Dec 19, 1994 - observations suggest that these regulatory proteins may all have .... RT-PCI for HSF1 (410bp) done in parallel a. [made from clone. (4); skeletal ...
Nucleic Acids Research, 1995, Vol. 23, No. 3
1995 Oxford University Press
467-474
Complex expression of murine heat shock transcription factors Maria Teresa Fiorenzal, Thomas Farkas Marianne Dissing', Dorthe Kolding2 and Vincenzo Zimarinol13,* ,
Department of Molecular Cell Biology, University of Copenhagen, Denmark, 2Statens Seruminstitut, Copenhagen, Denmark and 3Department of Biotechnology-HS Raffaele, Via Olgettina 60, 20132 Milano, Italy 1
Received September 23, 1994; Revised and Accepted December 19, 1994
ABSTRACT A central step in the transcriptional activation of heat shock genes is the binding of the heat shock factor (HSF) to upstream heat shock elements (HSEs). In vertebrates, HSF1 mediates the ubiquitous response to stress stimuli, while the role of a second HSE-binding factor, HSF2, is still unclear. In this work we show that both factors are expressed in a wide range of murine tissues and each exists as two splicing isoforms. Although HSFs are virtually ubiquitous proteins, their abundance is predominant in testis and variable among other tissues, indicating specific regulations of their expression. A low level of DNAbinding activity of HSF1, detected in many tissues, is probably physiological and is not explained by an anomalous regulation of one of the two isoforms. Our observations suggest that these regulatory proteins may all have roles in fully developed tissues. This possibility is not mutually exclusive of a role of HSF2 during cellular differentiation and tissue development [L. Sistonen, K. D. Sarge and R. 1. Morimoto (1994), Mol. Cell. Biol., 14, 2087-2099]. INTRODUCTION Cells of all organisms synthesize heat shock proteins (HSPs) which are implicated in the folding, assembly and transport of many other proteins (1,2). The expression of the genes encoding for HSPs is highly and transiently increased in response to elevated temperatures and to diverse physiological and experimental stimuli (3). In eukaryotes, a central step in the transcriptional induction of heat shock genes in response to external stimuli is the binding of the trimeric protein heat shock factor (HSF) to multiple copies of a conserved upstream sequence, the heat shock element (HSE), each defined as a minimum of three contiguous repeats of the pentanucleotide nGAAn arranged in alternating orientation (reviewed in 4). Previous studies have shown that, in the majority of organisms, the binding of HSF to HSEs is inducible (5) and have indicated trimerization of HSF subunits as the most likely mechanism for induction (4,6).
*
HSFs cloned from diverse species show substantial amino acid sequence divergence, although they all bear a conserved helixturn-helix type of DNA-binding domain (7,8) and an adjacent trimerization (leucine zipper-like) domain (9) in the N-terminal portion. Their C-terminal portion contains another leucine zipper which shows homologies among HSFs with inducible regulation of DNA-binding activity (4). While in yeasts HSF is encoded by a unique, single copy gene (10-13) and one gene has been isolated from Drosophila (14), higher eukaryotes (plants and vertebrates) contain multiple HSF-like genes encoding for divergent HSE-binding proteins (15-19). Two non-allelic and single copy genes highly conserved in humans, mice and chicken encode for HSF1 and HSF2. These two factors can both function as transcriptional activators (16,17,20) and can form HSE-binding homotrimers (20,2 1), but do not form hybrid trimers (6,22). We have previously characterized the cDNAs encoding for murine HSF1 and HSF2 (503 and 517 amino acids, respectively; 18) that share an overall amino acid identity of 38%, mostly due to homologies in the DNA-binding and trimerization domains. Beyond these regions their sequences are divergent, except for limited homologies in the C-terminal leucine zipper. This last region has been shown to be involved, in the respective human homologs, in the negative regulation of trimerization in HSF1 (6) and of nuclear translocation in HSF2 (23). Earlier experiments on primate and rodent cell lines have revealed that HSF1 exhibits the properties of a stress-inducible factor (6,21,24), while HSF2 is refractory to heat shock and other typical stress stimuli, as judged from their inability to induce DNA-binding activity. The activity of HSF2 is instead induced by hemin in human K562 erythroleukemia cells (22,25) and is also constitutively present in murine embryonic carcinoma lines and in embryonic stem cells (26,27). In this study, we have investigated the patterns of expression of murine HSF1 and HSF2. We report that both factors are virtually ubiquitous and each exists as two splicing isoforms. Two new forms are characterized by peptide insertions adjacent to the C-terminal leucine zipper that, at least in HSF1, may be part of a larger negative regulatory domain (4,6). In addition, both the overall abundance of HSF1 and HSF2 and the relative abundance of their isoforms are variable among tissues, suggesting that their expression is specifically regulated.
To whom correspondence should be addressed at: HS Raffaele, Department of Biotechnology (DIBIT), Room 2A2-39, Via Olgettina 60, 20132 Milano, Italy
468
Nucleic Acids Research, 1995, Vol. 23, No. 3
MATERIALS AND METHODS Mice
C57/Bl/6/j mice, purchased from Breeding and Research Centre Ltd, were sacrificed at the age of 6-7 weeks by cervical dislocation. Two or three adjacent organs were quickly dislodged and instantly transferred to liquid N2. Pools of tissues removed from three animals were processed for RNA or protein extraction. Cell culture, heat shock, harvesting and transfections L and COS-7 cells were grown in Dulbecco's Modified Eagle's medium supplemented with 5% fetal bovine serum at 37°C under a 5% CO2 atmosphere. For heat shock, cultures were submerged for 20 min in a 44°C water bath. Cells were collected after trypsinization, pelleted and frozen in dry ice. The calcium phosphate precipitation method (28) was used for transfection of COS-7 cells with pcDNAI-NEO (Invitrogen) carrying the murine HSFIaorHSF1,I3 cDNA. Cells (2.5 x 105) seeded in 3 cm dishes were transfected 24 h later with 0.5 jg of HSF expression plasmid, 3.5 jig of pSP70 (Promega) as carrier and 1 jg of pTKGH (human growth hormone reference plasmid; Allegro). The precipitate was washed off after 16 h and expression was allowed to occur for 42 h. Each experiment was done in triplicate. The complete ORF for HSF1 was reconstructed by introducing into HSF1a, after removal of nucleotides 1284-1465, a PCR fragment of 248 bp from the partial HSF1 cDNA clone, and the reconstituted ORF was sequenced.
RNA isolation and reverse transcription-PCR Total RNA was isolated by the method of Chomzynski and Sacchi (29). RNA pellets were dissolved in DNase I buffer and incubated for 15 min with RNase-free DNase 1 (20 U/ml) in the presence of RNasin. RNA concentrations were determined spectrophotometrically and samples with comparable A260:A280 ratios were selected for RT-PCR. RT-PCR was with the GeneAmp Thermostable rTth Reverse Transcriptase RNA PCR Kit (Perkin Elmer Cetus). The 123 bp ladder (GIBCO) was 32P-end labeled with T4 polynucleotide kinase. Oligonucleotide primer pairs: HSFI, sense (314-333) 5'-AAGTACTTCAAGCACAACAA-3', antisense (703-723) 5'-GAGATCAGGAACTGAATGAGC-3' and sense (1139-1159) 5'-ATCCTfCGAGAGAGCGAGCCT-3', antisense (1546-1567) 5'-CTCAGAGAAGTAGGAGCTCTCC -3'; HSF2, sense (421440) 5'-CTGTIGAATITCAGCATCCT-3', antisense (803-822) 5'-CAGTTGGTTCITITGACTATG-3' and sense (907-927) 5'-ATGTTACTGACGATAA TGTGG-3', antisense (1414-1434) 5'-ACTGGATAAGTTGT TTGTCTG-3'. Primers for murine ribosomal S16 protein (30) were: 5'-AGGAGCGATlTl GCTGGTGTGGA-3' (1451-1471, sense) and 5'-GCTACCAGGGCC`ITlfGAGATGGA-3' (1641-1621, antisense).
Over-production of murine HSF1 and HSF2 in E.coli, purification and antisera production Protein expression in E.coli BL21(DE3)LysS was induced with 0.4 mM isopropyl-p-thiogalactoside (IPTG) as described (31). Lysates were cleared by centrifugation at 100 000 g and sequentially chromatographed on Cibacron blue-Sepharose (Pharmacia) and HSE-Sepharose. Proteins were eluted with linear NaCl (0.2-1.0 M) gradients. Two mg of 70-90% homo-
geneous preparations of HSF were injected in each rabbit over a period of 8 months.
Iranscription-translation and heat shock in vitro RNAs were transcribed in vitro with T7 RNA polymerase and 1 jig was added to each translation reaction (50 g1) containing rabbit reticulocyte lysate (Promega). After translation at 30°C for 60 min, cycloheximide was added to 40 jM and lysates were spun at 100 000 g. Aliquots of 12 ,l were incubated at 30, 37 and 44°C for 20 min prior to gel shift assays. SDS-PAGE and Western blots Protein extracts (50 jg) were subjected to SDS-PAGE in 8% gels followed by semi-dry blotting onto nitrocellulose filters (BA-85; Schleicher & Schuell). Pre-stained 14C-labeled molecular size markers (14.3-200 kDa range; Amersham) were co-electrophoresed. Filters were stained with Ponceau S (Sigma) to confirm the transfer, blocked with 5% (w/v) milk proteins in TBST buffer (150mM NaCl, 0.1% Tween 20, 10mM Tris, pH 10.0), incubated for 60 min with anti-HSFI or anti-HSF2 (1:5000), washed, re-blocked and incubated with peroxidase-coupled anti-rabbit IgG. Filters were developed with the ECL system (Amersham). After decay of the light energy, filters were incubated with anti-a-tubulin monoclonal antibody (Sigma) and peroxidasecoupled anti-mouse IgG and developed again. Gel shift assays Protein extracts from cultured cells and from tissues were prepared as described in ref. 5. DNA-protein binding assays were with a consensus HSE (upper strand: 5'-GTCGACGGATCCGAGCGCGCCTCGAATGTTCTAGAAAAGG-3') labeled with 32P as described (5). Binding mixes (20 p1) contained 20 jg proteins, 10 jg poly(dI.dC-poly(dI.dC) (Pharmacia), 1.5 ng 32P-labeled HSE in binding buffer (10 mM HEPES, pH 7.9, 10 mM KCI, 50 mM NaCl, 1.5 mM MgCl2, 10% glycerol). Binding was at 4°C for 20 min. Where indicated, pre-immune and immune sera (anti-HSF1 1:3000, anti-HSF2 1:500) were added to the pre-formed complexes and incubated at 4°C for 20 min prior to gel loading. Electrophoresis was in 4% polyacrylamide gels (40:1), 0.5 x TBE at 10 V/cm in the cold.
RESULTS The mRNAs for HSF1 and HSF2 are transcribed in a wide range of tissues The mRNAs for HSF1 and HSF2 were detected by RT-PCR on total RNA isolated from nine tissues of 2-month-old mice and from the L fibroblast line. Amplifications with primers specific for 5' and 3' coding segments consistently showed that both mRNAs are transcribed in all tissues examined and in the L cells (Figs 1 and 2). The amplified products appeared to be specific, since they co-migrated with the corresponding products amplified from synthetic RNA and were not observed in reactions without the RT step or in complete reactions lacking the RNA input (not shown). These experiments also showed tissue variations of the abundance of the mRNAs for HSF1 and HSF2. Figures 1A and 2A show that the HSF1 signals were stronger in testis (lane 6), skeletal muscle (lane 5), heart and lung (lanes 8 and 9) andthatthey were weaker in liver (lane 3) andAkidney (lane 11).
Nucleic Acids Research, 1995, Vol. 23, No. 3
R 2
1
3
4
5
6
7
9
8
10
11
bp
469
2A and B). Furthermore, in separate experiments we determined that both smaller and larger RT-PCR products were amplified from cytoplasmic RNA, indicating that the species from which they originated are transported to the cytoplasm (not shown).
-615
-492
HSF1 .-p
_IJbiU
-369
-246
_
RUG
314
within the amplified segments (Fig. 3A and B). Furthermore, these were also recognized in the sequences of the human (and chicken) homologs. Their presence in the larger of the two RT-PCR products was confimed by diagnostic restrictions and by
Stop
regions
723
B 1
2
3
4
5
Identification of new transcripts encoding for HSF1 and HSF2 isoforms In an attempt to identify the origin of these new products, we sequenced various cDNA clones for murine HSFs which were isolated previously and not fully characterized (18). Two partial clones were identified which bore sequences identical to the previously characterized full-length cDNAs, except for an additional 66 (HSF1) and 54 (HSF2) nucleotides inserted in-frame and
6
7
8
9
1f
11
bp
-615
sequencing (Fig. 3C). Since murine HSF1 and HSF2 are each encoded by a single copy gene, the newly identified mRNA species most likely represent variants generated by differential processing of the primary transcripts. This possibility is validated by the observation that, in the sequence of the human HSF1 gene (G. Giorgi and C. Wu, personal communication), the 66 nucleotide region corresponds to a discrete exon. The same probably holds true for the variant form of the mRNA for HSF2, since the sequence of our partial murine clone, most probably originating from
-492
a
processing intermediate, shows the presence of a 5' splicing (GTAAGT) and bonafide intervening sequences on the
consensus
HSF 2
-*'-
3' side of the insert (Fig. 3B).
*ii i -246 RUG
amino
murine HSFs
4Z182Z
421
amino acid
822
um
which are
(18)
isoforms. The local
as
sequences
conserved in the
of HSF1
alignment
and HSF2[
of the
showed that the
inserts share limited homologies and, strikingly, they occur in the same position when the C-terminal leucine zippers are aligned.
Figure 1. RT-PCIR-amplified fragments specific for5'segments of the mRNAs for HSF1 (410 bp) (A) and HSF2 (402 bp) (B). Experiments in (A) and (B) were done in parallel amd exposed to the same X-ray film. Lanes: synthetic RNA [made from clone C12 (HSF1) orC9 (HSF2); 18](1); Lcells (2); liver (3); brain (4); skeletal musc-le (5); testis (6); ovary (7); heart (8); lung (9); spleen (10); kidney (1 1). Asteisks indicate minor products of unknown origin. The positions of moleecular size standards (123 bp DNA ladder) are indicated. Drawings: murinte HSF1 and HSF2 cDNAs (18). -+: primers for RT-PCR. Densitometric scainnings nornalized to the S16 signals (Fig. 2E) showed that -nni imILr4Pu wun 11I n-entivuPlu PQJ1 in t11thiU nniU R-fJiU1u,higsh the mRNA for Htar iu H1s uA 20-fuo rUVC Ye higher than in livi er and that the mRNA for HSF2 waLs 20-fold higher in testis than in liver. an
The newly identified murine transcripts essentially encode for isoforms characterized by the insertion of 22 (HSF1) or 18 acids, (HSF2) respective highly human and avian homologs (Fig. 3D). Hereafter we refer to these forms as isoforms and to the previously characterized forms of
i-
We also noticed that the 22 amino acid insert in HSF1I can be
viewed as part of a hydrophobic heptad repeat, contains three helix-breaking proline residues.
although it also
Furthermore, RT-PCRs in figure 2 showed variable ratios between the mRNAs for the a and , isoforms in many tissues. In testis, heart and spleen HSF1 was prevalent and HSF2,B was also prevalent in testis, while HSF2a was more prevalent than HSF2j in
brain, ovary and heart.
a
The HSF2 signals were also stronger in testis and skeletal muscle and showed various weaker intensities in the other tissues examined (Figs lB and 2B). While parallel experiments with primers specific for ribosomal S16 protein showed comparable amplifications, the variations in HSF signals most probably reflect tissue differences in the steady-state levels of the mRNAs that were detected under conditions of linear response to template concentration (Fig. 2C-E). Unexpectedly, the RT-PCRs with 3' primers resulted in amplification of additional products of slightly larger size (Fig.
Presence and complexities of HSF1 and HSF2 in murine tissues
To detect the presence of HSF1 and HSF2 in the tissues, immunoblots were incubated with specific polyclonal antisera raised against the proteins purified after over-production in E. coli (Figs 4A and 5A; see also Materials and Methods). The blots incubated with anti-HSF1 showed the strongest signal in testis and signals of various weaker intensities in brain, ovary, heart, lung and spleen (Fig. 4B, upper panel). HSF1 was below detection in liver and kidney, although very weak signals were visible after long exposure of films to chemiluminescence (not shown). HSF1 extracted from tissues and L cells was resolved as a cluster of closely spaced polypeptides with a slightly larger
470
Nucleic Acids Research, 1995, Vol. 23, No. 3 A
2
3
4
6
5
/
8
18 11
9
I
B
bp
2
3
4
5
6
8
9
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HSF1
--
-
[
492
HSF2
I8
11
bp
--- 615
-[O
-- 492
- 369
-- 369
RUG
Stop
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RUG
1139
1567
906
C
D
b
a
cd
e
f
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h
A
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Stop
: 934
-;
-6 1 5
-492
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S
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& a
H SF 2-
I[
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b
c
d
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Figure 2. RT-PCR-amplified fragments specific for 3' segments of the mRNAs for HSF1 (A) and HSF2 (B) are bracketed (for sizes see fig. 3C legend). Lanes and drawings are as in figure 1. (C) Tissue RNA (3 ,ug) stained with ethidium bromide after agarose gel electrophoresis. Lanes are as above. (D) RT-PCR amplifications from 2-fold dilution series: HSFI (above), RNA from testis (lanes a-d) and liver (lanes e-h); HSF2 (below), RNA from testis (lanes a-d ). Lanes a and e: 0.125 jg; b and f: 0.25 jg; c and g: 0.5 jg; d and h: 1.0 jg. (E) RT-PCR-amplified fragments specific for murine ribosomal protein S16.
molecular mass than HSFla translated in vitro (Fig. 4C). We speculate that this complexity is mostly due to the presence of various phosphorylated forms of HSFla and HSFID. This interpretation is supported by the results of phosphatase treatment of the extracts (M. T. Fiorenza, unpublished) and by the observation that each isoform is resolved as a cluster of phosphorylated polypeptides when individually expressed in transiently transfected COS7 cells (our work in progress; 32). In heat-shocked L cells (Fig. 4C) the electrophoretic mobility of HSF1 became slower, presumably owing to the additional and inducible phosphorylation previously described (21,33). The blots incubated with anti-HSF2 showed stronger signals in testis and brain and weaker but comparable signals in other tissues (Fig. 5B, upper panel). In L cells, HSF2 was resolved as two polypeptide bands which co-migrated with the in vitro translated protein (Fig. 5C); their electrophoretic mobility was not altered after heat shock, as was previously found in other rodent and human cell lines (21,27). Incubation of the filters with anti-a-tubulin helped to rule out major artifacts due to errors in sample loading or blotting (the liver extracts, which showed both low a-tubulin and HSF signals, were not visibly proteolyzed in Coomassie stained gels). In general the immunoblots did not seem to contradict the results of the RNA analysis with respect to both the abundance and the complexities of HSF1 and HSF2.
A limited DNA-binding activity of HSF1 is present in murine tissues
many
Gel shift assays revealed that many tissues contain a low level of specific HSE-binding activity that was resolved as a single complex migrating in the upper third of the gels (ovary showed an array of faster migrating complexes). The level of activity was intermediate between control and heat-shocked L cells and was higher in spleen, testis and brain than in heart and lung (Fig. 6A). The assays of liver and kidney extracts resulted in high backgrounds and very low specific signals (not shown). This limited HSE-binding activity may be physiological, although we cannot rule out that the level was slightly increased during the experiment. However, our conditions were as close as possible to physiological, since the interval between cervical dislocation and freezing was 1 min for most tissues and 4 min for brain; in addition the extracts were prepared and assayed in the cold to prevent in vitro induction of the activity (33,34). To find out which HSF is responsible for the activity, we used antisera to delay the electrophoretic mobility of HSF-HSE complexes (33). Since the purpose of this experiment was to separate two presumably antigenically related proteins under native conditions, we selected low antisera concentrations to obtain highly specific, although small, electrophoretic delays. Figure 6B shows the ability of antisera to specifically delay the
Nucleic Acids Research, 1995, Vol. 23, No. 3 murioe
A
HSFl
HSF1
length
full
471
cDNA
.....
7bp
194
HS71
murios partial
BstElI
cDNA
1316bp
I.
13671
CTGCTGGAC
mouse
1148
CTGCTGGACCTATTCAGCCCCTCGGTGACCATOCCCGACATGAGCCTGCCTGACCTGGACAGCAGCCTGOCCAGCATTCAGGAG
1231
human
1401
CTGCTGGACCTGTTCAGCCCCTCGGTGACCGTGCCCGACATGAGCCTGCCTGACCTTGACAGCAGCCTGGCCAGTATCCAAkGAG
1484
B
murios
1384
TTCAGGAG
HSF2
HSF2
length
full
..................................
cDNA
1972bp
curios
partial
.e. .l
HSF2
cDNA
1365bp
5 splicing consensus
mouse
13 25
GGTTGAT ............................TCTGAGAAT
1340
hurran
1258
GGTTGATCTTTTCACTAGTTCTGTGCAGATGAATCCCACAGATTACATrCAATAATACAAAA.TCTGAGAAT
1327
mouse
1 157:
GGTTGATCTTTTCACTAGTTCTGTGCAGATGAATCCCACAGATAACATCAATAATACAAGT
,AAG.TTTAATCCATGTTGCTCAGGACCCATAGCTATArCAAGTA GGAAGAGTCCTATTAAACATACAT
ACAGAAAGCATTTCCTATTCTTAC TTAAGTGTGTGGTTGAGATAGTAC
AGTGCTAG ATGGAAAAG.TAGCAG TTGGTTATGGGGTTAGTTTTTCCT TGT
1385
D
C
Iz06 40
615
615
C-TERMA
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LEUCINE
ZIPPER
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492 -4
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369
367
EKCLSVACLDKNELSDHLDAMDSNLDNLQTMLTSHGFSVDTSALLDLF5PSVTXPDKSLPDLDSSIASIQELLSPQEPPRP
447
mouse
HSF1p
367
EKCLSVACLDKNELSDHLDAMDSNLDNLQTMLTSHGFSVDTSALLD ............IQELLSPQEPPRP
425
mouse
HSFle
371
EKCLSVACLDKNELSDHLDAMDSNLDNLQTMLSSHGFSVDTSALLDLFSPSVTVPDKSLPDLDSSX.ASIQELLSPQEPPRP
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human
HSF1
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413
chick
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346
346
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ILNDNINLLGKVEI.LDYLDSIDCSLEDFQAMLSGROFSIDPOILVDLFTSSVQ6OIPTDNINNTKSENKGLEATKSSvV~QHV
426
mouse
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408
mouse
HSF2a
427
human
HSF2
45`7
chick
HSF2
LFSPSVTMPDMSLPDLDSSLAS
mouse
HOFl
LFTSSVQMNPTDNINNTK
mouse
HSF2
HSF25
6-insert
8-insert
HSFI and HSF2. (A) HSFl. Drawings: the full-length cDNA (18) and the newly characterized partial clone bearing an insert of (black box). The position of a BstEll site is indicated. Local aligniment: the sequence of the full-length cDNA shows a gap. corresponding to this gap the partial cDNA contains 66 nucleotides (boldface type) inserted in-frame and conserved, with minor changes, in human HSFlI (1 6). The 5' terminus of the partial cDNA clone corresponds to nucleotide 220 of the full-length cDNA. The BstElI site is underlined. (B) HSF2. Drawings: the full-length cDNA (1 8) and the newly
Figure 3.
New murine cDNAs for
66 nucleotides
SpeI site and ofa 5' splicing aligned with the sequence of human HSF2 (17). cDNA contains 54 nucleotides (boldface type) conserved, with minor changes, in human HSF2. The 5' terminus of the corresponding to the gap the partial cDNA corresponds to nucleotide 169 of full-length cDNA. The 54 nucleotide insert is followed by a 5' splicing consensus (GTAAGT) and 168 nucleotides of bona fide intervening sequence. An in-frame stop codon (TAG) is at position 1342. The SpeI site is underlined. Drawings are to scale. White boxes: non-coding sequence; gray boxes: coding sequence. (C) Restriction analysis of the RT-PCR products specific for 3' mRNA segments. HSF1I (left): restriction with BstEll splits the larger product (495 bp) into fragments of 251 and 244 bp, while the smaller product (429 bp) is not cut. HSF2 (right): restriction with Spel splits the larger product (582 bp) into fr-agments of 431 and 151 bp, while the smaller product (528 bp) is not cut. (D) Local alignments of the am-ino acid sequences of murine, human and characterized partial clone bearing an insert of 54nucleotides
consensus
avian
(19)
are
alignment: partial murine
indicated. Local
HSFI
(upper)
and HSF2
(lower);
inserts, and conserved in human and chick,
alignment
of the inserts of murine HSF1I
mobilities of in we
vitro-synthesized
(blackbox) and intervening sequences (dashed box). The positions of aunique
the sequence of the murine
full-length
cDNA shows
a
gap when
(1) and (0) indicate hydrophobic heptad repeats in the C-terminal leucine zipper. Amino acids encoded by the in-fr-ame
are
in boldface type. Small open circles:
and
hydrophobic heptad repeats.
Asterisks indicate the
prolines. Below:
position of
HSF20: (I) identities; (:) conservative substitutions.
HSFl and HSF2 at the dilutions
used. Under these conditions, incubations of antisera with the
pre-formed complexes showed that HSFI accounts for most of the activity in brain, heart, lung and spleen, while both HSF1I and HSF2 account for the activity in testis (Fig. 6C). The activity of HSF2 was below detection in the majority of the other extracts.
Inducible
DNA-binding activity
of
HSFlct
and
HSF10
in vitro and in vivo
Rabindran
tures,
et
HSF1I
al. have
proposed that,
is held back in
a
at
monomeric
interaction of C-terminal and N-terminal
physiological temperaformn by intramolecular zippers which antagon-
472
Nucleic Acids Research, 1995, Vol. 23, No. 3
B
R
C
in uitra translated I
HSFI
m
au
HSF2 *~~~~~~~~~~~~~~~~~~~
''
!: -,A
'L -: -2 ra-
~
m
ma
k ia
aC - X,
---
-I
C
E
C ua
a
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HSF1 _
.cells
r
a~
GJ.a
-69
m _ _
HSF I ox-tubulin _ [
As
.
to
w
0-[
oh
a% ow ea
.z
-46
Figure 4. Western blots with anti-HSFl and anti-a-tubulin. (A) Left to right: HSF1 (1, 2 and 3 pl) and HSF2 (3 gl) translated in reticulocyte lysate. (B) Above: tissue blot probed with anti-HSFI. Tissue extracts are indicated. Below: re-probing with anti-a-tubulin (55 kDa). Film exposure to cherniluminescence was for 10 min for HSFl and for 2 min for a-tubulin. Molecular mass markers: myosin, 200 kDa; phosphorylase b, 97.4 kDa; bovine serum albumin, 69 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.3 kDa. (C) Anti-HSF1-reactive polypeptides in testis and in control and heat-shocked L cells.
in
c
B
R
L
HSF2
L cells
a;
ultro translated HSFI
a
0
=-
m
Lo
-u
8'
i
cV. _
a
aIa M_
_i,
o
-97
HSFZN
ct-tubulin _.
AA_i,zw6
A _t
_
-69
HSF2
_
_
_
rE, -46
Figure 5. Western blots with anti-HSF2 and anti-a-tubulin. (A) Left to right: HSF2 (1, 2 and 3 p1) and HSF1 (3 gl) translated in reticulocyte lysate. (B) Above: tissue blot probed with anti-HSF2. Tissue extracts are indicated. Below: re-probing with anti-a-tubulin (55 kDa). (C) Anti-HSF2-reactive polypeptides in testis and in control and heat-shocked L cells. Film exposures and molecular mass markers are as in figure 4.
ize trimerization, thus inhibiting high affinity DNA-binding (4,6). In addition, these authors have shown that truncations ofthe polypeptide sequence downstream of the C-terminal zipper result in partial activation of DNA-binding, suggesting that this zipper may be part of a larger negative regulatory domain (6). Since the oa and [B isoforms are characterized by a variation in the sequence immediately downstream of the C-terminal zipper, we were prompted to compare their temperature regulation with respect to DNA-binding activity. For this, HSFla and HSF1I were individually expressed in rabbit reticulocyte lysate, in which HSFla was previously shown to exhibit heat-inducible activity (18), and in transiently transfected COS-7 cells. Figure 7 shows that, in both systems, HSF1a and HSF1[P exhibited a very similar temperature regulation, although protein over-production led to some activation at physiological temperatures (6,21) that was comparable for both isoforms. These results also suggest that the presence of a limited activity of HSF1 in the tissues is not due to the anomalous regulation of one of the two isoforms.
DISCUSSION The existence of multiple HSE-binding factors in the same mammalian species was not anticipated by earlier studies, except
for the observation of two activities, with distinctive developmental regulation, in murine embryonic carcinoma lines (26). Subsequently, cloning experiments have uncovered the genes for HSF1 and HSF2 in humans and mice, their avian homologs and a third avian gene encoding a yet distinct factor, HSF3 (19). Multiple HSE-binding factors have also been identified in plants (15). However, while in plants three factors all appear to mediate responses to temperature elevation (35), in vertebrates HSF1 and HSF2 exhibit functional diversification. In this study we have shown that murine HSF1 and HSF2 are virtually ubiquitous proteins. While this is consistent with the role of HSF1 in the response of any cell to stress stimuli, the observation that HSF2 is also ubiquitously expressed, together with the previous finding that diverse rodent and primate cell lines contain immunologically reactive HSF2 species (21,27), strongly suggests that the role of this second factor is not restricted. Our inability to detect the DNA-binding activity of HSF2 in the majority oftissues might be explained by a strong inhibition of the activity of this factor or most probably by limitations of our assay and probably a lower abundance of HSF2. We also note that other workers have been able to detect distinct HSF2 signals either in cell lines expressing higher levels of this protein (hemin-treated K562 or embryonic carcinoma and stem cells: 22,27) or upon
Nucleic Acids Research, 1995, Vol. 23, No. 3 A
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