Quantifying RNA synthesis at rate-limiting steps of transcription using nascent RNA-sequencing data

Nascent RNA-sequencing tracks transcription at nucleotide resolution. The genomic distribution of engaged transcription complexes, in turn, uncovers functional genomic regions. Here, we provide analytical steps to (1) identify transcribed regulatory elements de novo genome-wide, (2) quantify engaged transcription complexes at enhancers, promoter-proximal regions, divergent transcripts, gene bodies, and termination windows, and (3) measure distribution of transcription machineries and regulatory proteins across functional genomic regions. This protocol tracks engaged transcription complexes across functional genomic regions demonstrated in human K562 erythroleukemia cells.</p

Comparative interactomes of HSF1 in stress and disease reveal a role for CTCF in HSF1-mediated gene regulation

Heat shock transcription factor 1 (HSF1) orchestrates cellular stress protection by activating or repressing gene transcription in response to protein misfolding, oncogenic cell proliferation, and other environmental stresses. HSF1 is tightly regulated via intramolecular repressive interactions, post-ranslational modifications, and protein-protein interactions. How these HSF1 regulatory protein interactions are altered in response to acute and chronic stress is largely unknown. To elucidate the profile of HSF1 protein interactions under normal growth and chronic and acutely stressful conditions, quantitative proteomics studies identified interacting proteins in the response to heat shock or in the presence of a poly-glutamine aggregation protein cell-based model of Huntington's disease. These studies identified distinct protein interaction partners of HSF1 as well as changes in the magnitude of shared interactions as a function of each stressful condition. Several novel HSF1-interacting proteins were identified that encompass a wide variety of cellular functions, including roles in DNA repair, mRNA processing, and regulation of RNA polymerase II. One HSF1 partner, CTCF, interacted with HSF1 in a stress-inducible manner and functions in repression of specific HSF1 target genes. Understanding how HSF1 regulates gene repression is a crucial question, given the dysregulation of HSF1 target genes in both cancer and neurodegeneration. These studies expand our understanding of HSF1-mediated gene repression and provide key insights into HSF1 regulation via protein-protein interactions.Peer reviewe

HSFs drive transcription of distinct genes and enhancers during oxidative stress and heat shock

Reprogramming of transcription is critical for the survival under cellular stress. Heat shock has provided an excellent model to investigate nascent transcription in stressed cells, but the molecular mechanisms orchestrating RNA synthesis during other types of stress are unknown. We utilized PRO-seq and ChIP-seq to study how Heat Shock Factors, HSF1 and HSF2, coordinate transcription at genes and enhancers upon oxidative stress and heat shock. We show that pause-release of RNA polymerase II (Pol II) is a universal mechanism regulating gene transcription in stressed cells, while enhancers are activated at the level of Pol II recruitment. Moreover, besides functioning as conventional promoter-binding transcription factors, HSF1 and HSF2 bind to stress-induced enhancers to trigger Pol II pause-release from poised gene promoters. Importantly, HSFs act at distinct genes and enhancers in a stress type-specific manner. HSF1 binds to many chaperone genes upon oxidative and heat stress but activates them only in heat-shocked cells. Under oxidative stress, HSF1 localizes to a unique set of promoters and enhancers to trans-activate oxidative stress-specific genes. Taken together, we show that HSFs function as multi-stress-responsive factors that activate distinct genes and enhancers when encountering changes in temperature and redox state

Global SUMOylation on active chromatin is an acute heat stress response restricting transcription

ArticleBackground Cells have developed many ways to cope with external stress. One distinctive feature in acute proteotoxic stresses, such as heat shock (HS), is rapid post-translational modification of proteins by SUMOs (small ubiquitin-like modifier proteins; SUMOylation). While many of the SUMO targets are chromatin proteins, there is scarce information on chromatin binding of SUMOylated proteins in HS and the role of chromatin SUMOylation in the regulation of transcription. Results We mapped HS-induced genome-wide changes in chromatin occupancy of SUMO-2/3-modified proteins in K562 and VCaP cells using ChIP-seq. Chromatin SUMOylation was further correlated with HS-induced global changes in transcription using GRO-seq and RNA polymerase II (Pol2) ChIP-seq along with ENCODE data for K562 cells. HS induced a rapid and massive rearrangement of chromatin SUMOylation pattern: SUMOylation was gained at active promoters and enhancers associated with multiple transcription factors, including heat shock factor 1. Concomitant loss of SUMOylation occurred at inactive intergenic chromatin regions that were associated with CTCF-cohesin complex and SETDB1 methyltransferase complex. In addition, HS triggered a dynamic chromatin binding of SUMO ligase PIAS1, especially onto promoters. The HS-induced SUMOylation on chromatin was most notable at promoters of transcribed genes where it positively correlated with active transcription and Pol2 promoter-proximal pausing. Furthermore, silencing of SUMOylation machinery either by depletion of UBC9 or PIAS1 enhanced expression of HS-induced genes. Conclusions HS-triggered SUMOylation targets promoters and enhancers of actively transcribed genes where it restricts the transcriptional activity of the HS-induced genes. PIAS1-mediated promoter SUMOylation is likely to regulate Pol2-associated factors in HS.Publisher’s pd

Stress biology:Complexity and multifariousness in health and disease

Preserving and regulating cellular homeostasis in the light of changing environmental conditions or developmental processes is of pivotal importance for single cellular and multicellular organisms alike. To counteract an imbalance in cellular homeostasis transcriptional programs evolved, called the heat shock response, unfolded protein response, and integrated stress response, that act cell-autonomously in most cells but in multicellular organisms are subjected to cell-nonautonomous regulation. These transcriptional programs downregulate the expression of most genes but increase the expression of heat shock genes, including genes encoding molecular chaperones and proteases, proteins involved in the repair of stress-induced damage to macromolecules and cellular structures. Sixty-one years after the discovery of the heat shock response by Ferruccio Ritossa, many aspects of stress biology are still enigmatic. Recent progress in the understanding of stress responses and molecular chaperones was reported at the 12th International Symposium on Heat Shock Proteins in Biology, Medicine and the Environment in the Old Town Alexandria, VA, USA from 28th to 31st of October 2023.</p

CBP-HSF2 structural and functional interplay in Rubinstein-Taybi neurodevelopmental disorder

Rubinstein-Taybi syndrome (RSTS) is a neurodevelopmental disorder with unclear underlying mechanisms. Here, the authors unravel the contribution of a stress-responsive pathway to RSTS where impaired HSF2 acetylation, due to RSTS-associated CBP/EP300 mutations, alters the expression of neurodevelopmental players, in keeping with hallmarks of cell-cell adhesion defects.Patients carrying autosomal dominant mutations in the histone/lysine acetyl transferases CBP or EP300 develop a neurodevelopmental disorder: Rubinstein-Taybi syndrome (RSTS). The biological pathways underlying these neurodevelopmental defects remain elusive. Here, we unravel the contribution of a stress-responsive pathway to RSTS. We characterize the structural and functional interaction between CBP/EP300 and heat-shock factor 2 (HSF2), a tuner of brain cortical development and major player in prenatal stress responses in the neocortex: CBP/EP300 acetylates HSF2, leading to the stabilization of the HSF2 protein. Consequently, RSTS patient-derived primary cells show decreased levels of HSF2 and HSF2-dependent alteration in their repertoire of molecular chaperones and stress response. Moreover, we unravel a CBP/EP300-HSF2-N-cadherin cascade that is also active in neurodevelopmental contexts, and show that its deregulation disturbs neuroepithelial integrity in 2D and 3D organoid models of cerebral development, generated from RSTS patient-derived iPSC cells, providing a molecular reading key for this complex pathology.</p

Choosing the target loci : heat shock factors HSF1 and HSF2 as regulators of cell stress and development

Heat shock factors (HSFs) are an evolutionarily well conserved family of transcription factors that coordinate stress-induced gene expression and direct versatile physiological processes in eukaryote organisms. The essentiality of HSFs for cellular homeostasis has been well demonstrated, mainly through HSF1-induced transcription of heat shock protein (HSP) genes. HSFs are important regulators of many fundamental processes such as gametogenesis, metabolic control and aging, and are involved in pathological conditions including cancer progression and neurodegenerative diseases. In each of the HSF-mediated processes, however, the detailed mechanisms of HSF family members and their complete set of target genes have remained unknown. Recently, rapid advances in chromatin studies have enabled genome-wide characterization of protein binding sites in a high resolution and in an unbiased manner. In this PhD thesis, these novel methods that base on chromatin immunoprecipitation (ChIP) are utilized and the genome-wide target loci for HSF1 and HSF2 are identified in cellular stress responses and in developmental processes. The thesis and its original publications characterize the individual and shared target genes of HSF1 and HSF2, describe HSF1 as a potent transactivator, and discover HSF2 as an epigenetic regulator that coordinates gene expression throughout the cell cycle progression. In male gametogenesis, novel physiological functions for HSF1 and HSF2 are revealed and HSFs are demonstrated to control the expression of X- and Y-chromosomal multicopy genes in a silenced chromatin environment. In stressed human cells, HSF1 and HSF2 are shown to coordinate the expression of a wide variety of genes including genes for chaperone machinery, ubiquitin, regulators of cell cycle progression and signaling. These results highlight the importance of cell type and cell cycle phase in transcriptional responses, reveal the myriad of processes that are adjusted in a stressed cell and describe novel mechanisms that maintain transcriptional memory in mitotic cell division

PRO-IP-seq tracks molecular modifications of engaged Pol II complexes at nucleotide resolution

Abstract RNA Polymerase II (Pol II) is a multi-subunit complex that undergoes covalent modifications as transcription proceeds through genes and enhancers. Rate-limiting steps of transcription control Pol II recruitment, site and degree of initiation, pausing duration, productive elongation, nascent transcript processing, transcription termination, and Pol II recycling. Here, we develop Precision Run-On coupled to Immuno-Precipitation sequencing (PRO-IP-seq), which double-selects nascent RNAs and transcription complexes, and track phosphorylation of Pol II C-terminal domain (CTD) at nucleotide-resolution. We uncover precise positional control of Pol II CTD phosphorylation as transcription proceeds from the initiating nucleotide (+1 nt), through early (+18 to +30 nt) and late (+31 to +60 nt) promoter-proximal pause, and into productive elongation. Pol II CTD is predominantly unphosphorylated from initiation until the early pause-region, whereas serine-2- and serine-5-phosphorylations are preferentially deposited in the later pause-region. Upon pause-release, serine-7-phosphorylation rapidly increases and dominates over the region where Pol II assembles elongation factors and accelerates to its full elongational speed. Interestingly, tracking CTD modifications upon heat-induced transcriptional reprogramming demonstrates that Pol II with phosphorylated CTD remains paused on thousands of heat-repressed genes. These results uncover dynamic Pol II regulation at rate-limiting steps of transcription and provide a nucleotide-resolution technique for tracking composition of engaged transcription complexes

Inhibition of CDK12 elevates cancer cell dependence on P-TEFb by stimulation of RNA polymerase II pause release

P-TEFb and CDK12 facilitate transcriptional elongation by RNA polymerase II. Given the prominence of both kinases in cancer, gaining a better understanding of their interplay could inform the design of novel anti-cancer strategies. While down-regulation of DNA repair genes in CDK12-targeted cancer cells is being explored therapeutically, little is known about mechanisms and significance of transcriptional induction upon inhibition of CDK12. We show that selective targeting of CDK12 in colon cancer-derived cells activates P-TEFb via its release from the inhibitory 7SK snRNP. In turn, P-TEFb stimulates Pol II pause release at thousands of genes, most of which become newly dependent on P-TEFb. Amongst the induced genes are those stimulated by hallmark pathways in cancer, including p53 and NF-κB. Consequently, CDK12-inhibited cancer cells exhibit hypersensitivity to inhibitors of P-TEFb. While blocking P-TEFb triggers their apoptosis in a p53-dependent manner, it impedes cell proliferation irrespective of p53 by preventing induction of genes downstream of the DNA damage-induced NF-κB signaling. In summary, stimulation of Pol II pause release at the signal-responsive genes underlies the functional dependence of CDK12-inhibited cancer cells on P-TEFb. Our study establishes the mechanistic underpinning for combinatorial targeting of CDK12 with either P-TEFb or the induced oncogenic pathways in cancer.Peer reviewe