Abstract
BET bromodomain proteins are epigenetic readers required for oncogenic transcription activities, and BET inhibitors have been rapidly advanced into clinical trials. Understanding the effects of BET inhibition on other nuclear processes such as DNA replication will be important for future clinical applications. Here we show that BET inhibition causes replication stress in cancer and non-cancer cells due to a rapid burst in global RNA synthesis and interference of transcription with replication. We identify BRD4 as the main BET inhibitor target in this process and provide evidence that BRD4 inhibition causes transcription-replication interference through release of P-TEFb from its inhibitor HEXIM1, promoting RNA Polymerase II phosphorylation. Unusually, BET inhibitor-induced transcription-replication interference does not activate the classic ATM/ATR-dependent DNA damage response. We show however that they promote foci formation of the homologous recombination factor RAD51. Both HEXIM1 and RAD51 are required for BET inhibitor-induced fork slowing, but rescuing fork slowing by HEXIM1 or RAD51 depletion activate a DNA damage response. Our data support a new mechanism where BRD4 inhibition slows replication and suppresses DNA damage through concerted action of transcription and homologous recombination machineries. They shed new light on the roles of DNA replication and recombination in the action of this new class of cancer drugs.
Highlights
Members of the BET bromodomain-containing protein family bind to lysine-acetylated histone tails and regulate transcription by recruiting and activating positive transcription elongation factor b (P-TEFb)
P-TEFb can occur in two active complexes with BET protein BRD4 or the super elongation complex (SEC) and an inactive complex with 7SK-snRP (7SK RNA, HEXIM1, LARP7, and MEPCE)
We show that BET inhibition and loss of BRD4 cause rapid upregulation of RNA synthesis and transcription-dependent replication fork slowing in a pathway that depends on HEXIM1 and RAD51
Summary
Members of the BET bromodomain-containing protein family bind to lysine-acetylated histone tails and regulate transcription by recruiting and activating positive transcription elongation factor b (P-TEFb). P-TEFb can occur in two active complexes with BET protein BRD4 or the super elongation complex (SEC) and an inactive complex with 7SK-snRP (7SK RNA, HEXIM1, LARP7, and MEPCE). BRD4 activates P-TEFb by releasing it from 7SK-snRP and recruits active P-TEFb to gene promoters (Quaresma et al, 2016). BET proteins promote oncogenic transcription programs, and specific small-molecule inhibitors of BET bromodomains promise a targeted cancer treatment (Delmore et al, 2011). BET inhibition downregulates MYC protein levels and kills tumor cells independently of p53 (Da Costa et al, 2013). BET inhibitor responses can be MYC independent (Lockwood et al, 2012). The molecular mechanisms surrounding BET inhibitor action are still poorly understood, BET inhibitors are already undergoing clinical trials in a wide range of cancers (Andrieu et al, 2016; Fujisawa and Filippakopoulos, 2017)
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