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• W2306540679 abstract "•∼1,500 genes are up- and ∼8,000 are downregulated during the first hour of heat shock•HSF1 induces heat shock protein genes by increasing promoter-proximal pause release•Upon heat shock, SRF transiently induces immediate-early cytoskeletal genes•Broad repression during heat shock in mammals occurs by inhibiting pause release The heat shock response (HSR) is critical for survival of all organisms. However, its scope, extent, and the molecular mechanism of regulation are poorly understood. Here we show that the genome-wide transcriptional response to heat shock in mammals is rapid and dynamic and results in induction of several hundred and repression of several thousand genes. Heat shock factor 1 (HSF1), the “master regulator” of the HSR, controls only a fraction of heat shock-induced genes and does so by increasing RNA polymerase II release from promoter-proximal pause. Notably, HSF2 does not compensate for the lack of HSF1. However, serum response factor appears to transiently induce cytoskeletal genes independently of HSF1. The pervasive repression of transcription is predominantly HSF1-independent and is mediated through reduction of RNA polymerase II pause release. Overall, mammalian cells orchestrate rapid, dynamic, and extensive changes in transcription upon heat shock that are largely modulated at pause release, and HSF1 plays a limited and specialized role. The heat shock response (HSR) is critical for survival of all organisms. However, its scope, extent, and the molecular mechanism of regulation are poorly understood. Here we show that the genome-wide transcriptional response to heat shock in mammals is rapid and dynamic and results in induction of several hundred and repression of several thousand genes. Heat shock factor 1 (HSF1), the “master regulator” of the HSR, controls only a fraction of heat shock-induced genes and does so by increasing RNA polymerase II release from promoter-proximal pause. Notably, HSF2 does not compensate for the lack of HSF1. However, serum response factor appears to transiently induce cytoskeletal genes independently of HSF1. The pervasive repression of transcription is predominantly HSF1-independent and is mediated through reduction of RNA polymerase II pause release. Overall, mammalian cells orchestrate rapid, dynamic, and extensive changes in transcription upon heat shock that are largely modulated at pause release, and HSF1 plays a limited and specialized role. The evolutionarily conserved heat shock response (HSR) protects cells from the proteotoxic environment of heat stress. Elevated temperature and other stresses trigger the HSR, leading to rapid and robust induction of heat shock protein (HSP) genes (Hsps) (Lindquist, 1986Lindquist S. The heat-shock response.Annu. Rev. Biochem. 1986; 55: 1151-1191Crossref PubMed Google Scholar). HSPs are molecular chaperones responsible for maintaining protein homeostasis and are critical for survival during stress (Lindquist and Craig, 1988Lindquist S. Craig E.A. The heat-shock proteins.Annu. Rev. Genet. 1988; 22: 631-677Crossref PubMed Scopus (4414) Google Scholar). The HSR is orchestrated at the level of transcription by heat shock transcription factor (HSF) (Parker and Topol, 1984Parker C.S. Topol J. A Drosophila RNA polymerase II transcription factor binds to the regulatory site of an hsp 70 gene.Cell. 1984; 37: 273-283Abstract Full Text PDF PubMed Scopus (246) Google Scholar). Vertebrates have four Hsf genes, Hsf1–4. HSF1 is considered the master regulator of the HSR and is the ortholog of the sole Hsf gene in invertebrates. HSF2 is the ubiquitously expressed HSF1 paralog that interplays with HSF1 during HSR and is involved in developmental pathways (Sarge et al., 1991Sarge K.D. Zimarino V. Holm K. Wu C. Morimoto R.I. Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability.Genes Dev. 1991; 5: 1902-1911Crossref PubMed Scopus (299) Google Scholar). HSF3 and HSF4 show tissue-restricted expression, and their roles in HSR remain to be explored (Akerfelt et al., 2010Akerfelt M. Morimoto R.I. Sistonen L. Heat shock factors: integrators of cell stress, development and lifespan.Nat. Rev. Mol. Cell Biol. 2010; 11: 545-555Crossref PubMed Scopus (966) Google Scholar). HSF1 is constitutively expressed as an inactive monomer, but, upon HS, it trimerizes and binds to the inverted repeats of nGAAn pentamers, known as heat shock element (HSE), in the promoter of Hsps, such as Hsp70 (Perisic et al., 1989Perisic O. Xiao H. Lis J.T. Stable binding of Drosophila heat shock factor to head-to-head and tail-to-tail repeats of a conserved 5 bp recognition unit.Cell. 1989; 59: 797-806Abstract Full Text PDF PubMed Scopus (324) Google Scholar, Westwood et al., 1991Westwood J.T. Clos J. Wu C. Stress-induced oligomerization and chromosomal relocalization of heat-shock factor.Nature. 1991; 353: 822-827Crossref PubMed Scopus (310) Google Scholar). HSF1 then recruits co-factors that dramatically increase the transcription of Hsps (Akerfelt et al., 2010Akerfelt M. Morimoto R.I. Sistonen L. Heat shock factors: integrators of cell stress, development and lifespan.Nat. Rev. Mol. Cell Biol. 2010; 11: 545-555Crossref PubMed Scopus (966) Google Scholar). HSF1 also plays an important role in aging and longevity (Morley and Morimoto, 2004Morley J.F. Morimoto R.I. Regulation of longevity in Caenorhabditis elegans by heat shock factor and molecular chaperones.Mol. Biol. Cell. 2004; 15: 657-664Crossref PubMed Scopus (538) Google Scholar), protects organisms from obesity by regulating energy expenditure (Ma et al., 2015Ma X. Xu L. Alberobello A.T. Gavrilova O. Bagattin A. Skarulis M. Liu J. Finkel T. Mueller E. Celastrol Protects against Obesity and Metabolic Dysfunction through Activation of a HSF1-PGC1α Transcriptional Axis.Cell Metab. 2015; 22: 695-708Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), and reduces susceptibility to stress in elderly hearts (Locke and Tanguay, 1996Locke M. Tanguay R.M. Diminished heat shock response in the aged myocardium.Cell Stress Chaperones. 1996; 1: 251-260Crossref PubMed Scopus (116) Google Scholar). More importantly, cancer cells co-opt HSF1 to support malignancy (Dai et al., 2007Dai C. Whitesell L. Rogers A.B. Lindquist S. Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis.Cell. 2007; 130: 1005-1018Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar, Mendillo et al., 2012Mendillo M.L. Santagata S. Koeva M. Bell G.W. Hu R. Tamimi R.M. Fraenkel E. Ince T.A. Whitesell L. Lindquist S. HSF1 drives a transcriptional program distinct from heat shock to support highly malignant human cancers.Cell. 2012; 150: 549-562Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar), making its reduction in level or activity a potentially better target for cancer therapy than the several small inhibitory molecules against HSPs that are in ongoing clinical trials. In contrast, enhanced chaperone expression by activation of HSF1 can improve the prognosis of protein aggregate-related neurodegenerative disorders (Neef et al., 2011Neef D.W. Jaeger A.M. Thiele D.J. Heat shock transcription factor 1 as a therapeutic target in neurodegenerative diseases.Nat. Rev. Drug Discov. 2011; 10: 930-944Crossref PubMed Scopus (203) Google Scholar). This dichotomy in the roles of HSF1 in cancer and neurological diseases requires a deeper understanding of the precise molecular mechanism of HSF1-driven gene regulation before clinical application of HSF1-based therapeutic tools. Understanding the HSR-regulated networks of genes is also important to decipher how healthy cells maintain proteostasis. Earlier genome-wide studies using microarray and RNA sequencing (RNA-seq) indicated that additional genes besides Hsps are regulated during the HSR (Brown et al., 2014Brown J.B. Boley N. Eisman R. May G.E. Stoiber M.H. Duff M.O. Booth B.W. Wen J. Park S. Suzuki A.M. et al.Diversity and dynamics of the Drosophila transcriptome.Nature. 2014; 512: 393-399Crossref PubMed Scopus (417) Google Scholar, Trinklein et al., 2004Trinklein N.D. Murray J.I. Hartman S.J. Botstein D. Myers R.M. The role of heat shock transcription factor 1 in the genome-wide regulation of the mammalian heat shock response.Mol. Biol. Cell. 2004; 15: 1254-1261Crossref PubMed Scopus (261) Google Scholar). These assays measure stable mRNA and lack the temporal resolution to reveal the first-order transcriptional regulatory mechanisms. Transcription regulation consists of several steps, any one of which might regulate gene expression, including RNA polymerase II (Pol II) recruitment to the promoter, promoter-proximal pausing, release from the pause, and Pol II elongation (Fuda et al., 2009Fuda N.J. Ardehali M.B. Lis J.T. Defining mechanisms that regulate RNA polymerase II transcription in vivo.Nature. 2009; 461: 186-192Crossref PubMed Scopus (382) Google Scholar). Despite many efforts, the breadth of transcriptional regulation during HSR, the precise step(s) modulated, and the kinetics and dynamics of the regulation remain to be fully understood. Here we examine the HSR at the transcriptional level using precision nuclear run-on sequencing (PRO-seq) (Kwak et al., 2013Kwak H. Fuda N.J. Core L.J. Lis J.T. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing.Science. 2013; 339: 950-953Crossref PubMed Scopus (452) Google Scholar),- an assay that maps transcriptionally engaged Pol II at nucleotide resolution by sequencing of nascent RNA. PRO-seq measurements in mouse embryonic fibroblasts (MEFs) derived from HSF1 knockout (Hsf1−/−) mice, HSF1 and HSF2 double knockout (Hsf1&2−/−) mice, and their wild-type (WT) littermates (Lecomte et al., 2010Lecomte S. Desmots F. Le Masson F. Le Goff P. Michel D. Christians E.S. Le Dréan Y. Roles of heat shock factor 1 and 2 in response to proteasome inhibition: consequence on p53 stability.Oncogene. 2010; 29: 4216-4224Crossref PubMed Scopus (32) Google Scholar, McMillan et al., 1998McMillan D.R. Xiao X. Shao L. Graves K. Benjamin I.J. Targeted disruption of heat shock transcription factor 1 abolishes thermotolerance and protection against heat-inducible apoptosis.J. Biol. Chem. 1998; 273: 7523-7528Crossref PubMed Scopus (432) Google Scholar, McMillan et al., 2002McMillan D.R. Christians E. Forster M. Xiao X. Connell P. Plumier J.C. Zuo X. Richardson J. Morgan S. Benjamin I.J. Heat shock transcription factor 2 is not essential for embryonic development, fertility, or adult cognitive and psychomotor function in mice.Mol. Cell. Biol. 2002; 22: 8005-8014Crossref PubMed Scopus (65) Google Scholar) were compared with each other and to HSF1 chromatin immunoprecipitation sequencing (ChIP-seq) data to identify the genome-wide targets of HSF1 and its role in the HSR. A time course of PRO-seq and HSF1 ChIP-seq during heat shock (HS) revealed both primary and secondary transcriptional responses. We found HSF1 to be critical for induction of Hsps, other chaperones, and over 200 additional genes. However, the activation and repression of transcription during HSR are remarkably extensive, and the majority of these changes are HSF1-independent. Among these, a collection of cytoskeletal genes is transiently induced by a novel regulator of the HSR. Our analyses deciphered the mechanistic step in transcription where HSF1 acts to induce transcription as well as the HSF1-independent mechanism of global repression. Together, these comprehensive and highly sensitive analyses indicate that HSR is much more elaborate than previously appreciated and that regulators in addition to HSF1 are mobilized. To characterize the global changes in transcription associated with HSR and to understand the role of HSF1, we performed genome-wide PRO-seq assays on WT and Hsf1−/− MEFs. We prepared two biological replicates of PRO-seq libraries at 37°C (no heat shock [NHS]) and 2.5, 12, and 60 min after an instantaneous HS at 42°C (Figure 1A). The libraries were sequenced to high depth (Table S1) and mapped to the mouse genome (mm10). The biological replicates correlated well (Figure S1A; Table S2), and, as expected, the Hsf1−/− MEFs produced no HSF1 protein (Figure 1B) nor any PRO-seq reads in the deleted region of the Hsf1 gene (Figure S1B). Normalization of genomic libraries by conventional methods such as total mapped reads or ribosomal RNA reads are inadequate when dealing with significant changes in total transcription. Therefore, we devised a novel approach for normalizing the PRO-seq libraries using PRO-seq reads from the 3′ end of very long genes (>400 kb), the regions beyond the advancing or receding wave of Pol II even at the longest HS time point (60 min of HS) (Figures S1C and S1D). This normalization approach was validated using three different tests. First, the PRO-seq density after normalization in the 3′ ends of significantly upregulated and downregulated genes at 12 min of HS was unchanged, whereas the 5′ ends showed the expected change (Figure S1E; note that this includes a larger collection of genes than that used for normalization and excludes the genes used for normalization). Second, a set of genes identified as unaffected during HSR in MEFs using microarrays (Trinklein et al., 2004Trinklein N.D. Murray J.I. Hartman S.J. Botstein D. Myers R.M. The role of heat shock transcription factor 1 in the genome-wide regulation of the mammalian heat shock response.Mol. Biol. Cell. 2004; 15: 1254-1261Crossref PubMed Scopus (261) Google Scholar) showed no changes in PRO-seq density between NHS and HS conditions after normalization (Figure S1F). Third, a previously defined group of housekeeping genes (de la Grange et al., 2005de la Grange P. Dutertre M. Martin N. Auboeuf D. FAST DB: a website resource for the study of the expression regulation of human gene products.Nucleic Acids Res. 2005; 33: 4276-4284Crossref PubMed Scopus (71) Google Scholar) also showed no systematic deviation between HS and NHS conditions (Figure S1G). After normalization, genes that could be falsely detected in differential expression analysis because of transcription running past the 3′ end of upstream genes and internal transcription start site (TSS) or intronic enhancers were eliminated using discriminative Regulatory Element detection from GRO-seq (Danko et al., 2015Danko C.G. Hyland S.L. Core L.J. Martins A.L. Waters C.T. Lee H.W. Cheung V.G. Kraus W.L. Lis J.T. Siepel A. Identification of active transcriptional regulatory elements from GRO-seq data.Nat. Methods. 2015; 12: 433-438Crossref PubMed Scopus (105) Google Scholar; Figure S2A; Experimental Procedures). A substantial fraction of the transcriptome changes upon HS, and the number of genes detected and the levels of change progressively increase with time (Figure 1C; Figure S2B). Moreover, the kinetics and dynamics of change in transcription are remarkably diverse (Figure 1D). First, many Hsps are robustly and persistently induced in an HSF1-dependent manner (such as Hsph1 with ∼60-fold induction). Second, many genes are immediately and transiently induced (like Vcl), where the advancing wave of newly transcribing Pol II is particularly noticeable. This induction is independent of HSF1. Third, many genes show late induction (such as Ptprm), and the majority of these are also independent of HSF1. Fourth, a large fraction of the expressed genes (like Kif14) are significantly downregulated, and nearly all are independent of HSF1. Overall, DESeq2 identifies significant upregulation of 10% and downregulation of 55% of all active genes (Figure 1E). For the majority of these genes, the change in transcription measured by PRO-seq is recapitulated at the mRNA level, measured by RNA-seq (Shalgi et al., 2014Shalgi R. Hurt J.A. Lindquist S. Burge C.B. Widespread inhibition of posttranscriptional splicing shapes the cellular transcriptome following heat shock.Cell Rep. 2014; 7: 1362-1370Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), despite the fundamental difference between the two assays and mRNA stability being a part of RNA-seq measurement (Figure S2C). Detecting a change in RNA-seq requires a higher change in transcription than that required for PRO-seq because of the higher steady-state mRNA level compared with nascent RNA, leading to a diminished change in levels seen by RNA-seq relative to PRO-seq (Figures S2C and S2D). Overall, our results indicate that the gene regulation in response to HS occurs at the level of transcription and consists of multiple distinct regulatory programs that are captured here with high spatiotemporal resolution and sensitivity afforded by the PRO-seq assay. The number of genes showing similar regulation in both WT and Hsf1−/− MEFs is unexpectedly large (Figures 1E and 1F). The overlap reported in Figure 1F is likely an underestimation because many of the uniquely upregulated genes in WT MEFs are upregulated in Hsf1−/− MEFs and vice versa but do not meet the DESeq2 threshold (Figure S2E). The downregulated genes show even more overlap between WT and Hsf1−/− MEFs, indicating that transcriptional repression occurs by mechanisms that are largely HSF1-independent. Gene ontology (GO) analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (Huang et al., 2009Huang W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4: 44-57Crossref PubMed Scopus (25477) Google Scholar) shows, as expected, that genes upregulated only in WT MEFs are enriched for chaperones involved in protein folding and stress response (Figure S2F). Genes upregulated only in Hsf1−/− MEFs are enriched for ATPase and protein kinases, whereas those upregulated in both cell types are enriched for transcription factors (TFs) and protein kinases involved in developmental processes. Overall, more than 87% of genes regulated at 60 min of HS in the WT are similarly regulated in Hsf1−/− MEFs, indicating that HSF1-independent mechanisms mediate much of the widespread change in transcription upon HS. To understand the role of HSF1 binding during HSR, we performed HSF1 ChIP-seq in WT and Hsf1−/− MEFs at NHS, 12-min HS, and 60-min HS (Figure 2A). We optimized ChIP-seq parameters such as sonication (Figure S3A), cross-linkers and cross-linking duration (Figure S3B), and antibody concentrations (Figure S3C) and used two different antibodies (Table S4) recognizing different parts of HSF1 to maximize detection of HSF binding sites and to minimize false positive peaks (Chen et al., 2012Chen Y. Negre N. Li Q. Mieczkowska J.O. Slattery M. Liu T. Zhang Y. Kim T.-K. He H.H. Zieba J. et al.Systematic evaluation of factors influencing ChIP-seq fidelity.Nat. Methods. 2012; 9: 609-614Crossref PubMed Scopus (112) Google Scholar). Biological replicates correlated well (Figure S3D) and were combined, and ChIP-seq peaks were called using model-based analysis for ChIP-seq (MACS). As expected, we found prominent HSF1 peaks in the promoters of classical Hsps (for example, Hsph1), and the two antibodies generated similar ChIP-seq profiles (Figure 2B). The HSF1 peaks identified here are highly specific; 89% of the HSF1-bound sites contain canonical HSEs (p < 0.00001) (Figures S3E and S3F), and the fold enrichment of HSF1 peaks correlates with the motif match score of the HSE beneath the peaks (Figure S3G). Although HSF1 occupies some sites prior to HS, most sites are detectably bound only after HS (Figure 2C). The majority of HSF1 peaks are located far from the nearest TSSs (Figure 2D). However, all classical inducible Hsps have HSF1 binding within 1 kb upstream of their TSS (Figure S3H). Therefore, we defined a region 1 kb upstream and 500 bp downstream of the TSS as the promoter and examined the distribution of HSF1 binding on promoter, intragenic, and intergenic regions. The density of HSF1 peaks is highest in promoters; however, higher incidences of absolute binding events occur in intragenic and intergenic regions (Figure 2E). PRO-seq reveals that 17% of genes with HSF1 bound to their promoters undergo HSF1-dependent transcription induction upon HS (Figure 2F, magenta bars); however, 53% and 4% of genes with HSF1 bound on their promoters are repressed or unchanged upon HS independently of HSF1 (blue bars and purple bar, respectively). This demonstrates that promoter-bound HSF1 does not always induce transcription and may require a promoter to have additional features with which it can collaborate. Moreover, nearly all of the HSF1 promoter-bound repressed genes are also repressed in Hsf1−/− MEFs, indicating that promoter-bound HSF1 is not responsible for repression. Intriguingly, 13% of the HSF1 promoter-bound genes induced upon HS are also induced in Hsf1−/− MEFs (Figure 2F, green bar), indicating that some HSF1-bound genes do not require HSF1 for their induction. Similarly, promoter binding of HSF1 is not always necessary, even for genes showing HSF1-dependent transcription. Only ∼35% of genes that depend on HSF1 for induction upon HS have HSF1 bound at their promoters (Figure 2G). This indicates that HSF1 can exert its influence from a distance, presumably from an enhancer. Together, these observations show that the promoter-bound HSF1 is not responsible for the induction or repression of the majority of HS-regulated genes. Past studies have proposed that HSF1 bound in the gene body creates an obstacle to transcribing Pol II that leads to repression of transcription (Guertin and Lis, 2010Guertin M.J. Lis J.T. Chromatin landscape dictates HSF binding to target DNA elements.PLoS Genet. 2010; 6: e1001114Crossref PubMed Scopus (154) Google Scholar, Westwood et al., 1991Westwood J.T. Clos J. Wu C. Stress-induced oligomerization and chromosomal relocalization of heat-shock factor.Nature. 1991; 353: 822-827Crossref PubMed Scopus (310) Google Scholar). Here we find that transcription of genes that have HSF1 bound in the gene body is mostly regulated in an HSF1-independent manner (Figure S3I). Approximately 50% of these genes are repressed upon HS; however, this repression occurs in Hsf1−/− MEFs as well. To assess whether the gene body-bound HSF1 creates steric hindrance to transcribing Pol II, we examined PRO-seq density around HSF1 intragenic bound sites at 60 min of HS. We detect divergent transcription at these sites, a signature of enhancers (Core et al., 2014Core L.J. Martins A.L. Danko C.G. Waters C.T. Siepel A. Lis J.T. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers.Nat. Genet. 2014; 46: 1311-1320Crossref PubMed Scopus (374) Google Scholar), which was reduced in Hsf1−/− (Figure 2H). PRO-seq levels upstream of the HSF1 sites revealed no significant Pol II accumulation that would be expected from the steric hindrance created by the gene body-bound HSF1. Therefore, the HSF1 bound in the body of a gene is not an obstacle to transcription and does not contribute to repression during HS. The very early kinetics of induction of genes upon HS beyond the classic Drosophila Hsps (O’Brien and Lis, 1991O’Brien T. Lis J.T. RNA polymerase II pauses at the 5′ end of the transcriptionally induced Drosophila hsp70 gene.Mol. Cell. Biol. 1991; 11: 5285-5290Crossref PubMed Scopus (100) Google Scholar) have not been examined to date. Here we find that many genes in MEFs are significantly induced by 2.5 min of HS, and the majority of these early induced (EI) genes are HSF1-independent (Figure 3A). Induction of these genes continues to 12 min of HS, after which transcription declines to below basal levels (Figure 3B). GO analysis revealed that many of these EI genes, especially the HSF1-independent ones, encode proteins with a biological function related to cytoskeletal structure and function (Figure 3C). Dynamic rearrangement of the cytoskeleton in cells has been previously documented during HSR (Laszlo, 1992Laszlo A. The effects of hyperthermia on mammalian cell structure and function.Cell Prolif. 1992; 25: 59-87Crossref PubMed Scopus (123) Google Scholar), and cytoskeleton proteins are critical for survival during heat stress (Baird et al., 2014Baird N.A. Douglas P.M. Simic M.S. Grant A.R. Moresco J.J. Wolff S.C. Yates 3rd, J.R. Manning G. Dillin A. HSF-1-mediated cytoskeletal integrity determines thermotolerance and life span.Science. 2014; 346: 360-363Crossref PubMed Scopus (115) Google Scholar). Proteomics analysis also showed an increase in the level of some cytoskeletal proteins in nuclear extract after 2 hr of HS (Raychaudhuri et al., 2014Raychaudhuri S. Loew C. Körner R. Pinkert S. Theis M. Hayer-Hartl M. Buchholz F. Hartl F.U. Interplay of acetyltransferase EP300 and the proteasome system in regulating heat shock transcription factor 1.Cell. 2014; 156: 975-985Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). However, the extremely rapid and transient induction of selective cytoskeleton genes during HS has not been detected before. Because induction of these genes is predominantly HSF1-independent, we searched for TF-binding motifs in their promoters. Among the 1,200 TF-binding motifs examined, the serum response factor (SRF) binding motif is the most highly enriched (Figure 3D; Figure S4A). SRF is known to induce a class of genes known as immediate-early genes, which are rapidly and transiently induced in response to various extracellular stimuli (Schratt et al., 2001Schratt G. Weinhold B. Lundberg A.S. Schuck S. Berger J. Schwarz H. Weinberg R.A. Rüther U. Nordheim A. Serum response factor is required for immediate-early gene activation yet is dispensable for proliferation of embryonic stem cells.Mol. Cell. Biol. 2001; 21: 2933-2943Crossref PubMed Scopus (124) Google Scholar). SRF is also a known effector of the mitogen-activated protein (MAP) kinase pathway, which is often implicated in stress. Therefore, we examined SRF binding during HS in WT MEFs by ChIP-seq (Table S4). We found that the transiently induced genes upon HS that contain an SRF binding motif in their promoters are bound by SRF in a transient manner, mirroring the kinetics of transcription induction upon HS (Figures 3E and 3F). This finding strongly implicates SRF as a novel regulator of cytoskeletal genes during HSR. One striking feature of PRO-seq data in induced genes is a distinct wave of elongating Pol II that is in the midst of transcription (Figure 3G; Figure S4B). Here we used a three-state hidden Markov model (HMM) (Danko et al., 2013Danko C.G. Hah N. Luo X. Martins A.L. Core L. Lis J.T. Siepel A. Kraus W.L. Signaling pathways differentially affect RNA polymerase II initiation, pausing, and elongation rate in cells.Mol. Cell. 2013; 50: 212-222Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) to calculate the distance traveled by waves of newly released Pol II in EI genes. Our approach calculates the difference in PRO-seq density between time points in 50-bp windows throughout the gene and identifies the region (wave) with a difference in transcription (Figures S4C and S4D). PRO-seq wave measurements using this approach indicate that Pol II release from the pause region of EI genes occurs on average within the first minute and a half. The median distance traveled by PRO-seq wave at 2.5 min of HS is 2.7 kb (Figure S4E), and the average elongation rate of Pol II in EI genes is 2.6 kb/min, calculated using the length of the PRO-seq wave at 2.5 min of HS and 12 min of HS in genes induced at both time points (Figure 3H). Moreover, the lengths of PRO-seq waves at 2.5 min of HS in cytoskeleton genes and classical Hsps are very similar (Figure 3I), and the induction kinetics of HSF1-dependent and HSF1-independent genes are also highly analogous (Figure S4E). These findings demonstrate that transcription is induced very early during HSR and that the kinetics of induction are similar between HSF1-dependent (Hsps) and HSF1-independent (cytoskeleton) genes. Historically, transcriptionally induced genes have been the focus of HS studies. However, many more genes are transcriptionally repressed than induced upon HS, and this global repression is independent of HSF1 (Figure 4A). Genes undergoing repression display two distinct kinetics: some genes are gradually and consistently repressed over the course of HS (Pcdh18), but the majority are repressed only after 12-min HS (Cdkal1) (Figures 4B and 4C). The PRO-seq pattern following HS provides clues to how repression is mediated. Although the PRO-seq density decreases on the gene body, it increases on the 5′ end of repressed genes (Figure 4D; Figure S4F). This accumulation of Pol II in the first 100 bp downstream of the TSS (Figure 4E) indicates that the transcriptional repression in the majority of the downregulated genes is a result of reduced paused Pol II release into productive elongation. This extensive downregulation could, in principle, be a result of a thermally induced increase in transcription rate of elongating Pol II that would decrease Pol II density in the gene body. We calculated the Pol II elongation rate by comparing positions of the clearing wave of Pol II in downregulated genes at 12 min of HS and 60 min of HS. We found the average elongation rate of Pol II during HS to be 2.1 kb/min (Figure S4G), which is comparable with the elongation rate of Pol II under normal temperature in mouse embryonic stem cells (1.8–2.4 kb/min) (Jonkers et al., 2014Jonkers I. Kwak H. Lis J.T. Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons.eLife. 2014; 3: e02407Crossref Scopus (345) Google Scholar). Furthermore, the downregulation of transcription observed by PRO-seq is nicely reflected at the mRNA level, as measured by RNA-seq (Figure S2C, right panel). Therefore, the decrease in gene body PRO-seq reads is not the result of an increas" @default.
• W2306540679 created "2016-06-24" @default.
• W2306540679 creator A5024923108 @default.
• W2306540679 creator A5056279051 @default.
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• W2306540679 creator A5063117763 @default.
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• W2306540679 date "2016-04-01" @default.
• W2306540679 modified "2023-10-14" @default.
• W2306540679 title "Mammalian Heat Shock Response and Mechanisms Underlying Its Genome-wide Transcriptional Regulation" @default.
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