Dataset Information

DNA Damage Induces Dynamic Associations of BRD4/P-TEFb With Chromatin and Modulates Gene Transcription in a BRD4-Dependent and -Independent Manner.

ABSTRACT: The bromodomain-containing protein BRD4 has been thought to transmit epigenetic information across cell divisions by binding to both mitotic chromosomes and interphase chromatin. UV-released BRD4 mediates the recruitment of active P-TEFb to the promoter, which enhances transcriptional elongation. However, the dynamic associations between BRD4 and P-TEFb and BRD4-mediated gene regulation after UV stress are largely unknown. In this study, we found that BRD4 dissociates from chromatin within 30 min after UV treatment and thereafter recruits chromatin. However, P-TEFb binds tightly to chromatin right after UV treatment, suggesting that no interactions occur between BRD4 and P-TEFb within 30 min after UV stress. BRD4 knockdown changes the distribution of P-TEFb among nuclear soluble and chromatin and downregulates the elongation activity of RNA polymerase II. Inhibition of JNK kinase but not other MAP kinases impedes the interactions between BRD4 and P-TEFb. RNA-seq and ChIP assays indicate that BRD4 both positively and negatively regulates gene transcription in cells treated with UV stress. These results reveal previously unrecognized dynamics of BRD4 and P-TEFb after UV stress and regulation of gene transcription by BRD4 acting as either activator or repressor in a context-dependent manner.

SUBMITTER: Song Y

PROVIDER: S-EPMC7746802 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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DNA Damage Induces Dynamic Associations of BRD4/P-TEFb With Chromatin and Modulates Gene Transcription in a BRD4-Dependent and -Independent Manner.

Song Yawei Y Hu Gongcheng G Jia Jinping J Yao Mingze M Wang Xiaoshan X Lu Wenliang W Hutchins Andrew P AP Chen Jiekai J Ozato Keiko K Yao Hongjie H

Frontiers in molecular biosciences 20201204

The bromodomain-containing protein BRD4 has been thought to transmit epigenetic information across cell divisions by binding to both mitotic chromosomes and interphase chromatin. UV-released BRD4 mediates the recruitment of active P-TEFb to the promoter, which enhances transcriptional elongation. However, the dynamic associations between BRD4 and P-TEFb and BRD4-mediated gene regulation after UV stress are largely unknown. In this study, we found that BRD4 dissociates from chromatin within 30 mi ...[more]

PMID: 33344510

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Project description:RationaleSmall molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in preclinical models of heart failure (HF). However, since the inhibitors target BRD4 ubiquitously, it is unclear whether this chromatin reader protein functions in cell type-specific manner to control pathological myocardial fibrosis. Furthermore, the molecular mechanisms by which BRD4 stimulates the transcriptional program for cardiac fibrosis remain unknown.ObjectiveWe sought to test the hypothesis that BRD4 functions in a cell-autonomous and signal-responsive manner to control activation of cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart.Methods and resultsRNA-sequencing, mass spectrometry, and cell-based assays employing primary adult rat ventricular fibroblasts demonstrated that BRD4 functions as an effector of TGF-β (transforming growth factor-β) signaling to stimulate conversion of quiescent cardiac fibroblasts into Periostin (Postn)-positive cells that express high levels of extracellular matrix. These findings were confirmed in vivo through whole-transcriptome analysis of cardiac fibroblasts from mice subjected to transverse aortic constriction and treated with the small molecule BRD4 inhibitor, JQ1. Chromatin immunoprecipitation-sequencing revealed that BRD4 undergoes stimulus-dependent, genome-wide redistribution in cardiac fibroblasts, becoming enriched on a subset of enhancers and super-enhancers, and leading to RNA polymerase II activation and expression of downstream target genes. Employing the Sertad4 (SERTA domain-containing protein 4) locus as a prototype, we demonstrate that dynamic chromatin targeting of BRD4 is controlled, in part, by p38 MAPK (mitogen-activated protein kinase) and provide evidence of a critical function for Sertad4 in TGF-β-mediated cardiac fibroblast activation.ConclusionsThese findings define BRD4 as a central regulator of the pro-fibrotic cardiac fibroblast phenotype, establish a p38-dependent signaling circuit for epigenetic reprogramming in heart failure, and uncover a novel role for Sertad4. The work provides a mechanistic foundation for the development of BRD4 inhibitors as targeted anti-fibrotic therapies for the heart.

Project description:DNA damage activates a complex signaling network in cells that blocks cell cycle progression, recruits factors involved in DNA repair, and/or triggers programs that control senescence or programmed cell death. Alterations in chromatin structure are known to be important for the initiation and propagation of the DNA damage response, although the molecular details are unclear. We investigated the role of chromatin structure in the DNA damage response by monitoring multiple timedependent checkpoint signaling and response events with a high-content multiplex image-based RNAi screen of chromatin modifying and interacting genes. We discovered that Brd4, a double bromodomain-containing protein, functions as an endogenous inhibitor of DNA damage signaling by binding to acetylated histones at sites of open chromatin and altering chromatin accessibility. Loss of Brd4 or disruption of acetyl-lysine binding results in an increase in both the number and size of radiation-induced !H2AX nuclear foci while overexpression of a Brd4 splice isoform completely suppresses !H2AX formation, despite equivalent double strand break formation. Brd4 knock-down cells displayed altered chromatin structure, prolonged cell cycle checkpoint arrest and enhanced survival after irradiation, while overexpression of Brd4 isoform B results in enhanced radiationinduced lethality. Brd4 is the target of the t(15;19) chromosomal translocation in a rare form of cancer, NUT Midline Carcinoma. Acetyl lysine-bromodomain interactions of the Brd4-NUT fusion protein suppresses !H2AX foci in discrete nuclear compartments, rendering cells more radiosensitive, mimicking overexpression of Brd4 isoform B. NUT Midline Carcinoma is sensitive to radiotherapy, however tumor material from this rare cancer is scarce. We therefore investigated Brd4 expression in another human cancer commonly treated with radiotherapy, glioblastoma multiforme, and found that expression of Brd4 isoform B correlated specifically with treatment response to radiotherapy. These data implicate Brd4 as an endogenous insulator of DNA damage signaling through recognition of epigenetic modifications in chromatin and suggest that expression of the Brd4 in human cancer can modulate the clinical response to DNA-damaging cancer therapy. Two replicates each of U2OS cells expressing either Brd4 shRNA or a control shRNA

Dataset Information

DNA Damage Induces Dynamic Associations of BRD4/P-TEFb With Chromatin and Modulates Gene Transcription in a BRD4-Dependent and -Independent Manner.

Publications

DNA Damage Induces Dynamic Associations of BRD4/P-TEFb With Chromatin and Modulates Gene Transcription in a BRD4-Dependent and -Independent Manner.

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