BRD4 prevents the accumulation of R-loops and protects against transcription–replication collision events and DNA damage

Abstract

Proper chromatin function and maintenance of genomic stability depends on spatiotemporal coordination between the transcription and replication machinery. Loss of this coordination can lead to DNA damage from increased transcription-replication collision events. We report that deregulated transcription following BRD4 loss in cancer cells leads to the accumulation of RNA:DNA hybrids (R-loops) and collisions with the replication machinery causing replication stress and DNA damage. Whole genome BRD4 and γH2AX ChIP-Seq with R-loop IP qPCR reveals that BRD4 inhibition leads to accumulation of R-loops and DNA damage at a subset of known BDR4, JMJD6, and CHD4 co-regulated genes. Interference with BRD4 function causes transcriptional downregulation of the DNA damage response protein TopBP1, resulting in failure to activate the ATR-Chk1 pathway despite increased replication stress, leading to apoptotic cell death in S-phase and mitotic catastrophe. These findings demonstrate that inhibition of BRD4 induces transcription-replication conflicts, DNA damage, and cell death in oncogenic cells.

Inducing DNA damage through R-loops to kill cancer cells

“…10 Interestingly, we also found DNA damage throughout the gene body of DNMT1 following BRD4 loss. 9 As R-loops are key regulators of DNMT targeting to chromatin with important roles in cancer progression and treatment, 4 our findings hint that combining DNMT inhibitors, or other novel therapies that exacerbate TRCs and replication stress-induced DNA damage, with bromodomain inhibitors, could increase their therapeutic potential for cancer treatment. These findings that TRCs, DNA damage, and cell death result from R-loop accumulation following BRD4 inhibition suggests that therapeutic induction of R-loop persistence could be a generalizable approach for inducing oncogenic cell death.…”

“…6 We and others recently reported that the epigenetic reader BRD4, plays a critical role in maintaining R-loop homeostasis and preventing TRCs in a subset of cancer cells. [7][8][9] BRD4 and other members of the bromodomain-containing family of proteins, including BRD2 and BRD3, are transcriptional coactivators that recognize acetylated lysine residues on histones directly via their conserved tandem bromodomains. BRD4 in particular, is enriched at enhancer regions of the MYC oncogene, leading to the development of bromodomain inhibitors as a treatment for Myc-driven cancers.…”

“…We propose a model where loss of BRD4 function causes stalling of RNAPII elongation throughout entire gene bodies of a subset of BRD4-regulated genes. 9 This results in accumulation of R-loops, TRCs, and replication fork stress during S-phase, eventually leading to DNA damage and apoptosis as a consequence of mitotic catastrophe. 9 CONTACT Fred C. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.…”

“…Second, we noted that loss of BRD4 function suppressed expression of the ATR/Ck1 replication stress DNA damage response checkpoint mediator TopBP1, causing a paradoxical inactivation of the G 1 /S and G 2 /M checkpoints, and slippage of DNAdamaged cells into mitosis, where they died by mitotic catastrophe. 9 Third, BRD4 loss caused R-loop-induced DNA damage throughout the gene bodies of key R-loop processing genes, including DHX9, SRSF1, and SETX, steadily reducing the cellular stores of these proteins over time, thereby contributing to the inability of cells to resolve R-loops at 24-48 hours after bromodomain inhibition. Finally, we and others found that loss of BRD4 impairs transcription of several key genes -BRCA1, RAD51, and CtIP -involved in DNA repair by homologous recombination (HR, Figure 1d).…”

The Role of RNA in DNA Breaks, Repair and Chromosomal Rearrangements

“…Under normal conditions, RNAPII is phosphorylated at serine 2 by cyclin-dependent kinase 9 (CDK9) in complex with BET bromodomain protein 4 (BRD4). This modification appears to be important to proceed with transcription and to prevent TRC and DNA breaks [65,66]. Chromatin remodeling complexes, such as the one of Mec1, INO80 and PAF1 in budding yeast, can prevent TRC by removing RNAPII [67]; INO80 depletion in prostate cancer cells leads to increased formation of R-loops [68].…”