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Transcriptional Response to Stress in the Dynamic Chromatin Environment

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Transcriptional Response to Stress in the Dynamic Chromatin Environment Transcriptional response to stress in the dynamic PNAS PLUS 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 CELL BIOLOGY 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. www.pnas.org/cgi/doi/10.1073/pnas.1305275110 PNAS Early Edition | 1of10 Downloaded by guest on September 23, 2021 cell division. We discovered the broad range of molecular (mitochondrial ribosome protein 6), and DNAJB6 (Fig. 1C; 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
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