當細胞遇到複製壓力時會造成複製叉停滯（fork stalled），此時細胞啟動反向複製叉（fork reversal）機制，保護複製叉免於崩潰，並藉由同源重組（homologous recombination, HR）解決複製叉停滯問題。溴結構域和末端外結構域蛋白家族（Bromodomain and Extra-Terminal Domain, BET family）透過辨識組蛋白乙醯化和調控轉錄因子，在癌細胞中參與促癌因子的調控，使癌細胞增生，其中BRD4在BET family中研究最為廣泛，特別是含有長C端區域（C-terminal domain, CTD），和正轉錄因子（p-TEFb）結合以促進轉錄延伸。先前研究指出BRD4缺陷細胞削弱HR修復功能，也會使複製期的檢查點失效造成DNA損傷累積，然而BRD4是否在複製壓力下修復DNA損傷並參與反向複製叉的保護，再透過調控HR相關蛋白進行修復，尚未有明確的機制證實。在此我們發現使用BET 抑制劑Mivebresib或是降低BRD4，結合PARP抑制劑Olaparib聯合處理使細胞敏感性增加，下調BRD4也會使參與保護反向複製叉的蛋白FANCD2、NS、RAD51表達下降，利用DR-GFP檢測中證實BRD4參與HR修復路徑，在複製壓力下下調BRD4蛋白，證實BRD4保護停滯的複製叉。同時在BRD4缺陷細胞中處於複製壓力下，使DNA損傷修復能力降低，伴隨單股DNA暴露與複製叉崩潰的現象，綜合以上結果，我們證實BRD4透過調控HR相關蛋白參與反向複製叉的保護，並在DNA損傷修復中發揮重要作用。

Replication stress causes replication fork stalling, triggering the fork reversal mechanism to protect the stalled forks from collapse into DNA double-strand breaks (DSBs). The reversed forks are protected by BRCA1/BRCA2, components of homologous recombination (HR), and FANCD2, a component of Fanconi anemia pathway to prevent nascent DNA degradation by MRE11 nucleases. Current studies reveal that the reversal mechanism not only protect stalled forks from collapse, but also facilitates the restart of DNA replication through the HR mechanism. The Bromodomain and Extra-Terminal Domain (BET) family proteins, particularly BRD4, play a critical role in transcription regulation and are implicated in oncogenesis. Previous studies have shown that BET inhibitor (BETi) and (PARPi) have synergistical effects against cancer cells, indicating that BET proteins are involved in the HR pathway. Since several components of HR are also involved in fork protection, we want to test whether BRD4 is also involved in fork reversal mechanism. Here, we demonstrate that BRD4 depletion sensitizes cells to DNA damaging agents, shows defective DNA repair and impairs HR repair and fork protection by downregulating key proteins such as FANCD2 , HLTF , and RAD51.Our results underscore BRD4 as a pivotal regulator in maintaining genomic integrity under replication stress.

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