Transcriptional elongation control of hypoxic response.

The release of paused RNA polymerase II (RNAPII) from promoter-proximal regions is tightly controlled to ensure proper regulation of gene expression. The elongation factor PTEF-b is known to release paused RNAPII via phosphorylation of the RNAPII C-terminal domain by its cyclin-dependent kinase component, CDK9. However, the signal and stress-specific roles of the various RNAPII-associated macromolecular complexes containing PTEF-b/CDK9 are not yet clear. Here, we identify and characterize the CDK9 complex required for transcriptional response to hypoxia. Contrary to previous reports, our data indicate that a CDK9 complex containing BRD4 but not AFF1/4 is essential for this hypoxic stress response. We demonstrate that BRD4 bromodomains (BET) are dispensable for the release of paused RNAPII at hypoxia-activated genes and that BET inhibition by JQ1 is insufficient to impair hypoxic gene response. Mechanistically, we demonstrate that the C-terminal region of BRD4 is required for Polymerase-Associated Factor-1 Complex (PAF1C) recruitment to establish an elongation-competent RNAPII complex at hypoxia-responsive genes. PAF1C disruption using a small-molecule inhibitor (iPAF1C) impairs hypoxia-induced, BRD4-mediated RNAPII release. Together, our results provide insight into potentially targetable mechanisms that control the hypoxia-responsive transcriptional elongation.

Changing structures, changing paradigms: NELF helps regulate paused or elongating RNA polymerase II.

Using cryo-EM and biochemical methods, Su and Vos1 discover an alternative NELF structural state that enables transcription and switches NELF-RNA polymerase II (RNAPII) compatibility with other RNAPII-associated factors that regulate pausing, elongation, termination, and transcription-coupled DNA repair.

Chemoenzymatic Synthesis of 4,5-Dihydroxyisoleucine Fragment of α-Amanitin.

The ability of α-amanitin to potently inhibit RNA polymerase II (RNAP II) has elicited further research into its use as a novel payload for antibody-drug conjugates. Despite this promise, the de novo synthesis of α-amanitin is still a major challenge as it possesses an unusual bicyclic octapeptide structure that contains several oxidized amino acids, most notably 4,5-dihydroxy-l-isoleucine. Here, we report a concise chemoenzymatic synthesis of this key amino acid residue, which features two regioselective and diastereoselective enzymatic C-H oxidations on l-isoleucine.

Cooperative interaction of interferon regulatory factor -1 and bromodomain-containing protein 4 on RNA polymerase activation for intrinsic innate immunity.

Introduction: The human orthopneumovirus, Respiratory Syncytial Virus (RSV), is the causative agent of severe lower respiratory tract infections (LRTI) and exacerbations of chronic lung diseases. In immune competent hosts, RSV productively infects highly differentiated epithelial cells, where it elicits robust anti-viral, cytokine and remodeling programs. By contrast, basal cells are relatively resistant to RSV infection, in part, because of constitutive expression of an intrinsic innate immune response (IIR) consisting of a subgroup of interferon (IFN) responsive genes. The mechanisms controlling the intrinsic IIR are not known. Methods: Here, we use human small airway epithelial cell hSAECs as a multipotent airway stem cell model to examine regulatory control of an intrinsic IIR pathway. Results: We find hSAECs express patterns of intrinsic IIRs, highly conserved with pluri- and multi-potent stem cells. We demonstrate a core intrinsic IIR network consisting of Bone Marrow Stromal Cell Antigen 2 (Bst2), Interferon Induced Transmembrane Protein 1 (IFITM1) and Toll-like receptor (TLR3) expression are directly under IRF1 control. Moreover, expression of this intrinsic core is rate-limited by ambient IRF1• phospho-Ser 2 CTD RNA Polymerase II (pSer2 Pol II) complexes binding to their proximal promoters. In response to RSV infection, the abundance of IRF1 and pSer2 Pol II binding is dramatically increased, with IRF1 complexing to the BRD4 chromatin remodeling complex (CRC). Using chromatin immunoprecipitation in IRF1 KD cells, we find that the binding of BRD4 is IRF1 independent. Using a small molecule inhibitor of the BRD4 acetyl lysine binding bromodomain (BRD4i), we further find that BRD4 bromodomain interactions are required for stable BRD4 promoter binding to the intrinsic IIR core promoters, as well as for RSV-inducible pSer2 Pol II recruitment. Surprisingly, BRD4i does not disrupt IRF1-BRD4 interactions, but disrupts both RSV-induced BRD4 and IRF1 interactions with pSer2 Pol II. Conclusions: We conclude that the IRF1 functions in two modes- in absence of infection, ambient IRF1 mediates constitutive expression of the intrinsic IIR, whereas in response to RSV infection, the BRD4 CRC independently activates pSer2 Pol II to mediates robust expression of the intrinsic IIR. These data provide insight into molecular control of anti-viral defenses of airway basal cells.

RBM22 regulates RNA polymerase II 5' pausing, elongation rate, and termination by coordinating 7SK-P-TEFb complex and SPT5.

BACKGROUND: Splicing factors are vital for the regulation of RNA splicing, but some have also been implicated in regulating transcription. The underlying molecular mechanisms of their involvement in transcriptional processes remain poorly understood. RESULTS: Here, we describe a direct role of splicing factor RBM22 in coordinating multiple steps of RNA Polymerase II (RNAPII) transcription in human cells. The RBM22 protein widely occupies the RNAPII-transcribed gene locus in the nucleus. Loss of RBM22 promotes RNAPII pause release, reduces elongation velocity, and provokes transcriptional readthrough genome-wide, coupled with production of transcripts containing sequences from downstream of the gene. RBM22 preferentially binds to the hyperphosphorylated, transcriptionally engaged RNAPII and coordinates its dynamics by regulating the homeostasis of the 7SK-P-TEFb complex and the association between RNAPII and SPT5 at the chromatin level. CONCLUSIONS: Our results uncover the multifaceted role of RBM22 in orchestrating the transcriptional program of RNAPII and provide evidence implicating a splicing factor in both RNAPII elongation kinetics and termination control.

A fine-scale Arabidopsis chromatin landscape reveals chromatin conformation-associated transcriptional dynamics.

Plants, as sessile organisms, deploy transcriptional dynamics for adapting to extreme growth conditions such as cold stress. Emerging evidence suggests that chromatin architecture contributes to transcriptional regulation. However, the relationship between chromatin architectural dynamics and transcriptional reprogramming in response to cold stress remains unclear. Here, we apply a chemical-crosslinking assisted proximity capture (CAP-C) method to elucidate the fine-scale chromatin landscape, revealing chromatin interactions within gene bodies closely associated with RNA polymerase II (Pol II) densities across initiation, pausing, and termination sites. We observe dynamic changes in chromatin interactions alongside Pol II activity alterations during cold stress, suggesting local chromatin dynamics may regulate Pol II activity. Notably, cold stress does not affect large-scale chromatin conformations. We further identify a comprehensive promoter-promoter interaction (PPI) network across the genome, potentially facilitating co-regulation of gene expression in response to cold stress. Our study deepens the understanding of chromatin conformation-associated gene regulation in plant response to cold.

MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch.

The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.

Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release.

During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase (Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and is then released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.

Endogenous EWSR1 Exists in Two Visual Modalities That Reflect Its Associations with Nucleic Acids and Concentration at Sites of Active Transcription.

EWSR1 is a member of the FET family of nucleic acid binding proteins that includes FUS and TAF15. Here, we report the systematic analysis of endogenous EWSR1's cellular organization in human cells. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.

TANGO6 regulates cell proliferation via COPI vesicle-mediated RPB2 nuclear entry.

Coat protein complex I (COPI) vesicles mediate the retrograde transfer of cargo between Golgi cisternae and from the Golgi to the endoplasmic reticulum (ER). However, their roles in the cell cycle and proliferation are unclear. This study shows that TANGO6 associates with COPI vesicles via two transmembrane domains. The TANGO6 N- and C-terminal cytoplasmic fragments capture RNA polymerase II subunit B (RPB) 2 in the cis-Golgi during the G1 phase. COPI-docked TANGO6 carries RPB2 to the ER and then to the nucleus. Functional disruption of TANGO6 hinders the nuclear entry of RPB2, which accumulates in the cytoplasm, causing cell cycle arrest in the G1 phase. The conditional depletion or overexpression of TANGO6 in mouse hematopoietic stem cells results in compromised or expanded hematopoiesis. Our study results demonstrate that COPI vesicle-associated TANGO6 plays a role in the regulation of cell cycle progression by directing the nuclear transfer of RPB2, making it a potential target for promoting or arresting cell expansion.

Role of transcriptional cofactors in cardiovascular diseases.

Cardiovascular disease is a main cause of mortality in the world and the highest incidence of all diseases. However, the mechanism of the pathogenesis of cardiovascular disease is still unclear, and we need to continue to explore its mechanism of action. The occurrence and development of cardiovascular disease is significantly associated with genetic abnormalities, and gene expression is affected by transcriptional regulation. In this complex process, the protein-protein interaction promotes the RNA polymerase II to the initiation site. And in this process of transcriptional regulation, transcriptional cofactors are responsible for passing cues from enhancers to promoters and promoting the binding of RNA polymerases to promoters, so transcription cofactors playing a key role in gene expression regulation. There is growing evidence that transcriptional cofactors play a critical role in cardiovascular disease. Transcriptional cofactors can promote or inhibit transcription by affecting the function of transcription factors. It can affect the initiation and elongation process of transcription by forming complexes with transcription factors, which are important for the stabilization of DNA rings. It can also act as a protein that interacts with other proteins to affect the expression of other genes. Therefore, the aim of this overview is to summarize the effect of some transcriptional cofactors such as BRD4, EP300, MED1, EZH2, YAP, SIRT6 in cardiovascular disease and to provide a promising therapeutic strategy for the treatment of cardiovascular disease.

The Rtf1/Prf1-dependent histone modification axis counteracts multi-drug resistance in fission yeast.

RNA polymerase II transcription elongation directs an intricate pattern of histone modifications. This pattern includes a regulatory cascade initiated by the elongation factor Rtf1, leading to monoubiquitylation of histone H2B, and subsequent methylation of histone H3 on lysine 4. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate gene expression. To address this question, we investigated a drug resistance phenotype that characterizes defects in this axis in the model eukaryote Schizosaccharomyces pombe (fission yeast). The mutations caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU) that correlated with a reduced effect of HU on dNTP pools, reduced requirement for the S-phase checkpoint, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain of the RNA polymerase II large subunit Rpb1 led to similar phenotypes. Moreover, all the HU-resistant mutants also exhibited resistance to several azole-class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 C-terminal repeat domain in controlling fungal drug tolerance.

Disordered C-terminal domain drives spatiotemporal confinement of RNAPII to enhance search for chromatin targets.

Efficient gene expression requires RNA polymerase II (RNAPII) to find chromatin targets precisely in space and time. How RNAPII manages this complex diffusive search in three-dimensional nuclear space remains largely unknown. The disordered carboxy-terminal domain (CTD) of RNAPII, which is essential for recruiting transcription-associated proteins, forms phase-separated droplets in vitro, hinting at a potential role in modulating RNAPII dynamics. In the present study, we use single-molecule tracking and spatiotemporal mapping in living yeast to show that the CTD is required for confining RNAPII diffusion within a subnuclear region enriched for active genes, but without apparent phase separation into condensates. Both Mediator and global chromatin organization are required for sustaining RNAPII confinement. Remarkably, truncating the CTD disrupts RNAPII spatial confinement, prolongs target search, diminishes chromatin binding, impairs pre-initiation complex formation and reduces transcription bursting. The present study illuminates the pivotal role of the CTD in driving spatiotemporal confinement of RNAPII for efficient gene expression.

Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

Identification of a Novel Selective CDK9 Inhibitor for the Treatment of CRC: Design, Synthesis, and Biological Activity Evaluation.

Cyclin-dependent kinase 9 (CDK9) is a member of the transcription CDK subfamily. In this work, we preliminarily demonstrated the feasibility of CDK9 as a potent target of treatment for colorectal cancer, and a series of novel CDK9 inhibitors were rationally designed and synthesized based on the structure of AZD5438 (a pan CDKs inhibitor reported by AstraZeneca). A novel selective CDK9 inhibitor named CLZX-205, which possessed significant CDK9 inhibitory activity (IC50 = 2.9 nM) with acceptable pharmacokinetic properties and antitumor efficacy in vitro and in vivo, was developed. Research on the mechanism indicated that CLZX-205 could induce apoptosis in the HCT116 cell line by inhibiting phosphorylation of RNA polymerase II at Ser2, which resulted in the inhibition of apoptosis-related genes and proteins expression, and these results were validated at the cellular and tumor tissue levels. Currently, CLZX-205 is undergoing further research as a promising candidate for CRC treatment.

The role of O-GlcNAcylation in RNA polymerase II transcription.

Eukaryotic RNA polymerase II (RNAPII) is responsible for the transcription of the protein-coding genes in the cell. Enormous progress has been made in discovering the protein activities that are required for transcription to occur, but the effects of post-translational modifications (PTMs) on RNAPII transcriptional regulation are much less understood. Most of our understanding relates to the cyclin-dependent kinases (CDKs), which appear to act relatively early in transcription. However, it is becoming apparent that other PTMs play a crucial role in the transcriptional cycle, and it is doubtful that any sort of complete understanding of this regulation is attainable without understanding the spectra of PTMs that occur on the transcriptional machinery. Among these is O-GlcNAcylation. Recent experiments have shown that the O-GlcNAc PTM likely has a prominent role in transcription. This review will cover the role of the O-GlcNAcylation in RNAPII transcription during initiation, pausing, and elongation, which will hopefully be of interest to both O-GlcNAc and RNAPII transcription researchers.

Transcription elongation mechanisms of RNA polymerases I, II, and III and their therapeutic implications.

Transcription is a tightly regulated, complex, and essential cellular process in all living organisms. Transcription is comprised of three steps, transcription initiation, elongation, and termination. The distinct transcription initiation and termination mechanisms of eukaryotic RNA polymerases I, II, and III (Pols I, II, and III) have long been appreciated. Recent methodological advances have empowered high-resolution investigations of the Pols' transcription elongation mechanisms. Here, we review the kinetic similarities and differences in the individual steps of Pol I-, II-, and III-catalyzed transcription elongation, including NTP binding, bond formation, pyrophosphate release, and translocation. This review serves as an important summation of Saccharomyces cerevisiae (yeast) Pol I, II, and III kinetic investigations which reveal that transcription elongation by the Pols is governed by distinct mechanisms. Further, these studies illustrate how basic, biochemical investigations of the Pols can empower the development of chemotherapeutic compounds.

Molecular Mechanism of RNA Polymerase II Transcriptional Mutagenesis by the Epimerizable DNA Lesion, Fapy·dG.

Oxidative DNA lesions cause significant detrimental effects on a living species. Two major DNA lesions resulting from dG oxidation, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo) and formamidopyrimidine (Fapy·dG), are produced from a common chemical intermediate. Fapy·dG is formed in comparable yields under oxygen-deficient conditions. Replicative bypass of Fapy·dG in human cells is more mutagenic than that of 8-OxodGuo. Despite the biological importance of transcriptional mutagenesis, there are no reports of the effects of Fapy·dG on RNA polymerase II (Pol II) activity. Here we perform comprehensive kinetic studies to investigate the impact of Fapy·dG on three key transcriptional fidelity checkpoint steps by Pol II: insertion, extension, and proofreading steps. The ratios of error-free versus error-prone incorporation opposite Fapy·dG are significantly reduced in comparison with undamaged dG. Similarly, Fapy·dG:A mispair is extended with comparable efficiency as that of the error-free, Fapy·dG:C base pair. The α- and ß-configurational isomers of Fapy·dG have distinct effects on Pol II insertion and extension. Pol II can preferentially cleave error-prone products by proofreading. To further understand the structural basis of transcription processing of Fapy·dG, five different structures were solved, including Fapy·dG template-loading state (apo), error-free cytidine triphosphate (CTP) binding state (prechemistry), error-prone ATP binding state (prechemistry), error-free Fapy·dG:C product state (postchemistry), and error-prone Fapy·dG:A product state (postchemistry), revealing distinctive nucleotide binding and product states. Taken together, our study provides a comprehensive mechanistic framework for better understanding how Fapy·dG lesions impact transcription and subsequent pathological consequences.

Structural basis for RNA polymerase II ubiquitylation and inactivation in transcription-coupled repair.

During transcription-coupled DNA repair (TCR), RNA polymerase II (Pol II) transitions from a transcriptionally active state to an arrested state that allows for removal of DNA lesions. This transition requires site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase, a process that is facilitated by ELOF1 in an unknown way. Using cryogenic electron microscopy, biochemical assays and cell biology approaches, we found that ELOF1 serves as an adaptor to stably position UVSSA and CRL4CSA on arrested Pol II, leading to ligase neddylation and activation of Pol II ubiquitylation. In the presence of ELOF1, a transcription factor IIS (TFIIS)-like element in UVSSA gets ordered and extends through the Pol II pore, thus preventing reactivation of Pol II by TFIIS. Our results provide the structural basis for Pol II ubiquitylation and inactivation in TCR.

Maternal KLF17 controls zygotic genome activation by acting as a messenger for RNA Pol II recruitment in mouse embryos.

Initiation of timely and sufficient zygotic genome activation (ZGA) is crucial for the beginning of life, yet our knowledge of transcription factors (TFs) contributing to ZGA remains limited. Here, we screened the proteome of early mouse embryos after cycloheximide (CHX) treatment and identified maternally derived KLF17 as a potential TF for ZGA genes. Using a conditional knockout (cKO) mouse model, we further investigated the role of maternal KLF17 and found that it promotes embryonic development and full fertility. Mechanistically, KLF17 preferentially binds to promoters and recruits RNA polymerase II (RNA Pol II) in early 2-cell embryos, facilitating the expression of major ZGA genes. Maternal Klf17 knockout resulted in a downregulation of 9% of ZGA genes and aberrant RNA Pol II pre-configuration, which could be partially rescued by introducing exogenous KLF17. Overall, our study provides a strategy for screening essential ZGA factors and identifies KLF17 as a crucial TF in this process.