The bromodomain and extraterminal motif (BET) proteins are critical drug targets for diseases. The precise functions and relationship of BRD2 with other BET proteins remain elusive mechanistically. Here, we used acute protein degradation and quantitative genomic and proteomic approaches to investigate the primary functions of BRD2 in transcription. We report that BRD2 is required for TAF3-mediated Pol II initiation at promoters with low levels of H3K4me3 and for R-loop suppression during Pol II elongation. Single and double depletion revealed that BRD2 and BRD3 function additively, independently, or perhaps antagonistically in Pol II transcription at different promoters. Furthermore, we found that BRD2 regulates the expression of different genes during embryonic body differentiation processes by promoter priming in embryonic stem cells. Therefore, our results suggest complex interconnections between BRD2 and BRD3 at promoters to fine-tune Pol II initiation and elongation for control of cell state.

Heart failure with reduced ejection fraction (HFrEF) is associated with high mortality, highlighting an urgent need for new therapeutic strategies. As stress-activated cardiac signaling cascades converge on the nucleus to drive maladaptive gene programs, interdicting pathological transcription is a conceptually attractive approach for HFrEF therapy. Here, we demonstrate that CDK7/12/13 are critical regulators of transcription activation in the heart that can be pharmacologically inhibited to improve HFrEF. CDK7/12/13 inhibition using the first-in-class inhibitor THZ1 or RNAi blocks stress-induced transcription and pathologic hypertrophy in cultured rodent cardiomyocytes. THZ1 potently attenuates adverse cardiac remodeling and HFrEF pathogenesis in mice and blocks cardinal features of disease in human iPSC-derived cardiomyocytes. THZ1 suppresses Pol II enrichment at stress-transactivated cardiac genes and inhibits a specific pathologic gene program in the failing mouse heart. These data identify CDK7/12/13 as druggable regulators of cardiac gene transactivation during disease-related stress, suggesting that HFrEF features a critical dependency on transcription that can be therapeutically exploited.

The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs.

Cell behaviors are dictated by epigenetic and transcriptional programs. Little is known about how extracellular stimuli modulate these programs to reshape gene expression and control cell behavioral responses. Here, we interrogated the epigenetic and transcriptional response of endothelial cells to VEGFA treatment and found rapid chromatin changes that mediate broad transcriptomic alterations. VEGFA-responsive genes were associated with active promoters, but changes in promoter histone marks were not tightly linked to gene expression changes. VEGFA altered transcription factor occupancy and the distal epigenetic landscape, which profoundly contributed to VEGFA-dependent changes in gene expression. Integration of gene expression, dynamic enhancer, and transcription factor occupancy changes induced by VEGFA yielded a VEGFA-regulated transcriptional regulatory network, which revealed that the small MAF transcription factors are master regulators of the VEGFA transcriptional program and angiogenesis. Collectively these results revealed that extracellular stimuli rapidly reconfigure the chromatin landscape to coordinately regulate biological responses.

The CCCTC-binding factor (CTCF), which anchors DNA loops that organize the genome into structural domains, has a central role in gene control by facilitating or constraining interactions between genes and their regulatory elements(1,2). In cancer cells, the disruption of CTCF binding at specific loci by somatic mutation(3,4) or DNA hypermethylation(5) results in the loss of loop anchors and consequent activation of oncogenes. By contrast, the germ-cell-specific paralogue of CTCF, BORIS (brother of the regulator of imprinted sites, also known as CTCFL)(6), is overexpressed in several cancers(7-9), but its contributions to the malignant phenotype remain unclear. Here we show that aberrant upregulation of BORIS promotes chromatin interactions in ALK-mutated, MYCN-amplified neuroblastoma(10) cells that develop resistance to ALK inhibition. These cells are reprogrammed to a distinct phenotypic state during the acquisition of resistance, a process defined by the initial loss of MYCN expression followed by subsequent overexpression of BORIS and a concomitant switch in cellular dependence from MYCN to BORIS. The resultant BORIS-regulated alterations in chromatin looping lead to the formation of super-enhancers that drive the ectopic expression of a subset of proneural transcription factors that ultimately define the resistance phenotype. These results identify a previously unrecognized role of BORIS-to promote regulatory chromatin interactions that support specific cancer phenotypes.

Cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and selectively affects the expression of genes involved in the DNA damage response (DDR) and mRNA processing. Yet, the mechanisms underlying such selectivity remain unclear. Here we show that CDK12 inhibition in cancer cells lacking CDK12 mutations results in gene length-dependent elongation defects, inducing premature cleavage and polyadenylation (PCPA) and loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlates with an increased number of intronic polyadenylation sites, a feature especially prominent among DDR genes. Phosphoproteomic analysis indicated that CDK12 directly phosphorylates pre-mRNA processing factors, including those regulating PCPA. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition primarily due to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites.

Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes.

Abstract Key oncogenes can be misregulated in tumor cells by the acquisition of tumor-specific enhancers that direct gene expression through physical contacts (1,2). Identifying the full complement of enhancers regulating each oncogene could identify additional vulnerabilities in tumor expression programs, but remains challenging, because a given enhancer can regulate multiple genes and each of its targets may be genomically distant. Here we use DNA interaction data to construct gene regulatory landscapes for all expressed genes in a tumor cell and demonstrate that sets of enhancers can be assigned to these oncogenes using direct looping and insulated neighborhood data. A subset of these landscapes contains an exceptional amount of transcriptional apparatus, reminiscent of SEs (3), so we term this subset 3D-SEs. Well-characterized oncogenes, including c-Myc, acquire 3D-SEs in tumor cells where a c-Myc¬-associated linear SE was not identified, suggesting SE acquisition by c-Myc and other oncogenes has been underestimated. Each gene's regulatory network can also surprisingly extend beyond the confines of insulated neighborhoods and incorporate additional distal enhancers. Visualizing instances of 3D-SEs with microscopy demonstrates that they are components of liquid-liquid phase-separated bodies in cells, suggesting gene regulatory landscapes underpin these transcriptional condensates. The interactions comprising gene regulatory networks and their target genes thus extend previous interpretations of the targets of enhancers, signaling pathways, and disease-associated enhancer variants. References: 1. Hnisz, Abraham, Lee, et al. and Young, Cell 2013. 2. Mansour, Abraham, et al. and Young and Look, Science 2014. 3. Whyte, Orlando, Hnisz, Abraham, et al. and Young, Cell 2013. Citation Format: Brian J. Abraham, Yang Eric Guo, Denes Hnisz, Charles H. Li, Abraham S. Weintraub, Daniel S. Day, Jurian Schuijers, Tong Ihn Lee, Richard A. Young. Three-dimensional gene regulatory landscapes in normal and cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 971.

Transcriptional dysregulation of the MYC oncogene is among the most frequent events in aggressive tumor cells, and this is generally accomplished by acquisition of a super-enhancer somewhere within the 2.8 Mb TAD where MYC resides. We find that these diverse cancer-specific super-enhancers, differing in size and location, interact with the MYC gene through a common and conserved CTCF binding site located 2 kb upstream of the MYC promoter. Genetic perturbation of this enhancer-docking site in tumor cells reduces CTCF binding, super-enhancer interaction, MYC gene expression, and cell proliferation. CTCF binding is highly sensitive to DNA methylation, and this enhancer-docking site, which is hypomethylated in diverse cancers, can be inactivated through epigenetic editing with dCas9-DNMT. Similar enhancer-docking sites occur at other genes, including genes with prominent roles in multiple cancers, suggesting a mechanism by which tumor cell oncogenes can generally hijack enhancers. These results provide insights into mechanisms that allow a single target gene to be regulated by diverse enhancer elements in different cell types.

Recurrent somatic mutations in core components and modulators of the cohesin ring - a multimeric protein complex that forms a ring structure around DNA and provides spatial genome organization - have been identified across multiple cancer types, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), where they are associated with poor overall survival. Cohesin proteins are involved in sister chromatid cohesion, chromatin organization into loops, transcriptional activation, and DNA damage repair. The mechanisms underlying clonal expansion of these driver mutations are unknown and no therapies have selective efficacy in cohesin-mutant cancers. We sought to determine the effects of mutations in the most frequently mutated cohesin subunit, STAG2, on cohesin complex composition using immunoprecipitation followed by quantitative mass spectrometry (IP-MS), genetic dependencies of STAG2-mutant cells by genome-wide CRISPR screening, and mutant cohesin association with chromatin using chromatin immunoprecipitation followed by sequencing (ChIP-Seq). Our goal was to understand how these mutations contribute to cellular transformation and to identify possible therapeutic targets. Applying IP-MS in AML cell lines engineered with different STAG2 mutations, we identified and validated a switch from STAG2- to its paralog STAG1-containing cohesin complexes. In addition, we observed changes in the interaction of the mutant cohesin complex with proteins involved in DNA repair and replication, including PARP1, and RNA-mediated interaction with RNA splicing machinery, including SF3B family members. We next hypothesized that these cohesin-dependent alterations could lead to shifts in genetic dependencies. Using genome-scale CRISPR-Cas9 screens, we identified preferential dependency of STAG2-mutant cells on STAG1, consistent with our proteomics studies. We also found a striking concordance between additional cellular processes highlighted by IP-MS experiments and observed increased dependency of STAG2-mutant cells on DNA damage repair and mRNA processing. Therefore, STAG2 mutations lead to changes in cohesin complex structure and alter interactions with proteins involved in DNA damage, replication, and RNA modification, which become genetic dependencies in this context. Prompted by this concordance, we evaluated DNA replication, DNA damage and splicing in cohesin-mutant cells. We observed a 4-fold increase in replication fork stalling in STAG2-mutant cells, which was associated with accumulation of double strand DNA breaks and activation of the ATR and ATM DNA damage checkpoints. STAG2-mutant cells demonstrated ~100-fold increased sensitivity to the PARP inhibitor talazoparib, which was consistent across models of other cohesin-mutant subunits. In addition, cohesin-mutant cells showed aberrant splicing and increased sensitivity to treatment with SF3B1 inhibitors E7107 and H3B-8800. In aggregate, genetic or pharmacologic perturbation of DNA damage repair or splicing created a synthetic vulnerability for cohesin-mutant cells in vitro and in vivo. Finally, we explored how STAG1-containing complexes alter cohesin-mediated genome compartmentalization in cohesin-mutant cells. Using ChIP-Seq, we observed that STAG2 loss leads to a global decrease in cohesin binding to chromatin, including at sites of insulated neighborhood boundaries, with subsequent gene expression changes. Loss of cohesin binding was associated with increased enhancer activity and super-enhancer expansion in STAG2-mutant cells. In addition, we identified changes in the co-localization of the mutant cohesin complex with super-enhancer enriched factors, DNA damage repair and splicing machinery. These findings are consistent with a model in which wild type and mutant cohesin complexes, defined by their unique composition and patterns of chromatin binding and architecture, have differential abilities to maintain chromatin organization as it relates to spatial organization of super-enhancers, coactivators and transcription factors, as well as DNA damage repair and splicing machinery. Perturbation of any of these components, which have been recently proposed to form phase-separated nuclear bodies, creates vulnerabilities that may be exploited therapeutically with existing drugs in patients with cohesin-mutated malignancies. Abraham: Syros Pharmaceuticals: Equity Ownership. Seiler:H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. D'Andrea:Intellia Therapeutics: Consultancy; Cedilla Therpeutics: Consultancy, Equity Ownership; EMD-Serono: Consultancy, Research Funding; Sierra: Consultancy, Research Funding; Ideaya: Consultancy, Equity Ownership; Lilly: Consultancy, Research Funding; Formation Biologics: Consultancy. Young:Omega Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Camp4 Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.

Release of promoter-proximally paused RNA polymerase II (RNAPII) is a recently recognized transcriptional regulatory checkpoint. The biological roles of RNAPII pause release and the mechanisms by which extracellular signals control it are incompletely understood. Here we show that VEGF stimulates RNAPII pause release by stimulating acetylation of ETS1, a master endothelial cell transcriptional regulator. In endothelial cells, ETS1 binds transcribed gene promoters and stimulates their expression by broadly increasing RNAPII pause release. 34 VEGF enhances ETS1 chromatin occupancy and increases ETS1 acetylation, enhancing its binding to BRD4, which recruits the pause release machinery and increases RNAPII pause release. Endothelial cell angiogenic responses in vitro and in vivo require ETS1-mediated transduction of VEGF signaling to release paused RNAPII. Our results define an angiogenic pathway in which VEGF enhances ETS1-BRD4 interaction to broadly promote RNAPII pause release and drive angiogenesis.Promoter proximal RNAPII pausing is a rate-limiting transcriptional mechanism. Chen et al. show that this process is essential in angiogenesis by demonstrating that the endothelial master transcription factor ETS1 promotes global RNAPII pause release, and that this process is governed by VEGF.

GO biological process enrichment analysis for the top and bottom 25Â % of genes by PI for each cell type. (XLSX 23028 kb)

Abstract Super-enhancers are clusters of enhancers that drive expression of genes key to development and cancer. We recently showed that super-enhancers and their target genes occur within DNA loops bounded by CTCF proteins. These chromosome structures constrain the activities of enhancers to genes that occur within the same loop, and were thus called insulated neighborhoods. Recent studies have shown that signaling pathways converge on super-enhancers, but the effect of signaling-mediated gene activation has yet to be investigated in the context of insulated neighborhood structure and function. We used a model of Wnt-pathway activation in colon cancer to investigate Wnt-mediated activation of target genes within insulated neighborhoods. When the Wnt-pathway was activated through genetic deletion of the tumor suppressor Apc in intestinal organoids, all enhancers within insulated neighborhoods that contained Tcf4 binding sites were activated, including enhancers that were not considered to contribute to super-enhancers. Thus, Wnt-stimulation co-activated multiple enhancers that are not in linear proximity, yet cluster in 3D space within insulated neighborhoods. We conclude that insulated neighborhoods allow the formation of 3D enhancer clusters whose constituents can operate concertedly, and thus expand the definition of super-enhancers to incorporate 3D chromatin organization. This abstract is also presented as Poster A15. Citation Format: Jurian Schuijers, Daniel S. Day, Violaine Saint-Andre, Richard A. Young. 3D enhancer clusters in the response to Wnt-driven colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr PR01.

Oncogenes are activated through well-known chromosomal alterations such as gene fusion, translocation, and focal amplification. In light of recent evidence that the control of key genes depends on chromosome structures called insulated neighborhoods, we investigated whether proto-oncogenes occur within these structures and whether oncogene activation can occur via disruption of insulated neighborhood boundaries in cancer cells. We mapped insulated neighborhoods in T cell acute lymphoblastic leukemia (T-ALL) and found that tumor cell genomes contain recurrent microdeletions that eliminate the boundary sites of insulated neighborhoods containing prominent T-ALL proto-oncogenes. Perturbation of such boundaries in nonmalignant cells was sufficient to activate proto-oncogenes. Mutations affecting chromosome neighborhood boundaries were found in many types of cancer. Thus, oncogene activation can occur via genetic alterations that disrupt insulated neighborhoods in malignant cells.

Understanding how transcriptional enhancers control over 20,000 protein-coding genes tomaintain cell-type-specific gene expression programs in all human cells is a fundamental challenge in regulatory biology. Recent studies suggest that gene regulatory elements and their target genes generally occur within insulated neighborhoods, which are chromosomal loop structures formed by the interaction of two DNA sites bound by the CTCF protein and occupied by the cohesin complex. Here, we review evidence that insulated neighborhoods provide for specific enhancer-gene interactions, are essential for both normal gene activation and repression, form a chromosome scaffold that is largely preserved throughout development, and are perturbed by genetic and epigenetic factors in disease. Insulated neighborhoods are a powerful paradigm for gene control that provides new insights into development and disease.

Background For many genes, RNA polymerase II stably pauses before transitioning to productive elongation. Although polymerase II pausing has been shown to be a mechanism for regulating transcriptional activation, the extent to which it is involved in control of mammalian gene expression and its relationship to chromatin structure remain poorly understood. Results Here, we analyze 85 RNA polymerase II chromatin immunoprecipitation (ChIP)-sequencing experiments from 35 different murine and human samples, as well as related genome-wide datasets, to gain new insights into the relationship between polymerase II pausing and gene regulation. Across cell and tissue types, paused genes (pausing index > 2) comprise approximately 60 % of expressed genes and are repeatedly associated with specific biological functions. Paused genes also have lower cell-to-cell expression variability. Increased pausing has a non-linear effect on gene expression levels, with moderately paused genes being expressed more highly than other paused genes. The highest gene expression levels are often achieved through a novel pause-release mechanism driven by high polymerase II initiation. In three datasets examining the impact of extracellular signals, genes responsive to stimulus have slightly lower pausing index on average than non-responsive genes, and rapid gene activation is linked to conditional pause-release. Both chromatin structure and local sequence composition near the transcription start site influence pausing, with divergent features between mammals and Drosophila . Most notably, in mammals pausing is positively correlated with histone H2A.Z occupancy at promoters. Conclusions Our results provide new insights into the contribution of RNA polymerase II pausing in mammalian gene regulation and chromatin structure.

Understanding how transcriptional enhancers control over 20,000 protein-coding genes to maintain cell-type-specific gene expression programs in all human cells is a fundamental challenge in regulatory biology. Recent studies suggest that gene regulatory elements and their target genes generally occur within insulated neighborhoods, which are chromosomal loop structures formed by the interaction of two DNA sites bound by the CTCF protein and occupied by the cohesin complex. Here, we review evidence that insulated neighborhoods provide for specific enhancer-gene interactions, are essential for both normal gene activation and repression, form a chromosome scaffold that is largely preserved throughout development, and are perturbed by genetic and epigenetic factors in disease. Insulated neighborhoods are a powerful paradigm for gene control that provides new insights into development and disease.