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.
Highlights
Proper chromatin function and maintenance of genomic stability depends on spatiotemporal coordination between the transcription and replication machinery
We previously reported a novel role for BRD4 in insulating the chromatin against radiation-induced DNA damage response signaling in oncogenic cells[21]
To further explore our previous finding that BRD4 is involved in regulating the DNA damage response in oncogenic cells[21], we treated cells with the prototypical bromodomain and extraterminal domain (BET) bromodomain inhibitor JQ122 and assayed for changes in DNA damage response signaling using immunofluorescence (IF) and western blotting for γH2AX, a marker of DNA damage signaling and DSB23
Summary
BRD4 bromodomain inhibition causes DNA damage and apoptosis. To further explore our previous finding that BRD4 is involved in regulating the DNA damage response in oncogenic cells[21], we treated cells with the prototypical BET bromodomain inhibitor JQ122 and assayed for changes in DNA damage response signaling using immunofluorescence (IF) and western blotting for γH2AX, a marker of DNA damage signaling and DSB23. Together, these data indicate that deregulated transcription caused by degradation of BET bromodomain proteins leads to DNA damage in S-phase as a result of collisions between the transcription and replication machinery. As shown, the modest increase in G1 and G2/M populations and reduction in the S-phase population caused by JQ1 treatment was abrogated by RNase H1 expression, which partially rescued the JQ1-induced block in cell proliferation (Fig. 9d) These results suggest that removal of transcriptional roadblocks caused by the accumulation of R-loops following BET bromodomain protein loss leads to resolution of TRCs and restoration of normal cell cycle kinetics
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