ABSTRACT
Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1 µm), but burst size and frequency are enhanced when the condensate moves in proximity (<1 µm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting.
Subject(s)
Gene Expression Regulation , SOXB1 Transcription Factors , Super Enhancers , Transcription, Genetic , DNA/genetics , Enhancer Elements, Genetic , SOXB1 Transcription Factors/genetics , Animals , Mice , Embryonic Stem Cells/metabolism , Microscopy/methodsABSTRACT
Ultraviolet (UV) irradiation and other genotoxic stresses induce bulky DNA lesions, which threaten genome stability and cell viability. Cells have evolved two main repair pathways to remove such lesions: global genome nucleotide excision repair (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER). The modes by which these subpathways recognize DNA lesions are distinct, but they converge onto the same downstream steps for DNA repair. Here, we first summarize the current understanding of these repair mechanisms, specifically focusing on the roles of stalled RNA polymerase II, Cockayne syndrome protein B (CSB), CSA and UV-stimulated scaffold protein A (UVSSA) in TC-NER. We also discuss the intriguing role of protein ubiquitylation in this process. Additionally, we highlight key aspects of the effect of UV irradiation on transcription and describe the role of signaling cascades in orchestrating this response. Finally, we describe the pathogenic mechanisms underlying xeroderma pigmentosum and Cockayne syndrome, the two main diseases linked to mutations in NER factors.
Subject(s)
Cockayne Syndrome , Humans , Cockayne Syndrome/genetics , Cockayne Syndrome/metabolism , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , Transcription, Genetic , DNA Repair , DNA Damage , DNA/genetics , DNA/metabolism , Carrier Proteins/metabolismABSTRACT
A fundamental feature of cellular growth is that total protein and RNA amounts increase with cell size to keep concentrations approximately constant. A key component of this is that global transcription rates increase in larger cells. Here, we identify RNA polymerase II (RNAPII) as the limiting factor scaling mRNA transcription with cell size in budding yeast, as transcription is highly sensitive to the dosage of RNAPII but not to other components of the transcriptional machinery. Our experiments support a dynamic equilibrium model where global RNAPII transcription at a given size is set by the mass action recruitment kinetics of unengaged nucleoplasmic RNAPII to the genome. However, this only drives a sub-linear increase in transcription with size, which is then partially compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to scale mRNA amounts with cell size.
Subject(s)
Cell Size , RNA Polymerase II , Transcription, Genetic , Feedback , RNA Polymerase II/metabolism , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
In eukaryotes, transcription of protein-coding genes requires the assembly at core promoters of a large preinitiation machinery containing RNA polymerase II (RNAPII) and general transcription factors (GTFs). Transcription is potentiated by regulatory elements called enhancers, which are recognized by specific DNA-binding transcription factors that recruit cofactors and convey, following chromatin remodeling, the activating cues to the preinitiation complex. This review summarizes nearly five decades of work on transcription initiation by describing the sequential recruitment of diverse molecular players including the GTFs, the Mediator complex, and DNA repair factors that support RNAPII to enable RNA synthesis. The elucidation of the transcription initiation mechanism has greatly benefited from the study of altered transcription components associated with human diseases that could be considered transcription syndromes.
Subject(s)
RNA Polymerase II/metabolism , Regulatory Sequences, Nucleic Acid , Transcription Factor TFIID/genetics , Transcription Factor TFIIH/genetics , Transcription Initiation, Genetic/physiology , DNA Repair/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Mediator Complex/genetics , Mediator Complex/metabolism , Mutation , Promoter Regions, Genetic , RNA Polymerase II/genetics , SyndromeABSTRACT
Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.
Subject(s)
Cyclin-Dependent Kinase 9/metabolism , Molecular Targeted Therapy , Neoplasms/drug therapy , Neoplasms/genetics , Protein Phosphatase 2/metabolism , RNA-Binding Proteins/metabolism , Transcription, Genetic , Tumor Suppressor Proteins/metabolism , Animals , Cell Line, Tumor , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Humans , Mice, Inbred NOD , Phosphorylation , Protein Binding , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , Substrate SpecificityABSTRACT
Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30-35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase "clamp head loop" and the TFIIF "charged region" that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems.
Subject(s)
DNA/chemistry , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Initiation, Genetic , Amino Acid Sequence , Cryoelectron Microscopy , DNA/ultrastructure , Models, Biological , Models, Molecular , Nucleic Acid Conformation , Promoter Regions, Genetic , RNA Polymerase II/ultrastructure , Sequence Deletion , Transcription Factor TFIIH , Transcription Factors, TFII/metabolismABSTRACT
In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.
Subject(s)
DNA Damage , RNA Polymerase II/metabolism , DNA Repair , HEK293 Cells , Humans , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription, Genetic , Ubiquitination , Ultraviolet RaysABSTRACT
Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.
Subject(s)
DNA Repair/physiology , RNA Polymerase II/metabolism , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , DNA/metabolism , DNA Damage/physiology , DNA Helicases/metabolism , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , Female , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA Polymerase II/genetics , UbiquitinationABSTRACT
Accurate regulation of mRNA termination is required for correct gene expression. Here, we describe a role for SCAF4 and SCAF8 as anti-terminators, suppressing the use of early, alternative polyadenylation (polyA) sites. The SCAF4/8 proteins bind the hyper-phosphorylated RNAPII C-terminal repeat domain (CTD) phosphorylated on both Ser2 and Ser5 and are detected at early, alternative polyA sites. Concomitant knockout of human SCAF4 and SCAF8 results in altered polyA selection and subsequent early termination, leading to expression of truncated mRNAs and proteins lacking functional domains and is cell lethal. While SCAF4 and SCAF8 work redundantly to suppress early mRNA termination, they also have independent, non-essential functions. SCAF8 is an RNAPII elongation factor, whereas SCAF4 is required for correct termination at canonical, distal transcription termination sites in the presence of SCAF8. Together, SCAF4 and SCAF8 coordinate the transition between elongation and termination, ensuring correct polyA site selection and RNAPII transcriptional termination in human cells.
Subject(s)
RNA Polymerase II/metabolism , RNA, Messenger/biosynthesis , RNA-Binding Proteins/metabolism , Serine-Arginine Splicing Factors/metabolism , Transcription Elongation, Genetic , Transcription Termination, Genetic , HEK293 Cells , Humans , Poly A/genetics , Poly A/metabolism , Protein Domains , RNA Polymerase II/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Serine-Arginine Splicing Factors/geneticsABSTRACT
Insulin receptor (IR) signaling is central to normal metabolic control and dysregulated in prevalent chronic diseases. IR binds insulin at the cell surface and transduces rapid signaling via cytoplasmic kinases. However, mechanisms mediating long-term effects of insulin remain unclear. Here, we show that IR associates with RNA polymerase II in the nucleus, with striking enrichment at promoters genome-wide. The target genes were highly enriched for insulin-related functions including lipid metabolism and protein synthesis and diseases including diabetes, neurodegeneration, and cancer. IR chromatin binding was increased by insulin and impaired in an insulin-resistant disease model. Promoter binding by IR was mediated by coregulator host cell factor-1 (HCF-1) and transcription factors, revealing an HCF-1-dependent pathway for gene regulation by insulin. These results show that IR interacts with transcriptional machinery at promoters and identify a pathway regulating genes linked to insulin's effects in physiology and disease.
Subject(s)
Gene Expression Regulation , Genome-Wide Association Study , Receptor, Insulin/metabolism , Animals , Cell Line, Tumor , Chromatin/metabolism , Gene Expression Regulation/drug effects , Host Cell Factor C1/antagonists & inhibitors , Host Cell Factor C1/genetics , Host Cell Factor C1/metabolism , Humans , Insulin/metabolism , Insulin/pharmacology , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic , Protein Binding , Protein Subunits/metabolism , RNA Interference , RNA Polymerase II/metabolism , RNA, Small Interfering/metabolism , Receptor, Insulin/chemistry , Signal Transduction/drug effectsABSTRACT
Although cell metabolism has been established as a major regulator of eukaryotic gene expression, the mechanisms underlying this regulation are still being uncovered. Recent years have seen great advances in our understanding of biochemical mechanisms of metabolic regulation of transcription and chromatin. Prime examples include insights into how nutrients and cellular energy status regulate synthesis of ribosomal RNAs by RNA polymerases I and III during ribosome biogenesis and how a variety of enzymes that catalyze modifications of histones in chromatin are regulated by the levels of certain metabolites. This volume of the Annual Review of Biochemistry includes a set of reviews describing these and other advances in understanding aspects of the metabolic regulation of RNA polymerases I and III transcription and chromatin.
Subject(s)
Chromatin/metabolism , Transcription, Genetic , Animals , DNA-Directed RNA Polymerases/metabolism , HumansABSTRACT
The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal â¼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.
Subject(s)
Alternative Splicing/radiation effects , DNA Helicases/genetics , RNA, Untranslated/genetics , Transcription, Genetic , Ultraviolet Rays , Cell Line , Exons , Humans , RNA Polymerase II/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Elongation, Genetic/radiation effects , Transcription Initiation, Genetic/radiation effectsABSTRACT
Chromatin architecture is fundamental in regulating gene expression. To investigate when spatial genome organization is first established during development, we examined chromatin conformation during Drosophila embryogenesis and observed the emergence of chromatin architecture within a tight time window that coincides with the onset of transcription activation in the zygote. Prior to zygotic genome activation, the genome is mostly unstructured. Early expressed genes serve as nucleation sites for topologically associating domain (TAD) boundaries. Activation of gene expression coincides with the establishment of TADs throughout the genome and co-localization of housekeeping gene clusters, which remain stable in subsequent stages of development. However, the appearance of TAD boundaries is independent of transcription and requires the transcription factor Zelda for locus-specific TAD boundary insulation. These results offer insight into when spatial organization of the genome emerges and identify a key factor that helps trigger this architecture.
Subject(s)
Chromatin/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Genome, Insect , Transcriptional Activation , Zygote/metabolism , Animals , Drosophila Proteins/metabolism , Embryo, Nonmammalian/metabolism , Genes, Essential , Nuclear Proteins , RNA Polymerase II/metabolism , Time Factors , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Cell size and growth are intimately related across the evolutionary scale, but whether cell size is important to attain maximal growth or fitness is still an open question. We show that growth rate is a non-monotonic function of cell volume, with maximal values around the critical size of wild-type yeast cells. The transcriptome of yeast and mouse cells undergoes a relative inversion in response to cell size, which we associate theoretically and experimentally with the necessary genome-wide diversity in RNA polymerase II affinity for promoters. Although highly expressed genes impose strong negative effects on fitness when the DNA/mass ratio is reduced, transcriptomic alterations mimicking the relative inversion by cell size strongly restrain cell growth. In all, our data indicate that cells set the critical size to obtain a properly balanced transcriptome and, as a result, maximize growth and fitness during proliferation.
ABSTRACT
The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape in vitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs. We suggest that promoter escape occurs in three major steps. First, the growing RNA displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, the rewinding of the transcription bubble coincides with the eviction of TFIIA, TFIIB, and TBP. Third, the binding of DSIF and NELF facilitates TFIIE and TFIIH dissociation, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of RNA Pol II transcription.
Subject(s)
Phosphoproteins , Promoter Regions, Genetic , RNA Polymerase II , TATA-Box Binding Protein , Transcription Factor TFIIB , Transcription Factors , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Humans , Transcription Factor TFIIB/metabolism , Transcription Factor TFIIB/genetics , TATA-Box Binding Protein/metabolism , TATA-Box Binding Protein/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Transcription Initiation, Genetic , Transcription Factor TFIIH/metabolism , Transcription Factor TFIIH/genetics , Transcription Factor TFIIH/chemistry , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Protein Binding , Transcription Factor TFIIA/metabolism , Transcription Factor TFIIA/genetics , Transcription, Genetic , Transcription Elongation, Genetic , RNA/metabolism , RNA/genetics , Transcription Factors, TFII/metabolism , Transcription Factors, TFII/geneticsABSTRACT
Metazoan gene expression regulation involves pausing of RNA polymerase (Pol II) in the promoter-proximal region of genes and is stabilized by DSIF and NELF. Upon depletion of elongation factors, NELF appears to accompany elongating Pol II past pause sites; however, prior work indicates that NELF prevents Pol II elongation. Here, we report cryoelectron microscopy structures of Pol II-DSIF-NELF complexes with NELF in two distinct conformations corresponding to paused and poised states. The paused NELF state supports Pol II stalling, whereas the poised NELF state enables transcription elongation as it does not support a tilted RNA-DNA hybrid. Further, the poised NELF state can accommodate TFIIS binding to Pol II, allowing for Pol II reactivation at paused or backtracking sites. Finally, we observe that the NELF-A tentacle interacts with the RPB2 protrusion and is necessary for pausing. Our results define how NELF can support pausing, reactivation, and elongation by Pol II.
Subject(s)
Nuclear Proteins , RNA Polymerase II , Animals , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Cryoelectron Microscopy , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Facilitates chromatin transcription (FACT) is a histone chaperone that supports transcription through chromatin in vitro, but its functional roles in vivo remain unclear. Here, we analyze the in vivo functions of FACT with the use of multi-omics analysis after rapid FACT depletion from human cells. We show that FACT depletion destabilizes chromatin and leads to transcriptional defects, including defective promoter-proximal pausing and elongation, and increased premature termination of RNA polymerase II. Unexpectedly, our analysis revealed that promoter-proximal pausing depends not only on the negative elongation factor (NELF) but also on the +1 nucleosome, which is maintained by FACT.
Subject(s)
Chromatin , High Mobility Group Proteins , Nucleosomes , Promoter Regions, Genetic , RNA Polymerase II , Transcription, Genetic , Transcriptional Elongation Factors , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Humans , Transcriptional Elongation Factors/metabolism , Transcriptional Elongation Factors/genetics , Chromatin/metabolism , Chromatin/genetics , Nucleosomes/metabolism , Nucleosomes/genetics , High Mobility Group Proteins/metabolism , High Mobility Group Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , HeLa Cells , Chromatin Assembly and Disassembly , HEK293 Cells , Transcription Elongation, Genetic , Transcription Termination, GeneticABSTRACT
RNA polymerase II (RNA Pol II) can backtrack during transcription elongation, exposing the 3' end of nascent RNA. Nascent RNA sequencing can approximate the location of backtracking events that are quickly resolved; however, the extent and genome-wide distribution of more persistent backtracking are unknown. Consequently, we developed a method to directly sequence the extruded, "backtracked" 3' RNA. Our data show that RNA Pol II slides backward more than 20 nt in human cells and can persist in this backtracked state. Persistent backtracking mainly occurs where RNA Pol II pauses near promoters and intron-exon junctions and is enriched in genes involved in translation, replication, and development, where gene expression is decreased if these events are unresolved. Histone genes are highly prone to persistent backtracking, and the resolution of such events is likely required for timely expression during cell division. These results demonstrate that persistent backtracking can potentially affect diverse gene expression programs.
Subject(s)
RNA Polymerase II , RNA , Humans , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA/genetics , Transcription, Genetic , DNA-Directed RNA Polymerases/geneticsABSTRACT
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , RNA Polymerase II , Transcription, Genetic , Transcriptional Elongation Factors , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Animals , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Transcriptional Elongation Factors/metabolism , Transcriptional Elongation Factors/genetics , HSP70 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/genetics , Positive Transcriptional Elongation Factor B/metabolism , Positive Transcriptional Elongation Factor B/genetics , Promoter Regions, Genetic , CRISPR-Cas Systems , Transcription Factors/metabolism , Transcription Factors/genetics , Polytene Chromosomes/genetics , Polytene Chromosomes/metabolism , Gene Expression Regulation , Phosphorylation , Protein Binding , Heat-Shock Response/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Nucleosomes/metabolism , Nucleosomes/geneticsABSTRACT
Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.