoter. Evidence for symmetric and dynamic interactions. J. Biol. Chem. 261:7934-7940. 42. Sorger, P. K., M. J. Lewis, and H. R. B. Pelham. 1987. Heat shock factor is regulated differently in yeast and HeLa cells. Nature (London) 329:81-84. 43. Sorger, P. K., and H. C. M. Nelson. 1989. Trimerization of a yeast transcriptional activator via a coiled-coil motif. Cell 59:807-813. 44. Sorger, P. K., and H. R. Pelham. 1988. Yeast heat shock factor is an essential DNA-binding protein that exhibits temperaturedependent phosphorylation. Cell 54:855-864. 45. Sturm, R., T. Baumruker, R. Franza, and W. Herr. 1987. A 100 kD HeLa cell octamer binding protein (OBP100) interacts differently with two separate octamer-related sequences within the SV40 enhancer. Genes Dev. 1:1147-1160. 46. Taylor, I. C. A., W. Solomon, B. M. Weiner, E. Paucha, M. Bradley, and R. E. Kingston. 1989. Stimulation of human heat

MOL. CELL. BIOL.

47.

48.

49.

50. 51.

52.

53. 54. 55.

56. 57. 58. 59. 60. 61. 62.

shock protein 70 promoter in vitro by simian virus 40 large T antigen. J. Biol. Chem. 264:16160-16164. Theodorakis, N. G., D. J. Zand, P. T. Kotzbauer, G. T. WilLiams, and R. I. Morimoto. 1989. Hemin-induced transcriptional activation of the HSP70 gene during erythroid maturation in K562 cells is due to a heat shock factor-mediated stress response. Mol. Cell. Biol. 9:3166-3173. Tullius, T. D., and B. A. Dombrowski. 1986. Hydroxyl radical "footprinting": high resolution information about DNA-protein contacts and applications to lac repressor and Cro protein. Proc. Natl. Acad. Sci. USA 83:5469-5473. Tullius, T. D., B. A. Dombrowski, M. E. A. Churchill, and L. Kam. 1987. Hydroxyl radical footprinting: a high resolution method for mapping protein-DNA contacts. Methods Enzymol. 155:537-558. Varshavsky, A. 1987. Electrophoretic assay for DNA-binding proteins. Methods Enzymol. 151:551-565. Vieira, J., and J. Messing. 1987. Production of single-stranded plasmid DNA. Methods Enzymol. 153:3-11. Vinson, C. R., P. B. Sigler, and S. L. McKnight. 1989. Scissorsgrip model for DNA recognition by a family of leucine zipper proteins. Science 246:911-916. Wagner, J., and W. D. Morgan. Unpublished data. Weiderrecht, G., D. Seto, and C. S. Parker. 1988. Isolation of the gene encoding the S. cerevisiae heat shock transcription factor. Cell 54:841-853. Williams, G. T., T. K. McClanahan, and R. I. Morimoto. 1989. Ela transactivation of the human HSP70 promoter is mediated through the basal transcriptional complex. Mol. Cell. Biol. 9:2574-2587. Wu, B. J., H. C. Hurst, N. C. Jones, and R. I. Morimoto. 1986. The ElA 13S product of adenovirus 5 activates transcription of the cellular human HSP70 gene. Mol. Cell. Biol. 6:2994-2996. Wu, B. J., R. E. Kingston, and R. I. Morimoto. 1986. Human HSP70 promoter contains at least two distinct regulatory domains. Proc. Natl. Acad. Sci. USA 83:629-633. Wu, C., S. Wilson, B. Walker, I. Dawid, T. Paisley, V. Zimarino, and H. Ueda. 1987. Purification and properties of Drosophila heat shock activator protein. Science 238:1247-1253. Xiao, H., and J. T. Lis. 1988. Germline transformation used to define key features of heat shock response elements. Science 239:1139-1142. Zimarino, V., C. Tsai, and C. Wu. 1990. Complex modes of heat shock factor activation. Mol. Cell. Biol. 10:752-759. Zimarino, V., and C. Wu. 1987. Induction of sequence specific binding of Drosophila heat shock activator protein without protein synthesis. Nature (London) 327:727-730. Zolier, M. J., and M. Smith. 1984. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and single-stranded DNA template. DNA 3:479-488.