-
Molecular Cell Dec 2022Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues, making it difficult to define the...
Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues, making it difficult to define the mechanism of transcription factor function using slow genetic methods. We used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO1, to define the mechanism by which it regulates gene expression programs. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses and integrating CUT&RUN, ATAC-seq, and eRNA analysis with deep proteomic analysis, we defined PAX3-FOXO1 function at a small network of direct transcriptional targets. PAX3-FOXO1 degradation impaired RNA polymerase pause release and transcription elongation at most regulated gene targets. Moreover, the activity of PAX3-FOXO1 at enhancers controlling this core network was surprisingly selective, affecting single elements in super-enhancers. This combinatorial analysis indicated that PAX3-FOXO1 was continuously required to maintain chromatin accessibility and enhancer architecture at regulated enhancers.
Topics: Proteomics; Regulatory Sequences, Nucleic Acid; Base Sequence; DNA-Directed RNA Polymerases; Chromatin Immunoprecipitation Sequencing; Transcription Factors
PubMed: 36395771
DOI: 10.1016/j.molcel.2022.10.025 -
Molecular Cell May 2021The metazoan-specific acetyltransferase p300/CBP is involved in activating signal-induced, enhancer-mediated transcription of cell-type-specific genes. However, the...
The metazoan-specific acetyltransferase p300/CBP is involved in activating signal-induced, enhancer-mediated transcription of cell-type-specific genes. However, the global kinetics and mechanisms of p300/CBP activity-dependent transcription activation remain poorly understood. We performed genome-wide, time-resolved analyses to show that enhancers and super-enhancers are dynamically activated through p300/CBP-catalyzed acetylation, deactivated by the opposing deacetylase activity, and kinetic acetylation directly contributes to maintaining cell identity at very rapid (minutes) timescales. The acetyltransferase activity is dispensable for the recruitment of p300/CBP and transcription factors but essential for promoting the recruitment of TFIID and RNAPII at virtually all enhancers and enhancer-regulated genes. This identifies pre-initiation complex assembly as a dynamically controlled step in the transcription cycle and reveals p300/CBP-catalyzed acetylation as the signal that specifically promotes transcription initiation at enhancer-regulated genes. We propose that p300/CBP activity uses a "recruit-and-release" mechanism to simultaneously promote RNAPII recruitment and pause release and thereby enables kinetic activation of enhancer-mediated transcription.
Topics: Acetylation; Animals; Biocatalysis; Chromatin; Down-Regulation; Enhancer Elements, Genetic; Histone Deacetylases; Histones; Lysine; Mice; Models, Biological; Nuclear Proteins; Protein Binding; RNA Polymerase II; Transcription Factor TFIID; Transcription Factors; Transcription Initiation, Genetic; p300-CBP Transcription Factors
PubMed: 33765415
DOI: 10.1016/j.molcel.2021.03.008 -
Molecular Cell Nov 2021Transcription progression is governed by multitasking regulators including SPT5, an evolutionarily conserved factor implicated in virtually all transcriptional steps...
Transcription progression is governed by multitasking regulators including SPT5, an evolutionarily conserved factor implicated in virtually all transcriptional steps from enhancer activation to termination. Here we utilize a rapid degradation system and reveal crucial functions of SPT5 in maintaining cellular and chromatin RNA polymerase II (Pol II) levels. Rapid SPT5 depletion causes a pronounced reduction of paused Pol II at promoters and enhancers, distinct from negative elongation factor (NELF) degradation resulting in short-distance paused Pol II redistribution. Most genes exhibit downregulation, but not upregulation, accompanied by greatly impaired transcription activation, altered chromatin landscape at enhancers, and severe Pol II processivity defects at gene bodies. Phosphorylation of an SPT5 linker at serine 666 potentiates pause release and is antagonized by Integrator-PP2A (INTAC) targeting SPT5 and Pol II, while phosphorylation of the SPT5 C-terminal region links to 3' end termination. Our findings position SPT5 as an essential positive regulator of global transcription.
Topics: Animals; Antigens, Differentiation, B-Lymphocyte; Chromatin; Chromosomal Proteins, Non-Histone; Enhancer Elements, Genetic; Fibroblasts; Genome; HEK293 Cells; Histocompatibility Antigens Class II; Humans; Mice; Mutation; Nuclear Proteins; Phosphorylation; Promoter Regions, Genetic; RNA Polymerase II; RNA-Seq; Regulatory Sequences, Nucleic Acid; Transcription, Genetic; Transcriptional Activation; Transcriptional Elongation Factors
PubMed: 34534457
DOI: 10.1016/j.molcel.2021.08.029 -
Nature Genetics Aug 2023How enhancers control target gene expression over long genomic distances remains an important unsolved problem. Here we investigated enhancer-promoter communication by...
How enhancers control target gene expression over long genomic distances remains an important unsolved problem. Here we investigated enhancer-promoter communication by integrating data from nucleosome-resolution genomic contact maps, nascent transcription and perturbations affecting either RNA polymerase II (Pol II) dynamics or the activity of thousands of candidate enhancers. Integration of new Micro-C experiments with published CRISPRi data demonstrated that enhancers spend more time in close proximity to their target promoters in functional enhancer-promoter pairs compared to nonfunctional pairs, which can be attributed in part to factors unrelated to genomic position. Manipulation of the transcription cycle demonstrated a key role for Pol II in enhancer-promoter interactions. Notably, promoter-proximal paused Pol II itself partially stabilized interactions. We propose an updated model in which elements of transcriptional dynamics shape the duration or frequency of interactions to facilitate enhancer-promoter communication.
Topics: RNA Polymerase II; Enhancer Elements, Genetic; Promoter Regions, Genetic; Transcription, Genetic
PubMed: 37430091
DOI: 10.1038/s41588-023-01442-7 -
Nature Communications May 2022c-MYC (MYC) is a major driver of prostate cancer tumorigenesis and progression. Although MYC is overexpressed in both early and metastatic disease and associated with...
c-MYC (MYC) is a major driver of prostate cancer tumorigenesis and progression. Although MYC is overexpressed in both early and metastatic disease and associated with poor survival, its impact on prostate transcriptional reprogramming remains elusive. We demonstrate that MYC overexpression significantly diminishes the androgen receptor (AR) transcriptional program (the set of genes directly targeted by the AR protein) in luminal prostate cells without altering AR expression. Analyses of clinical specimens reveal that concurrent low AR and high MYC transcriptional programs accelerate prostate cancer progression toward a metastatic, castration-resistant disease. Data integration of single-cell transcriptomics together with ChIP-seq uncover an increase in RNA polymerase II (Pol II) promoter-proximal pausing at AR-dependent genes following MYC overexpression without an accompanying deactivation of AR-bound enhancers. Altogether, our findings suggest that MYC overexpression antagonizes the canonical AR transcriptional program and contributes to prostate tumor initiation and progression by disrupting transcriptional pause release at AR-regulated genes.
Topics: Cell Line, Tumor; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Male; Prostate; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc; Receptors, Androgen
PubMed: 35562350
DOI: 10.1038/s41467-022-30257-z -
Molecular Cell May 2023Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger pausing by stabilizing RNA polymerase (RNAP) swiveling and inhibiting DNA...
Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger pausing by stabilizing RNA polymerase (RNAP) swiveling and inhibiting DNA translocation. The N-terminal domain (NGN) of the only universal transcription factor, NusG/Spt5, modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs enhance pauses, whereas anti-pausing NusGs suppress pauses. Little is known about pausing and NusG in the human pathogen Mycobacterium tuberculosis (Mtb). We report that MtbNusG is pro-pausing. MtbNusG captures paused, swiveled RNAP by contacts to the RNAP protrusion and nontemplate-DNA wedged between the NGN and RNAP gate loop. In contrast, anti-pausing Escherichia coli (Eco) NGN contacts the MtbRNAP gate loop, inhibiting swiveling and pausing. Using CRISPR-mediated genetics, we show that pro-pausing NGN is required for mycobacterial fitness. Our results define an essential function of mycobacterial NusG and the structural basis of pro- versus anti-pausing NusG activity, with broad implications for the function of all NusG orthologs.
Topics: Humans; Transcription Factors; Transcription, Genetic; Mycobacterium tuberculosis; Escherichia coli Proteins; DNA-Directed RNA Polymerases; Escherichia coli; DNA; Peptide Elongation Factors
PubMed: 37116494
DOI: 10.1016/j.molcel.2023.04.007 -
Bone Research Jun 2023As the major cell precursors in osteogenesis, mesenchymal stem cells (MSCs) are indispensable for bone homeostasis and development. However, the primary mechanisms...
As the major cell precursors in osteogenesis, mesenchymal stem cells (MSCs) are indispensable for bone homeostasis and development. However, the primary mechanisms regulating osteogenic differentiation are controversial. Composed of multiple constituent enhancers, super enhancers (SEs) are powerful cis-regulatory elements that identify genes that ensure sequential differentiation. The present study demonstrated that SEs were indispensable for MSC osteogenesis and involved in osteoporosis development. Through integrated analysis, we identified the most common SE-targeted and osteoporosis-related osteogenic gene, ZBTB16. ZBTB16, positively regulated by SEs, promoted MSC osteogenesis but was expressed at lower levels in osteoporosis. Mechanistically, SEs recruited bromodomain containing 4 (BRD4) at the site of ZBTB16, which then bound to RNA polymerase II-associated protein 2 (RPAP2) that transported RNA polymerase II (POL II) into the nucleus. The subsequent synergistic regulation of POL II carboxyterminal domain (CTD) phosphorylation by BRD4 and RPAP2 initiated ZBTB16 transcriptional elongation, which facilitated MSC osteogenesis via the key osteogenic transcription factor SP7. Bone-targeting ZBTB16 overexpression had a therapeutic effect on the decreased bone density and remodeling capacity of Brd4 Prx1-cre mice and osteoporosis (OP) models. Therefore, our study shows that SEs orchestrate the osteogenesis of MSCs by targeting ZBTB16 expression, which provides an attractive focus and therapeutic target for osteoporosis. Without SEs located on osteogenic genes, BRD4 is not able to bind to osteogenic identity genes due to its closed structure before osteogenesis. During osteogenesis, histones on osteogenic identity genes are acetylated, and OB-gain SEs appear, enabling the binding of BRD4 to the osteogenic identity gene ZBTB16. RPAP2 transports RNA Pol II from the cytoplasm to the nucleus and guides Pol II to target ZBTB16 via recognition of the navigator BRD4 on SEs. After the binding of the RPAP2-Pol II complex to BRD4 on SEs, RPAP2 dephosphorylates Ser5 at the Pol II CTD to terminate the transcriptional pause, and BRD4 phosphorylates Ser2 at the Pol II CTD to initiate transcriptional elongation, which synergistically drives efficient transcription of ZBTB16, ensuring proper osteogenesis. Dysregulation of SE-mediated ZBTB16 expression leads to osteoporosis, and bone-targeting ZBTB16 overexpression is efficient in accelerating bone repair and treating osteoporosis.
PubMed: 37280207
DOI: 10.1038/s41413-023-00267-8 -
Molecular Cell May 2023Gene expression in metazoans is controlled by promoter-proximal pausing of RNA polymerase II, which can undergo productive elongation or promoter-proximal termination....
Gene expression in metazoans is controlled by promoter-proximal pausing of RNA polymerase II, which can undergo productive elongation or promoter-proximal termination. Integrator-PP2A (INTAC) plays a crucial role in determining the fate of paused polymerases, but the underlying mechanisms remain unclear. Here, we establish a rapid degradation system to dissect the functions of INTAC RNA endonuclease and phosphatase modules. We find that both catalytic modules function at most if not all active promoters and enhancers, yet differentially affect polymerase fate. The endonuclease module induces promoter-proximal termination, with its disruption leading to accumulation of elongation-incompetent polymerases and downregulation of highly expressed genes, while elongation-competent polymerases accumulate at lowly expressed genes and non-coding elements, leading to their upregulation. The phosphatase module primarily prevents the release of paused polymerases and limits transcriptional activation, especially for highly paused genes. Thus, both INTAC catalytic modules have unexpectedly general yet distinct roles in dynamic transcriptional control.
Topics: RNA Polymerase II; Phosphoric Monoester Hydrolases; Gene Expression Regulation; Transcriptional Activation; Up-Regulation; Transcription, Genetic
PubMed: 37080207
DOI: 10.1016/j.molcel.2023.03.022 -
Molecular Cell Jul 2023Nuclear receptor-binding SET-domain protein 1 (NSD1), a methyltransferase that catalyzes H3K36me2, is essential for mammalian development and is frequently dysregulated...
Nuclear receptor-binding SET-domain protein 1 (NSD1), a methyltransferase that catalyzes H3K36me2, is essential for mammalian development and is frequently dysregulated in diseases, including Sotos syndrome. Despite the impacts of H3K36me2 on H3K27me3 and DNA methylation, the direct role of NSD1 in transcriptional regulation remains largely unknown. Here, we show that NSD1 and H3K36me2 are enriched at cis-regulatory elements, particularly enhancers. NSD1 enhancer association is conferred by a tandem quadruple PHD (qPHD)-PWWP module, which recognizes p300-catalyzed H3K18ac. By combining acute NSD1 depletion with time-resolved epigenomic and nascent transcriptomic analyses, we demonstrate that NSD1 promotes enhancer-dependent gene transcription by facilitating RNA polymerase II (RNA Pol II) pause release. Notably, NSD1 can act as a transcriptional coactivator independent of its catalytic activity. Moreover, NSD1 enables the activation of developmental transcriptional programs associated with Sotos syndrome pathophysiology and controls embryonic stem cell (ESC) multilineage differentiation. Collectively, we have identified NSD1 as an enhancer-acting transcriptional coactivator that contributes to cell fate transition and Sotos syndrome development.
Topics: Animals; Humans; Nuclear Proteins; Chromatin; Sotos Syndrome; Histone Methyltransferases; Transcription Factors; Cell Differentiation; Mammals; Histone-Lysine N-Methyltransferase
PubMed: 37402365
DOI: 10.1016/j.molcel.2023.06.007 -
Molecular Cancer Mar 2023Diffuse large B-cell lymphoma (DLBCL) exhibits significant genetic heterogeneity which contributes to drug resistance, necessitating development of novel therapeutic...
Diffuse large B-cell lymphoma (DLBCL) exhibits significant genetic heterogeneity which contributes to drug resistance, necessitating development of novel therapeutic approaches. Pharmacological inhibitors of cyclin-dependent kinases (CDK) demonstrated pre-clinical activity in DLBCL, however many stalled in clinical development. Here we show that AZD4573, a selective inhibitor of CDK9, restricted growth of DLBCL cells. CDK9 inhibition (CDK9i) resulted in rapid changes in the transcriptome and proteome, with downmodulation of multiple oncoproteins (eg, MYC, Mcl-1, JunB, PIM3) and deregulation of phosphoinotiside-3 kinase (PI3K) and senescence pathways. Following initial transcriptional repression due to RNAPII pausing, we observed transcriptional recovery of several oncogenes, including MYC and PIM3. ATAC-Seq and ChIP-Seq experiments revealed that CDK9i induced epigenetic remodeling with bi-directional changes in chromatin accessibility, suppressed promoter activation and led to sustained reprograming of the super-enhancer landscape. A CRISPR library screen suggested that SE-associated genes in the Mediator complex, as well as AKT1, confer resistance to CDK9i. Consistent with this, sgRNA-mediated knockout of MED12 sensitized cells to CDK9i. Informed by our mechanistic findings, we combined AZD4573 with either PIM kinase or PI3K inhibitors. Both combinations decreased proliferation and induced apoptosis in DLBCL and primary lymphoma cells in vitro as well as resulted in delayed tumor progression and extended survival of mice xenografted with DLBCL in vivo. Thus, CDK9i induces reprogramming of the epigenetic landscape, and super-enhancer driven recovery of select oncogenes may contribute to resistance to CDK9i. PIM and PI3K represent potential targets to circumvent resistance to CDK9i in the heterogeneous landscape of DLBCL.
Topics: Animals; Mice; Apoptosis; Cell Line, Tumor; Epigenesis, Genetic; Lymphoma, Large B-Cell, Diffuse; Phosphatidylinositol 3-Kinases; Transcription Factors; Cyclin-Dependent Kinase 9; Drug Resistance, Neoplasm
PubMed: 36998071
DOI: 10.1186/s12943-023-01762-6