Targeting transcription in heart failure via CDK7/12/13 inhibition.

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.

BORIS promotes chromatin regulatory interactions in treatment-resistant cancer cells.

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 elements1,2. In cancer cells, the disruption of CTCF binding at specific loci by somatic mutation3,4 or DNA hypermethylation5 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 cancers7-9, 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 neuroblastoma10 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.

CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation.

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.

A dynamic and integrated epigenetic program at distal regions orchestrates transcriptional responses to VEGFA.

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.

VEGF amplifies transcription through ETS1 acetylation to enable angiogenesis.

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.

Activation of proto-oncogenes by disruption of chromosome neighborhoods.

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.

Interrogating translational efficiency and lineage-specific transcriptomes using ribosome affinity purification.

Transcriptional profiling is a useful strategy to study development and disease. Approaches to isolate RNA from specific cell types, or from specific cellular compartments, would extend the power of this strategy. Previous work has shown that isolation of genetically tagged ribosomes (translating ribosome affinity purification; TRAP) is an effective means to isolate ribosome-bound RNA selectively from transgene-expressing cells. However, widespread application of this technology has been limited by available transgenic mouse lines. Here we characterize a TRAP allele (Rosa26(fsTRAP)) that makes this approach more widely accessible. We show that endothelium-specific activation of Rosa26(fsTRAP) identifies endothelial cell-enriched transcripts, and that cardiomyocyte-restricted TRAP is a useful means to identify genes that are differentially expressed in cardiomyocytes in a disease model. Furthermore, we show that TRAP is an effective means for studying translational regulation, and that several nuclear-encoded mitochondrial genes are under strong translational control. Our analysis of ribosome-bound transcripts also shows that a subset of long intergenic noncoding RNAs are weakly ribosome-bound, but that the majority of noncoding RNAs, including most long intergenic noncoding RNAs, are ribosome-bound to the same extent as coding transcripts. Together, these data show that the TRAP strategy and the Rosa26(fsTRAP) allele will be useful tools to probe cell type-specific transcriptomes, study translational regulation, and probe ribosome binding of noncoding RNAs.

A dynamic H3K27ac signature identifies VEGFA-stimulated endothelial enhancers and requires EP300 activity.

Histone modifications are now well-established mediators of transcriptional programs that distinguish cell states. However, the kinetics of histone modification and their role in mediating rapid, signal-responsive gene expression changes has been little studied on a genome-wide scale. Vascular endothelial growth factor A (VEGFA), a major regulator of angiogenesis, triggers changes in transcriptional activity of human umbilical vein endothelial cells (HUVECs). Here, we used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to measure genome-wide changes in histone H3 acetylation at lysine 27 (H3K27ac), a marker of active enhancers, in unstimulated HUVECs and HUVECs stimulated with VEGFA for 1, 4, and 12 h. We show that sites with the greatest H3K27ac change upon stimulation were associated tightly with EP300, a histone acetyltransferase. Using the variation of H3K27ac as a novel epigenetic signature, we identified transcriptional regulatory elements that are functionally linked to angiogenesis, participate in rapid VEGFA-stimulated changes in chromatin conformation, and mediate VEGFA-induced transcriptional responses. Dynamic H3K27ac deposition and associated changes in chromatin conformation required EP300 activity instead of altered nucleosome occupancy or changes in DNase I hypersensitivity. EP300 activity was also required for a subset of dynamic H3K27ac sites to loop into proximity of promoters. Our study identified thousands of endothelial, VEGFA-responsive enhancers, demonstrating that an epigenetic signature based on the variation of a chromatin feature is a productive approach to define signal-responsive genomic elements. Further, our study implicates global epigenetic modifications in rapid, signal-responsive transcriptional regulation.