Transcriptional response to stress in the dynamic chromatin environment of cycling and mitotic cells

Anniina Vihervaaraa,b, Christian Sergeliusa, Jenni Vasaraa,b, Malin A. H. Bloma,b, Alexandra N. Elsinga,b, Pia Roos-Mattjusa, and Lea Sistonena,b,1

aDepartment of Biosciences, Åbo Akademi University, 20520 Turku, Finland; and bTurku Centre for Biotechnology, University of Turku and Åbo Akademi University, 20520 Turku, Finland

Edited by Susan Lindquist, Whitehead Institute for Biomedical Research, Cambridge, MA, and approved July 18, 2013 (received for review March 23, 2013) Heat shock factors (HSFs) are the master regulators of transcrip- rapid posttranslational modifications (PTMs), and HSF2 is pre- tion under protein-damaging conditions, acting in an environment dominantly regulated at the level of expression (22, 23). where the overall transcription is silenced. We determined the The rapid and robust chaperone expression has served as a genomewide transcriptional program that is rapidly provoked by model for inducible transcriptional responses (24). However, the HSF1 and HSF2 under acute stress in human cells. Our results previous studies have almost exclusively concentrated on the revealed the molecular mechanisms that maintain cellular homeo- expression of a handful of HSP genes in unsynchronized cell stasis, including HSF1-driven induction of polyubiquitin genes, as populations (17, 25–28). Currently, comprehensive knowledge well as HSF1- and HSF2-mediated expression patterns of cocha- on the target genes for HSF1 and HSF2 and their cooperation perones, transcriptional regulators, and signaling molecules. We during stress responses is missing. Moreover, the cell cycle pro- characterized the genomewide transcriptional response to stress gression creates profoundly different environments for tran- also in mitotic cells where the chromatin is tightly compacted. We scription depending on whether the chromatin undergoes found a radically limited binding and transactivating capacity of replication or division or whether the cell resides in the gap HSF1, leaving mitotic cells highly susceptible to proteotoxicity. In phases. For transcription factors, the synthesis phase provides an contrast, HSF2 occupied hundreds of loci in the mitotic cells and opportunity to access the transiently unwound DNA, whereas in localized to the condensed chromatin also in meiosis. These results mitosis, most factors are excluded from the condensed chromatin highlight the importance of the cell cycle phase in transcriptional (29–32). Importantly, throughout the cell cycle progression, responses and identify the specific mechanisms for HSF1 and HSF2 in transcriptional orchestration. Moreover, we propose that HSF2 epigenetic cues are required to maintain the cellular identity and is an epigenetic regulator directing transcription throughout cell fate (33). cycle progression. In this study, we investigated the genomewide transcriptional response that is provoked in the acute phase of heat stress in ChIP-seq | ENCODE | human genome | proteostasis freely cycling cells and in cells arrested in mitosis. We charac- terized the transactivator capacities and the genomewide target loci for HSF1 and HSF2 and analyzed chromatin landmarks at ells exposed to proteotoxic conditions provoke a rapid and the HSF target sites. By comparing transcriptional responses in transient response to maintain homeostasis. The stress re- C cycling versus mitotic cells, we determined the ability of mitotic sponse induces profound cellular adaptation as cytoskeleton and cells to respond to proteotoxic insults and the capacity of tran- membranes are reorganized, cell cycle progression is stalled, and scription factors to interact with chromatin that is condensed for the global transcription and translation are silenced (1, 2). De- spite the silenced chromatin environment, the stressed cell Significance mounts a transcriptional program that involves induction of genes coding for heat shock proteins (HSPs). HSPs are molec- ular chaperones and proteases that assist in protein folding and We determined the transcriptional program that is rapidly maintain cellular structures and molecular functions (3). provoked to counteract heat-induced stress and uncovered the Heat shock factor 1 (HSF1) is an evolutionarily well-conserved broad range of molecular mechanisms that maintain cellular homeostasis under hostile conditions. Because transcriptional transcription factor that is rapidly activated by stress and abso- responses are directed in the complex chromatin environment lutely required for the stress-induced HSP expression (4). Ab- that undergoes dramatic changes during the cell cycle pro- errant HSF1 levels are associated with stress sensitivity, aging, fi – gression, we identi ed the genomewide transcriptional re- neurodegenerative diseases, and cancer (5 9). Instead of a single sponse to stress also in cells where the chromatin is condensed HSF in yeasts and invertebrates, vertebrates contain a family of for mitotic division. Our results highlight the importance of the – four members, HSF1 4. HSF2 and HSF4 are involved in corti- cell cycle phase in provoking cellular responses and identify cogenesis, spermatogenesis, and formation of sensory epithe- molecular mechanisms that direct transcription during the lium, and they have primarily been considered as developmental progression of the cell cycle. factors (10–14). HSF1 and HSF2 share high sequence homology of the DNA-binding and oligomerization domains and are able Author contributions: A.V. and L.S. designed research; A.V., J.V., M.A.H.B., and A.N.E. to form heterotrimers at the chromatin (15, 16). Moreover, performed research; A.V. and C.S. performed computational data analysis; A.V., C.S., P.R.-M., and L.S. analyzed data; and A.V. and L.S. wrote the paper. HSF2 participates in the regulation of stress-responsive genes The authors declare no conflict of interest. and is required for proper protein clearance also at febrile temperatures (17, 18). Although HSF1 and HSF2 have been This article is a PNAS Direct Submission. shown to interplay on the heat shock elements (HSEs) of the Freely available online through the PNAS open access option. target loci, their impacts on transcription of chaperone genes are Data deposition: The high-throughput sequencing data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo remarkably different; HSF1 is a potent inducer of transcription, (accession no. GSE43579). HSPs whereas HSF2 is a poor transactivator of on heat stress 1To whom correspondence should be addressed. E-mail: lea.sistonen@abo.fi. – (19 21). HSF1 and HSF2 are also subjected to distinct regulatory This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mechanisms, because HSF1 is a stable protein that undergoes 1073/pnas.1305275110/-/DCSupplemental.

E3388–E3397 | PNAS | Published online August 19, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1305275110 Downloaded by guest on October 1, 2021 cell division. We discovered the broad range of molecular (mitochondrial ribosome protein 6), and DNAJB6 (Fig. 1C; PNAS PLUS mechanisms that maintain cellular homeostasis in stressed cells Dataset S1). Enrichments of HSF1 and HSF2 on selected target and provide unique mechanistic insights into the regulation of genes are illustrated in Fig. 1 C–F. gene expression during the cell cycle progression. Our results The HSPA1/HSP70 locus was strongly bound by HSF1 and revealed the cooperation of HSF1 and HSF2 in orchestrating HSF2 in heat-treated cycling and mitotic cells (Fig. 1C), whereas gene expression in stressed cycling cells and identified their DUSP1 (dual specific phosphatase 1) was occupied by HSF1 and profoundly distinct capacities to coordinate transcription in cells HSF2 in cycling cells only, demonstrating the importance of the where the chromatin is compacted for cell division. cell cycle phase in transcriptional responses (Fig. 1D). In Fig. 1 E and F, the capacity of HSF1 and HSF2 to bind their individual Results target loci is indicated with promoters of GBA (glucosidase β Genomewide Identification of Target Sites for HSF1 and HSF2 in acid) and MLL (myeloid/lymphoid or mixed-lineage leukemia). Cycling and Mitotic Cells. ChIP coupled to massively parallel se- Intriguingly, HSF2 occupied the promoter of MLL in unstressed quencing (ChIP-seq) is a powerful method that enables genome- mitotic cells, although in cycling cells the binding was strictly wide mapping of protein binding sites in a high-resolution and induced by heat shock (Fig. 1F). Promoter-proximally paused unbiased manner (34–36). We used ChIP-seq to characterize the RNA polymerase II (RNPII) was originally identified at the binding sites for HSF1 and HSF2 in cycling and mitotic cells that HSP70 promoter (40, 41), and it is estimated to poise ∼30% of were either untreated or heat treated for 30 min at 42 °C. As the human genes for rapid or synchronous activation (42). We in- model system, we chose human K562 erythroleukemia cells, where vestigated the status of RNPII at selected HSF target promoters HSF1 and HSF2 levels and regulatory mechanisms are well using an existing ChIP-seq data on RNPII (wgEncodeEH000616; characterized (17, 20, 37), and chromatin landmarks have been Snyder Laboratory, Yale University). ChIP cannot determine identified by the Encyclopedia of DNA Elements (ENCODE) transcriptional engagement, but recent global-run-on sequencing consortium (38). The efficiency of cell cycle arrest was improved (GRO-seq) experiments revealed that the majority of promoter- by collecting the cells in S-phase before nocodazole treatment associated polymerases are transcriptionally engaged and com- (39). Histograms of cells based on the DNA content are shown in petent for elongation (43). Accordingly, we refer to RNPII Fig. 1A,confirming the mitotic arrest (5% of cells in G1 and 85% whose enrichment on a promoter site exceeds at least five times in G2/M) compared with freely cycling cells (45% in G1 and 15% the overall signal on the gene as paused RNPII (see below). In in G2/M). For sequencing, 10 PCR-verified ChIP-replicates were Fig. 1 C–F, the distribution of RNPII is visualized. collected per sample, and two controls, IgG and input, were in- cluded (Fig. S1). HSF1 and HSF2 Recognize Similar Consensus DNA Sequences but The ChIP-seq provided high-resolution maps of HSF1 and Display Distinct Binding Profiles in the Human Genome. Binding of HSF2 target loci in the human genome (Dataset S1; Gene Ex- HSF2 to target genes on stress has been considered to occur in pression Omnibus accession no. GSE43579). Under optimal an HSF1-dependent manner (16, 17). However, we identified growth conditions in cycling cells, 45 target sites were identified target genes that are specific for either HSF1 or HSF2 (Fig. 1 E for HSF1 and 148 for HSF2. On acute stress, both the number of and F; Fig. S2A; Dataset S1). In heat-treated cycling cells, HSF1 the target sites (1242 for HSF1 and 899 for HSF2) and the fold and HSF2 shared a majority of their target sites, but under op- enrichments of the targets were considerably higher, indicating timal growth conditions and in mitosis, they displayed strikingly CELL BIOLOGY a rapid recruitment of HSF1 and HSF2 to their target loci in different localization to the human genome (Fig. S2A). Despite heat-stressed cycling cells (Fig. 1B; Dataset S1). In mitosis, the their distinct binding sites, HSF1 and HSF2 recognized similar ability of HSFs to bind chromatin was clearly different; HSF2 HSEs both in cycling and mitotic cells (Fig. S2B), demonstrating occupied 50 loci under optimal conditions and 545 loci on acute that the DNA sequence alone is not sufficient to determine the stress. In contrast, HSF1 interacted only with the promoter of binding site for HSF1 vs. HSF2. HSPA1B/HSP70.2 in the absence of stress, and with 35 loci on Given that promoters and exons account for <2% of the hu- heat stress (Fig. 1B; Dataset S1). Although effectively excluded man genome (Human Genome Project), HSF1 and HSF2 were from the dividing chromatin, HSF1 displayed prominent heat- enriched on gene promoters and protein coding sequences (Fig. induced occupancy on certain loci, including promoters of S3A). For example, 19% of HSF1 and 22% of HSF2 target loci HSPA1A/HSP70.1, HSPA1B/HSP70.2, HSPH1/HSP110, MRPS6 were found within promoters in heat-treated cycling cells (Fig.

Fig. 1. Binding sites for HSF1 and HSF2 in cycling and mitotic K562 cells. (A) Histograms of cycling and mitotic cells based on the DNA content. (B)(Upper) Number of HSF1 (red) and HSF2 (green) target loci in cycling and mitotic cells. (Lower) Fold enrichment over input of each identified HSF1 (red) and HSF2 (green) target locus. The dashed gray line indicates fold en- richment five, which was set as cutoff criterion for HSF target sites. (C–F) Enrichments of HSF1 (red) and HSF2 (green) on in- dicated target genes in cycling and mitotic cells. (C) HSPA1A and HSPA1B promoters are bound by HSF1 and HSF2 in cycling and mitotic cells. (D) DUSP1 promoter harbors prominent HSF1 and HSF2 enrichments in stressed cycling cells only. (E) GBA is an HSF1-specific and (F) MLL is an HSF2-specific target pro- moter. Distribution of RNPII in nontreated cycling cells (blue) is recovered from the ENCODE project (wgEncodeEH000616; Snyder Laboratory, Yale University). The scale for each HSF or input sample is set to 0–100, except at the HSPA1 locus, where, due to high enrichments, the scale is set to 0–250 in cycling and 0–125 in mitotic cells. C, control; HS, heat shock.

Vihervaara et al. PNAS | Published online August 19, 2013 | E3389 Downloaded by guest on October 1, 2021 S3A). On average, the enrichments of HSF1 and HSF2 were ChIP-seq enabled unbiased analyses of HSF1 and HSF2 on every higher on promoter regions than on coding sequences (Fig. S3B), chaperone gene encoded in the human genome and revealed but prominent HSF1 and HSF2 targets were found also within HSF occupancy on 70% of HSP, 90% of chaperonin, and 13% of introns, exons, and intergenic regions as illustrated with PRKCA DNAJ genes (Table 1; Table S1). A majority of the HSF-targeted and PRKCB (protein kinase C α and β, respectively), KCNN1 chaperone genes were bound by both HSF1 and HSF2 on pro- (calcium-activated channel N1), and an intergenic region up- moters that contained paused RNPII (Table 1; Table S1). stream of HSPB1/HSP27 (Fig. S3C). Exceptions were the small HSPs HSPB2/HSP27-2, HSPB5/ CRYAB, and HSPB9, whose promoters lacked paused RNPII HSF1 and HSF2 Bind Selected Chaperone Gene Promoters. HSF1 and (Table 1). To address the impact of HSF1 and HSF2 on tran- HSF2 are best known for their stress-related regulatory functions scription, we depleted either HSF1 or HSF2, or both factors on certain chaperone genes, such as HSPA1A/HSP70.1, HSPC/ together, by shRNA-mediated degradation and analyzed a set HSP90, HSPB1/HSP27, and DNAJB1/HSP40 (4, 17, 26, 27). The of HSF-targeted chaperone genes for their mRNA expression

Table 1. HSF1 and HSF2 occupancy on HSPA, HSPB, HSPC, HSPH, and chaperonin genes HSF1 HSF2

Cycling Mitosis Cycling Mitosis Binding Paused Family Gene Alias C HS C HS C HS C HS site RNPII*

HSPA HSPA1A HSP70-1 2.8 40.4 2.5 16.3 5.2 25.6 3.3 10.8 Promoter Yes HSPA1B HSP70-2 4.3 45.1 5.3 20.5 5.4 26.8 5.4 15.5 Promoter Yes HSPA1L HSP70-1L 2.8 40.4 2.5 16.3 5.2 25.6 3.3 10.8 Promoter Yes HSPA2 HSPA3 ———— — ——— — No HSPA5 BIP, GRP78 ———— — ——— — Yes HSPA6 HSP70B’—15.2 —— — 7.3 —— Promoter Yes HSPA7 HSP70 — 3.0 —— — ——— Promoter No HSPA8 HSC70 4.3 29.5 2.8 5.3 20.2 26.8 8.3 12.3 Promoter Yes HSPA9 GRP75 — 6.6 —— — 5.9 —— Promoter Yes HSPA12A FLJ13874 ———— — ——— — No HSPA12B RP23-32L15.1 ———— — ——— — No HSPA13 Stch ———— — ——— — Yes HSPA14 HSP70-4 ———— — ——— — Yes HSPB HSPB1 HSP27-1 — 23.8 — 6.3 — 16.5 3.5 5.6 Promoter Yes HSPB2 HSP27-2 — 14.1 —— —10.4 —— Promoter No HSPB3 HSPL27 ———— — ——— — No HSPB4 CRYAA ———— — ——— — No HSPB5 CRYAB — 14.1 —— —10.4 —— Promoter No HSPB6 HSP20 ———— — ——— — No HSPB7 cvHSP ———— — ——— — No HSPB8 HSP22 ———— — ——— — No HSPB9 FLJ27437 — 12.3 — 3.5 7.0 19.0 — 6.0 Promoter No HSPB10 ODF1 ———— — ——— — NA HSPB11 HSP16.2 ———— — —3.1 — Promoter Yes HSPC HSPC1 HSP90AA1 — 13.2 — 6.0 — 10.5 3.2 5.3 Promoter Yes HSPC2 HSP90AA2 ———— — ——— — NA HSPC3 HSP90AB1 — 23.8 — 8.2 6.8 14.1 — 9.9 Promoter Yes HSPC4 HSP90B1 ———— — ——— — Yes HSPC5 TRAP1, HSP90L ———— — ——— — Yes HSPD HSPD1 HSP60, GROEL 5.8 43.0 3.5 15.1 17.8 33.4 6.0 9.5 Promoter Yes HSPE HSPE1 HSP10, GROES 5.8 43.0 3.5 15.1 17.8 33.4 6.0 9.5 Promoter Yes HSPH HSPH1 HSP105/110 — 32.6 — 8.8 — 19.0 — 6.3 Promoter Yes HSPH2 HSPA4 — 22.5 — 4.2 — 20.9 — 4.2 Promoter Yes HSPH3 HSPA4L — 9.1 — 4.2 — 5.5 — 3.5 Promoter Yes HSPH4 HYOU1 ———— — ——— — Yes TRiC CCT1 TCP1, CCTA 3.5 31.8 — 4.6 8.5 21.5 — 5.2 Promoter Yes CCT2 CCTB — 8.7 —— — 6.3 —— Prom/Int Yes CCT3 CCTG — 8.5 —— — 8.5 3.9 — Prom/Int Yes CCT4 CCTD — 12.4 — 3.9 4.7 8.5 —— Promoter Yes CCT5 CCTE — 14.0 — 2.7 — 15.3 — 4.9 Promoter Yes CCT6A CCTZ ———— — 5.7 —— Promoter Yes CCT6B CCTZ2 ———— — ——— — Yes CCT7 CCTH — 12.5 — 3.5 — 11.4 — 3.9 Promoter Yes CCT8 CCTQ — 5.0 —— — 7.1 —— Promoter Yes

Fold enrichments ≥2 are shown. The nomenclature of HSPs is according to ref. 85. C, control; HS, heat shock; NA, not analyzed; Prom/Int, promoter or intron depending on the transcript variant. *The RNPII density signal is recovered from ENCODE (wgEncodeEH000616; Snyder Laboratory, Yale University).

E3390 | www.pnas.org/cgi/doi/10.1073/pnas.1305275110 Vihervaara et al. Downloaded by guest on October 1, 2021 during heat shock (Fig. 2 A and B; Fig. S4A). In freely cycling HSP90 protein complex (49), showed a threefold heat-induced PNAS PLUS cells, HSPH1/HSP110, HSPH2, and DNAJB6 were identified as expression that required the presence of HSF1 (Fig. 2D). CDC37 unique HSF-regulated genes whose heat-induced expression was and p23 are involved in client protein recruitment and matura- strictly dependent on HSF1 (Fig. 2B; Fig. S4A). CCT1 (chaper- tion, and they inhibit the HSP90 ATPase cycle (48, 50, 51). We onin 1) has previously been shown to be regulated by HSFs (44), did not detect any significant change in CDC37 mRNA levels which we corroborated in K562 cells (Fig. 2B). The HSPH1/ during the 2-h heat shock but found a twofold HSF1-dependent HSP110 mRNA expression rapidly increased to sevenfold, increase in p23 mRNA (Fig. 2D). Curiously, HSF1 and HSF2 whereas the levels of DNAJB6 and CCT1 mRNA doubled bound the promoters of AHSA1 and PTGES3, but the first intron during the acute heat stress (Fig. 2B). DNAJB6 is the most po- of the noninducible CDC37 (Fig. 2C). We conclude that in ad- tent chaperone in inhibiting protein aggregation, and HSPH1/ dition to controlling the set of chaperones induced by acute heat HSP110 remodels the HSP70-HSP40 machinery to solubilize stress, HSFs also determine the expression patterns of cocha- and refold aggregated proteins in metazoan cells (45, 46). These perones, which are critical for the recognition and fate of the results indicate a considerably wider repertoire of HSF-induced clients, as well as for the chaperoning efficacy. chaperones than earlier anticipated and highlight the importance of managing aggregated proteins under proteotoxic conditions. Stress-Generated Demand for Protein Clearance Is Mitigated by HSF1- Dependent Ubiquitin Expression. On heat stress, damaged and HSF1 Is Required for Heat-Induced Cochaperone Expression. As aggregated proteins accumulate and generate an increased de- shown in Fig. 2C and Dataset S1, HSF1 and HSF2 occupied mand for protein clearance. The vast majority of proteins des- many cochaperone genes, which have not previously been dem- tined for degradation are marked by ubiquitin, which is encoded onstrated to be under control of HSFs. Most cochaperones by four genes in humans (52). As revealed by ChIP-seq, HSF1 possess intrinsic chaperone activity but are termed cochaperones and HSF2 selectively bound to the promoters of polyubiquitin because they determine the activities of the HSP70 and/or genes UBB and UBC but were absent on the monoubiquitin HSP90 protein complexes (47, 48). As summarized in Fig. 2C, genes RPS27A and UBA52 (Fig. 3A). Transcription of UBB or HSF1 and HSF2 showed a strong tendency to bind cochaperone UBC enables a prompt increase in the ubiquitin levels because gene promoters that contain the paused RNPII. We determined their corresponding mRNAs encode a precursor for three and the mRNA levels of the HSP90 cochaperones AHSA1 (encoding nine ubiquitin proteins, respectively (Fig. 3B). In comparison, AHA1), CDC37, and PTGES3 (encoding p23) in the presence transcription of RPS27A or UBA52 would generate one ribo- and absence of HSF1 or HSF2 (Fig. 2D). AHA1, which stim- somal protein per each ubiquitin produced (Fig. 3B). In heat- ulates the ATPase cycle and is the most potent activator of the treated cycling cells, the mRNA levels of UBB and UBC rapidly CELL BIOLOGY

Fig. 2. HSFs define the set of chaperones and cochaperones induced in response to acute heat stress. (A) HSF1 and HSF2 protein levels during the 2-h heat stress in scrambled-trans- fected (Scr), HSF1-depleted (shHSF1), and HSF2-depleted (shHSF2) cycling cells. HSF2 levels decline on heat stress as previously reported (25). (B) mRNA levels of chaperones HSPH1, HSPH2, DNAJB6, and chaperonin CCT1 during heat stress in the presence or absence of HSF1 or HSF2. (C) Cochaperone genes bound by HSF1 and HSF2 in cycling cells. Fold enrichments over input as detected with ChIP-seq are indicated. (D) mRNA levels of HSP90 cochaperones AHA1, CDC37, and p23 in nontreated and heat-treated cycling cells with or without HSF1 or HSF2. In B and D, SDs of a minimum of three biological replicates are shown. The statistical analyses were conducted with two-tailed independent Student t test and asterisks denote statistical significance between indicated scrambled-transfected and HSF- depleted cells. *P < 0.1; **P < 0.05; ***P < 0.01.

Vihervaara et al. PNAS | Published online August 19, 2013 | E3391 Downloaded by guest on October 1, 2021 Fig. 3. HSF1 induces polyubiquitin gene expression in cycling cells. (A) HSF1 and HSF2 bind to polyubiquitin (UBB and UBC) but not to monoubiquitin (RPS27A, UBA52) genes as shown by ChIP-seq. (B) Schematic illustration of the four ubiquitin coding genes in the human genome. (C and D) Heat stress leads to increased expression of polyubiquitin but not monoubiquitin genes in cycling cells, and the heat-induced ubiquitin expres- sion is abolished in HSF1-depleted cells. The mRNA levels and statistical significances were analyzed as in Fig. 2.

increased to two- and fourfold, respectively, whereas the levels of studied genes, but it disrupted the expression kinetics of MLL RPS27A and UBA52 mRNA remained unchanged (Fig. 3 C and (Fig. 4B). Moreover, absence of HSF2 caused more than a two- D). Depletion of HSF1 severely compromized the heat-induced fold increase in mRNA levels of BANF1 and PRKCA under ubiquitin expression in cycling cells (Fig. 3D), revealing that optimal growth conditions (Fig. 4B). HSF1 localization to the promoters of polyubiquitin genes is required for the cell to generate adequate levels of ubiquitin Mitosis Inhibits Transcriptional Activation and Renders Chromatin mRNA under proteotoxic stress. Inaccessible for HSF1. HSF1 bound to 35 target loci in heat-trea- ted mitotic cells, mainly on the promoters of chaperones and HSF1- and HSF2-Driven Transcriptional Programming Extends to translational components (Fig. 5 A and B; Datasets S1 and S2). It Genes Encoding Transcriptional and Translational Regulators, Cell is noteworthy that HSF1 was unable to bind to 1,207 loci, which Cycle Determinants, and Core Signaling Components. To investigate it occupied in heat-stressed cycling cells (Fig. 5B). Among the the biological processes associated with HSF1 and HSF2 target genes that lacked HSF1 in stressed mitotic cells were UBB and genes in acute stress, we performed gene ontology analysis using UBC (Fig. S4B), and subsequently, the ubiquitin gene expression Database for Annotation, Visualization and Integrated Discovery remained unchanged during heat treatment of mitotic cells (Fig. (DAVID) (53). Because heat shock leads to a rapid transcriptional S4C). In contrast to the radically limited capacity of HSF1 to response, we sought to understand whether HSFs are recruited to interact with the mitotic chromatin, HSF2 occupied hundreds of genomic loci that harbor RNPII before stress (wgEncodeE- target loci both in cycling and mitotic cells (Figs. 1B and 5B; H000529; Iyer Laboratory, University of Texas) and whether the Dataset S1). However, depending on the phase of the cell cycle, presence or absence of RNPII characterizes HSF target genes HSF2 localized to a distinct set of targets (Fig. 5 A and B; associated with a specific function. Interestingly, 90% of HSF1- or Datasets S1 and S2). We confirmed that depletion of HSF1 or HSF2-targeted loci on promoters were occupied by RNPII, and at HSF2 did not interfere with synchronization of cells to mitosis the gene bodies, approximately half of the HSF1 or HSF2 target (Fig. 5 C and D) and investigated whether binding of HSF1 or sites harbored RNPII (Fig. 4A). HSF2 leads to induced transcription of their target genes in In heat-shocked cycling cells, HSF1 and HSF2 co-occupied mitotic cells (Fig. 5E; Fig. S4D). Although HSF1 was a potent promoters of chaperones and cochaperones, transcriptional and transactivator in cycling cells (Figs. 2–4), no significant induction translational regulators, and mediators of cell cycle progression of any of the studied HSF1 and HSF2 target genes, HSPH1/ (Fig. 4A; Dataset S2). On the gene bodies, HSF1 occupied HSP110, HSPH2, NUDC, DNAJB6, or MRPS6, was detected in RNPII-containing loci of genes that mediate transcriptional re- mitosis (Fig. 5E; Fig. S4D). These results indicate that besides pression, methylation, and inhibition of mRNA metabolism. As a displaying an impaired ability to bind to the mitotic chromatin, comparison, HSF2 localized to the coding sequences of genes the occupancy of HSF1 at promoters does not induce tran- involved in activation of diverse cellular processes, specifically on scription in the mitotic environment, where the promoters likely the sites that lacked RNPII (Fig. 4A; Dataset S2). Among the lack RNPII and the condensed chromatin generates barriers for promoter-targeted genes, we verified the mRNA levels of tran- transcriptional initiation and elongation (30). Moreover, mitotic scriptional regulators TAF7 (TATA-box associated factor 7) and cells have been shown to be highly susceptible to heat-induced MLL, translational components EEF1G (eukaryote elongation stress (54, 55), which is in agreement with increased cell death factor 1γ) and MRPS6, as well as mitotic factors NUDC (nuclear that we observed in heat-treated mitotic cells (Fig. S4E). distribution C homolog) and BANF1 (barrier to autointegration The mRNA levels of the HSF2-specific target gene, MLL, factor 1). Among the genes that harbored HSF on the coding remained relatively constant in the mitotic cells exposed to acute sequence, mRNA levels of CTCF (CCCTC-binding factor) and heat stress. However, depletion of HSF2 led to increased MLL PRKCA were investigated. In response to heat stress, an HSF1- expression under nonstress conditions (Fig. 5E), suggesting that dependent increase to 3-fold was detected for TAF7, 2-fold for HSF2 either inhibits transcription or modifies the chromatin en- EEF1G, and 1.5-fold for NUDC (Fig. 4B). Depletion of HSF2 vironment of MLL. To elucidate the role of HSF2 in mitosis, when did not hamper the heat-inducible expression of any of the the overall transcription is silenced (30), we monitored the expres-

E3392 | www.pnas.org/cgi/doi/10.1073/pnas.1305275110 Vihervaara et al. Downloaded by guest on October 1, 2021 PNAS PLUS CELL BIOLOGY

Fig. 4. HSFs control a broad range of cellular processes during stress. (A) Gene ontology analyses of HSF1 and HSF2 target genes in heat-treated cycling cells in respect to genomic region and to presence or absence of RNPII. Promoter is defined as −1,200 to +300 bp from the TSS, and gene body is coding sequence from +301 bp to the end of the gene. HSF1-specific target gene groups are indicated in red, HSF2-specific target gene groups are in green, and their shared target gene groups are in blue. (Lower) Fractions of HSF1 or HSF2 target sites that localize to RNPII-occupied sites. RNPII-containing sites in nontreated cycling cells are recovered from the ENCODE project (wgEncodeEH000529; Iyer Laboratory, University of Texas). (B) mRNA levels of HSF target genes during heat shock. mRNA levels and statistical significances were analyzed as in Fig. 2.

sion kinetics of MLL after the non–heat-shocked cells had been target sites in heat-treated mitotic cells to DNaseI hypersensitive released from the mitotic arrest (Fig. 6A). In scrambled-transfected regions in mitosis (wgEncodeEH003472; Crawford Laboratory, cells, the mRNA levels of MLL gradually doubled as the cells Duke University) and found that open chromatin is a pre- progressed from mitosis to G1 phase (Fig. 6A). These results are requisite for HSF1 binding, as 97% of its target sites occurred in agreement with a previous study where the MLL protein levels within DNaseI sensitive regions (Fig. 6B). Instead, 42% of HSF2 were shown to increase after mitosis (56). However, the post- targets in mitosis were found within open chromatin (Fig. 6B). mitotic induction of MLL expression in HSF2-depleted cells was Although HSF2 was able to bind both to DNaseI hypersensitive only minute and showed delayed kinetics (Fig. 6A). As a control, and insensitive regions, this did not explain why HSF1 was absent we used DUSP1, which was not bound by HSFs in the mitotic from 195 DNaseI hypersensitive loci that contained HSF2. Next, cells (Fig. 1D). DUSP1 is a heat-inducible gene (57), and no we addressed whether the exclusion of HSF1 from the mitotic elevation in DUSP1 mRNA expression was detected after the chromatin is mediated by inhibiting the intrinsic DNA-binding mitotic release, nor did the absence of HSF2 affect the DUSP1 capacity of HSF1 or by rendering the chromatin inaccessible for expression during cell cycle progression (Fig. 6A). In conclusion, HSF1. As analyzed with oligonucleotide-mediated pull-down, the ability of HSF2 to bind to the MLL promoter in mitosis (Fig. both HSF1 and HSF2 bound to the exogenous HSE-containing 1D) and to direct its postmitotic expression (Fig. 6A) indicates oligonucleotides in cycling and mitotic heat-treated cells (Fig. the involvement of HSF2 in restoring the transcription of MLL 6C). Curiously, HSF2 bound to DNA also in untreated cycling after the mitotic silencing. cells, and its levels declined in mitosis (Fig. 6C). Besides binding The binding of HSF2 to mitotic chromatin (Figs. 1B and 5B; to the exogenous HSE, hyperphosphorylation of HSF1 was Fig. S2A) argued that a great number of HSEs are accessible for detected in mitosis (Fig. 6C), indicating HSF1 transactivating transcription factors during cell division. We compared the HSF capacity (22). These results demonstrate that HSF1 was indeed

Vihervaara et al. PNAS | Published online August 19, 2013 | E3393 Downloaded by guest on October 1, 2021 maintain homeostasis, highlighting the myriad of different cellular processes that are orchestrated under protein-damaging con- ditions. In stressed freely cycling cells, HSFs induced the ex- pression of polyubiquitin genes, which is a rapid means to produce a large amount of ubiquitin (Fig. 3). Ubiquitin is a small signaling molecule with versatile functions in the cell, and it is required for the proteasomal degradation of damaged proteins (52). Polyubiquitin genes have been shown to be induced on exposure to heat (52, 62), and here we provide evidence for the direct involvement of HSF1 and HSF2 in the regulation of pol- yubiquitin gene expression. HSFs also defined the expression of chaperones (Table 1; Table S1; Fig. 2B) and cochaperones (Fig. 2 C and D) that facilitate protein folding. Previously, a subset of chaperone genes has been used as a model for induced tran- scriptional responses, but here we characterized the complete set of human chaperones that are transcriptionally regulated by HSF1 and HSF2 (Table 1; Table S1). Importantly, we discovered that HSF1 and HSF2 also determine the cochaperones that are in- duced during heat stress, demonstrating that HSFs orchestrate the

Fig. 5. HSF2 interacts with mitotic chromatin. (A) Gene ontology analyses of HSF1 and HSF2 target loci in mitosis. The genomic regions were defined as in Fig. 4. (B) The number of HSF1 (red) and HSF2 (green) target loci in cycling and mitotic heat-treated cells. (C) HSF1 and HSF2 protein levels in scrambled- transfected (Scr), HSF1-depleted (shHSF1), and HSF2-depleted (shHSF2) mi- totic cells during heat stress. (D) Histograms of scrambled-transfected (Scr), HSF1-depleted (shHSF1), and HSF2-depleted (shHSF2) mitotic cells after a 1-h heat treatment, indicating that neither transfection nor lack of HSF1 or HSF2 compromises the synchronization of cells to G2/M. (E) mRNA levels of HSPH1, HSPH2, NUDC, and MLL in mitotic cells with or without HSF1 or HSF2. mRNA levels and statistical significances were analyzed as in Fig. 2.

capable of binding to DNA in mitosis, but its access to chromatin was dramatically reduced from 1,242 sites in heat-treated cycling cells to only 35 sites in cells arrested to mitosis. An intriguing congruency has been reported during meiotic division in mouse spermatocytes, where HSF1 was shown to bind to the exogenous HSE on heat stress but no binding to the HSP70 promoter could be detected (11, 58–60). Because HSF2- Fig. 6. Chromatin compaction during mitosis and meiosis renders DNA in- deficient mice display meiotic defects (61), we investigated by accessible for HSF1. (A) mRNA levels of MLL and DUSP1 in the presence (Scr) confocal microscopy the subcellular localization of HSF1 and or absence (shHSF2) of HSF2. The analyses were conducted in non–heat HSF2 in metaphase spermatocytes. HSF2 was abundant at the shock conditions at indicated times points after releasing the cells from fi chromatin in meiotic divisions, whereas HSF1, albeit highly ex- mitotic arrest. mRNA levels and statistical signi cances were analyzed as in Fig. 2. (Right) Progression of cells from mitotic arrest is illustrated with FACS pressed, was detected only outside the dividing chromatin (Fig. profiles. (B) Fraction of HSF1 or HSF2 target sites in mitosis that occur within 6D). These results suggest a common mechanism in cell division DNaseI hypersensitive regions. The DNaseI sensitive regions in G2/M are re- that selectively allows HSF2 to bind mitotic and meiotic chro- covered from the ENCODE project (wgEncodeEH003472; Crawford Labora- matin from which HSF1 is efficiently excluded. tory, Duke University). (C) Oligonucleotide-mediated pull-down in cycling and mitotic cells. Bound fraction indicates binding of HSF1 or HSF2 to the Discussion HSE-containing oligonucleotide (+). The scrambled oligonucleotide sequence − Characterization of the genomewide binding sites and trans- ( ) lacks an HSE. WB indicates the total HSF1 and HSF2 protein levels in the respective samples. (D) Single sections of confocal microscope images activating capacities for HSF1 and HSF2 discovered the extent showing localization of HSF1 (red) and HSF2 (green) in mouse male germ of HSF-mediated transcriptional reprogramming under acute cells during meiotic division I. DNA (stained with Hoechst) is shown in blue. heat stress. The results uncovered molecular mechanisms that (Scale bar, 100 μm.)

E3394 | www.pnas.org/cgi/doi/10.1073/pnas.1305275110 Vihervaara et al. Downloaded by guest on October 1, 2021 whole machinery that maintains proteostasis. Beyond the protein in driving gene expression. The versatility of cellular processes PNAS PLUS quality control, HSFs directed genes encoding transcriptional and directed by HSF1 and HSF2 underscore the importance of translational regulators, mitotic determinants, and core compo- establishing their specific activators and inhibitors, PTM sig- nents of various signaling pathways (Fig. 4; Dataset S2). For ex- natures, and chromatin-associated factors that direct HSFs to ample, TAF7 has been suggested to function as a transcriptional their individual target loci. checkpoint regulator by inhibiting RNPII phosphorylation at the promoter (63, 64), whereas BANF1 and NUDC are key regulators Is HSF2 an Epigenetic Regulator of Cell Fate? An unexpected finding in mitosis required for correct spindle assembly, cytokinesis, and of our studies was the ability of HSF2 to interact with the di- nuclear envelope assembly (65–68). The stress response has dra- viding chromatin where HSF1 was hardly detected (Figs. 1B and matic effects on cellular physiology, but the basic mechanisms of 6D). HSF2 has been reported to bind HSP promoters in mitosis the adaptation processes such as the transcriptional and trans- and suggested to mediate bookmarking of stress-responsive lational silencing or the stalling of the cell cycle progression are genes (74, 75). We identified both HSF1 and HSF2 on chaper- not understood. The characterized HSF1- and HSF2-driven one genes in mitosis, including promoters of HSPA1A/HSP70.1, transcriptional reprogramming will aid in elucidating the detailed HSPA1B/HSP70.2, HSPH1/HSP110, and HSPE1/HSP10 (Fig. mechanisms that rapidly adjust cellular processes during stress, as 1C; Dataset S1). However, we did not detect HSF1-induced well as clarify how the processes are reestablished once favorable transcription in mitosis (Fig. 5E; Fig. S4D), indicating an im- conditions for proliferation are again restored. paired ability of dividing cells to provoke transcriptional re- sponses. HSF1 has been shown to recruit chromatin modifiers Functions of HSF1 and HSF2 Are Tightly Interlinked, but Their Effects and facilitate RNPII loading (76), which could explain why HSF1 on Promoters Are Profoundly Distinct. Previous genomewide stud- localized to 25 target gene promoters in heat-treated mitotic ies in yeast (69), fly (70, 71), and humans (8, 72) identified the cells without inducing transcription (Fig. 5; Fig. S4D; Dataset HSE for HSF1, showing a striking conservation of the HSF1- S1). The unbiased analyses by ChIP-seq identified a broad range recognized DNA sequence over evolution. Our analyses cor- of mitotic target genes for HSF2 both under optimal growth and roborated the importance of inverted nGAAn pentamers for stress conditions (Fig. 5A; Datasets S1 and S2). One prominent B HSF1 binding both in cycling and mitotic cells (Fig. S2 ). By unique HSF2 target gene in mitosis is MLL, a trithorax homolog revealing the HSF2-targeted HSEs in a genomewide scale, we and a transcriptional coactivator that has been found to interact fi expected to nd a major difference between the DNA sequences with promoters in mitotic human cells, marking the genes for preferred by HSF1 vs. HSF2. Despite their unique target sites in early activation in G1 (77, 78). We discovered that HSF2 con- B E F B A the human genome (Figs. 1 , , and and 5 ; Fig. S2 ), HSF1 trols the cell cycle phase–dependent expression of MLL, which is B and HSF2 recognized a staggeringly similar HSE (Fig. S2 ). prerequisite for an efficient induction of MLL after mitotic si- Earlier footprinting studies have revealed that HSF2 is able to lencing (Fig. 6A). Moreover, recent computational analyses bind shorter HSEs than HSF1 (19, 37). The ability of HSF2 to found MLL target promoters to be enriched with HSEs (77), bind less extensive HSEs could provide an advantage when arguing for either cooperation or competition of MLL and HSFs accessing HSEs in the compacted chromatin regions. The over- fi on the genome. Several studies have addressed how the memory lapping genomic coordinates and highly similar binding pro les of cell fate is maintained during mitosis when most of the tran- of HSF1 and HSF2 at the shared target sites (Figs. 1 C and D and CELL BIOLOGY A C scriptional regulators are erased from the chromatin (33). To this 3 ; Fig. S3 ; Dataset S1) suggest their intimate interplay in tran- end, the capacity of HSF2 to bind mitosis-specific target genes scriptional regulation. The resolution of our ChIP-seq analyses, both under optimal growth conditions and on stress (Figs. 1B and however, cannot distinguish HSF1-HSF2 heterotrimers from 5B; Dataset S1) indicates that HSF2 directs gene expression homotrimers on adjacent pentamers of the same or nearby HSE. throughout cell cycle progression. Cancer cells display a nononcogene addiction to HSF1 as a re- Genomewide analyses in high resolution enable broad views sult of ameliorated proteotoxic stress (5, 73). Moreover, the on the interplay of transcriptional regulators and chromatin state binding profile of HSF1 in nontransformed cells radically differs in different cell types, cell cycle phases, and in response to various from that in cancer cells with high malignant potential (8). Be- stimuli. In this study, we characterized the HSF1- and HSF2- cause K562 cells are an erythroleukemia cell line and express high driven transcriptional response to acute stress in cycling and mi- basal levels of HSPs, HSF1 could have cancer-specific target genes totic cells. Our results revealed the molecular mechanisms that also in these cells. However, the low fold enrichments and the maintain cellular homeostasis in cycling cells and identified limited number of HSF1 target sites in nonstressed K562 cells a dramatically impaired capacity of mitotic cells to provoke tran- (Fig. 1B) are in accordance with the HSF1 binding profiles in scriptional responses, even when challenged with proteotoxicity. nontransformed cells and in cells with low malignant potential (8). We characterized the complete set of chaperones, cochaperones, The detailed analyses of target gene expression revealed HSF1 and ubiquitin genes that is directed by HSFs under acute stress as a potent transactivator that responds instantly to stress (Figs. 1B and 2–4). Also HSF2 rapidly localized to the target sites (Fig. and highlighted the various processes that are transcriptionally 1B), but it did not enhance transcription during 2 h of heat stress reprogrammed in proteotoxic conditions. We uncovered the (Figs. 2–4). HSF1 and HSF2 show similar recruitment kinetics to strikingly distinct mechanisms for HSF1 and HSF2 in orchestrat- the HSP70 promoter, but HSF2 is gradually degraded from the ing transcription and discovered HSF2 as an epigenetic regulator HSE after 30 min of heat stress (25), indicating the need for that controls transcription throughout cell cycle progression. Fu- delicate regulation of the HSF1-HSF2 composition at the pro- ture studies, expanding beyond acute heat shock, will establish moter. Our preliminary results show an HSF2-mediated effect whether cells are able to epigenetically remember past environ- on transcription during prolonged stress when HSF2 levels have mental exposures, such as stress. F fi already declined (Fig. S4 ) (25) and suggest that HSF2 modi es Materials and Methods the chromatin landscape for transcriptional responses. HSF2 is also involved in many developmental processes when the epi- Cell Culture, Synchronization of Cells to Mitosis, and Heat Treatments. Human K562 erythroleukemia cells were maintained at 37 °C in a humidified 5% CO2 genetic features of the cells are determined (10, 12, 61), and it atmosphere and cultured in RPMI medium (Sigma) containing 10% (vol/vol) has been shown to affect the chromatin compaction and integrity FCS, 2 mM L-glutamate, and streptomycin/penicillin. Cells were arrested to (10). These results argue for an HSF2-mediated establishment of mitosis using thymidine and nocodazole (39). Briefly, after 16 h in 2 mM the transcriptional environment and indicate profoundly distinct thymidine (Sigma), the cells were washed with PBS to allow cell cycle pro- mechanisms for HSF1 and HSF2 during their dynamic interplay gression for 8 h. The cells were collected in S-phase by a second thymidine

Vihervaara et al. PNAS | Published online August 19, 2013 | E3395 Downloaded by guest on October 1, 2021 treatment for 24 h. After cell cycle progression for 5 h, the cells were scribed earlier (17) using ABI Prism 7900 (Applied Biosystems). The primers arrested in mitosis with nocodazole (100 ng/mL; Fluka) for 12 h. The noco- were purchased from Oligomer (Helsinki) and the probes were purchased dazole was removed, and the cells were harvested, allowed to proceed the from Roche Applied Science. Primer and probe sequences are listed in Table cell cycle at 37 °C, or heat treated. The heat shock treatments were con- S3. Relative quantities of the target gene mRNAs were normalized to ducted in a water bath at 42 °C. The DNA content of the cells was de- GAPDH, and the fold inductions were calculated against the respective termined by propidium iodide (PI) staining (40 μg/mL; Sigma) and mRNA level in nontreated cells. All reactions were made in triplicate for fluorescence-mediated counting by FACSCalibur (BD Biosciences). The FACS samples derived from at least three biological replicates. SDs were calculated histograms were generated using Cell Quest Pro-6.0 (BD Biosciences) and and are shown in the graphs. An independent two-tailed Student t test was Flowing Software 2.5 (Turku Centre for Biotechnology). The error bars in used to determine the P value when comparing mRNA levels between statistical analyses indicate SDs. scrambled-transfected and shHSF1- or shHSF2-transfected cells.

ChIP. HSF1- and HSF2-bound DNA fragments were isolated by ChIP using Western Blotting. Cells were lysed with buffer C [25% (vol/vol) glycerol, 20 mM fi a previously described protocol (11, 17) and the following ChIP-veri ed Hepes, pH 7.4, 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, 0.5 mM PMSF, and antibodies: HSF1 (Spa-901; Enzo) and HSF2 (17). IgG (sc-2027; Santa Cruz) 0.5 mM DTT], and the protein concentration in the soluble fraction was was used as a negative antibody and AcH4 (06-866; Upstate) was used as measured using Bradford analysis. Twenty micrograms of total protein was a positive antibody. PCR primers are listed in Table S2. boiled in Laemmli sample buffer and subjected to SDS/PAGE followed by trans- fer to nitrocellulose membrane (Protran nitrocellulose; Schleicher & Schuell). High-Throughput Sequencing and Data Analysis. For sequencing, 10 ChIP Proteins were analyzed with primary antibodies against HSF1 (Spa-901; Enzo), replicates per sample were collected. Sequencing libraries were generated HSF2 (3E2; Upstate), and β-TUBULIN (ab6046; Abcam). The secondary anti- using New England BioLabs NEBNext kits, and adapters and primers were bodies were HRP conjugated (GE Healthcare), and the blots were developed from a Bioo Scientific AIR DNA Barcodes kit. Template amplification and using an enhanced chemiluminescence method (ECL kit; GE Healthcare). cluster generation were performed using the cBot and Truseq SR Cluster kit for cBot v, and 36 nucleotides were sequenced with Illumina Genome Ana- Oligonucleotide-Mediated Pull-Down Assay. The oligonucleotide-mediated lyzer IIx using v5 TruSeq SBS sequencing kits. After quality trim and removal of pull-down assay was performed as described previously (83) using bio- duplicates, the reads were mapped to the human genome (hg19) with Bowtie tinylated oligonucleotides (Oligomer). The double-stranded HSE contained (79). The peaks were called with MACS 1.4 (80), and a minimum fold en- the sequence 5′-biotin-TCG ACT AGA AGC TTC TAG AAG CTT CTA G-3′ and richment of five times over input was set as a cutoff criterion for target sites. the scrambled control 5′-biotin-AAC GAC GGT CGC TCC GCC TGG CT-3′. Any site that exceeded the cutoff in the negative IgG sample was discarded. Buffer C extracts of 100–400 μg total protein were annealed to oligonucle- otide (0.5 μM) in binding buffer [20 mM Tris·HCl, pH 7.5, 100 mM NaCl, 2 mM Identification of HSF1- and HSF2-Targeted DNA Sequences and Genomic EDTA, and 10% (vol/vol) glycerol] containing salmon sperm DNA (0.5 μg/μL). Regions and Functional Annotation of Target Genes. The consensus DNA The samples were precleared, and the oligonucleotides were precipitated sequences for HSF1 and HSF2 were identified by motif analysis of large DNA with a 50% (vol/vol) slurry of UltraLink streptavidin beads (Pierce). Bound datasets (MEME-ChIP) (81) using a 120-bp region centered on the summit fractions were washed three times with binding buffer containing 0.1% point. HSF1 and HSF2 target sites were annotated to genomic regions using Triton X-100. DNA-bound proteins were eluted with Laemmli buffer and exon and intron coordinates provided by RefSeq and by defining a core detected by SDS/PAGE and Western blotting. promoter to span from −1,200 to +300 bp from the transcriptional start site (TSS). Fifty percent of peak length was centered on the summit point, and Immunofluorescence of HSF1 and HSF2 in Dividing Male Spermatocytes. Testes peaks that fell on exon-intron boundaries are indicated as exons. Biological of 60- to 80-d-old C57BL/6N mice were isolated, fixed in 4% (wt/vol) para- processes associated with HSF1 or HSF2 target genes in cycling and mitotic formaldehyde, and sectioned to 4 μm. All mice were handled according to cells were analyzed with DAVID (53), which uses Fisher’s exact test for cal- the institutional animal care policies of the Åbo Akademi University (Turku, culation of the P value for enriched gene ontology terms. The density signals Finland). Confocal immunofluorescence analyses were performed as pre- of HSF1, HSF2, and RNPII on the human genome were visualized with the viously described (11) using primary antibodies against HSF1 and HSF2 (20) Integrative Genomics Viewer (82). and secondary antibodies conjugated to Alexa 488 or Alexa 568 (Invitrogen). The DNA was stained with Hoechst 33342 (H-1399; Molecular Probes). Depletion of HSF1 and HSF2 with RNAi by shRNA. HSF1 and HSF2 were de- Images for all channels were sequentially captured from a single confocal pleted from the cells using shRNA constructs ligated into pSUPER vectors section using a Zeiss Meta510 confocal microscope. For analyzing the meiotic (Oligoengine) as previously described (17). The vector-encoded oligonu- divisions, stage XII of the mouse seminiferous cycle was selected, and di- cleotides were specific for HSF1 (GCT CAT TCA GTT CCT GAT C) or HSF2 (CAG viding spermatocytes in meiotic division I were imaged. The channels were GCG AGT ACA ACA GCA T). A scrambled sequence (GCG CGC TTT GTA GGA merged using ImageJ (84). TTC G) was used as a control. The shRNA constructs were transfected into cells by electroporation (970 μF, 220 mV), and the cells were incubated for ACKNOWLEDGMENTS. We thank Michael J. Guertin for insightful discus- 72 h prior to harvesting. Synchronization of cells to mitosis was initiated sions and for sharing his expertise in bioinformatics. The Functional after a 7-h recovery from the transfection, allowing for simultaneous sample Genomics Unit (University of Helsinki) is acknowledged for performing the preparation of the cycling and mitotic cells. high-throughput sequencing, Jukka Lehtonen for expert assistance in computational analyses, and Diana M. Toivola, Johanna K. Björk, and Eva Henriksson for constructive advice during manuscript preparation. This work Quantitative Real-Time RT-PCR. RNA was isolated using the RNeasy kit (Qia- was financially supported by the Academy of Finland, the Sigrid Jusélius μ gen), and 1 g of total RNA was DNaseI treated (Promega) and reverse Foundation, the Finnish Cancer Organizations, Åbo Akademi University transcribed with Moloney murine leukemia virus reverse transcriptase RNase Foundation, the Tor, Joe, and Pentti Borg Foundation, BioCenter Finland, H(–) (Promega). Real-time RT-PCR reactions were prepared and run as de- and the Turku Doctoral Program of Biomedical Sciences (A.V. and A.N.E.).

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