Acetyltransferase p300 collaborates with chromodomain helicase DNA-binding protein 4 (CHD4) to facilitate DNA double-strand break repair
Acetyltransferase p300 collaborates with chromodomain helicase DNA-binding protein 4 (CHD4) to facilitate DNA double-strand break repair
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乙酰转移酶 p300 与染色质结构域解旋酶 DNA 结合蛋白 4 (CHD4) 合作促进 DNA 双链断裂修复
DOI：
10.1093/mutage/gev075
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发表时间：
2016-03-01
期刊：
影响因子：
2.7
通讯作者：
Zeng, Xianlu
中科院分区：
文献类型：
--
作者：
Qi, Wenjing;Chen, Hongyu;Zeng, Xianlu
染色质重塑对于修复DNA损伤和维持基因组完整性至关重要。以往的研究报道，组蛋白乙酰转移酶p300和ATP依赖的染色质重塑chromodomain解旋酶DNA结合蛋白4（CHD 4）的功能，分别在DNA双链断裂（DSB）修复。但它们相互作用的生理意义仍然难以捉摸。在这里，我们发现p300和CHD 4都被募集到DSB的位点。它们的消融导致DSB修复受损，并使细胞对激光和抗癌药物依托泊苷敏感。使用DR-GFP和EJ 5-GFP报告系统，我们发现p300或CHD 4的敲除损害同源重组（HR）修复，但不损害非同源末端连接（NHEJ）修复。此外，p300或CHD 4敲低分别抑制了复制蛋白A（RPA），HR的关键蛋白，DSB网站的招聘。此外，免疫荧光结果表明，敲低p300减少了CHD 4在DSB位点的募集。反过来，CHD 4敲低也减少了p300组装。免疫沉淀和蛋白质pull down实验表明，p300与CHD 4在DNA损伤位点发生了物理相互作用，这种相互作用依赖于CHD 4的chromodomain和ATPase/helicase结构域以及p300的CH 2、Bd和HAT结构域。这些结果表明，p300和CHD 4可以在DSB位点协同发挥作用，并提供了一个新的见解，详细的染色质重塑蛋白之间的串扰。
Chromatin remodelling is critical for repairing DNA damage and maintaining genomic integrity. Previous studies have reported that histone acetyltransferase p300 and ATP-dependent chromatin remodeler chromodomain helicase DNA-binding protein 4 (CHD4) functions, respectively, in DNA double-strand breaks (DSBs) repair. But the physiological significance of their interaction remains elusive. Here, we showed that p300 and CHD4 were both recruited to the sites of DSBs. Their ablation led to impaired DSBs repair and sensitised cells to laser and the anti-cancer drug, etoposide. Using DR-GFP and EJ5-GFP reporter systems, we found that knockdown of p300 or CHD4 impaired the homologous recombination (HR) repair but no the non-homologous end joining (NHEJ) repair. Furthermore, p300 or CHD4 knockdown respectively suppressed the recruitment of replication protein A (RPA), a key protein for HR, to the DSB sites. In addition, immunofluorescence results showed that knockdown of p300 reduced the recruitment of CHD4 at DSB sites. In turn, CHD4 knockdown also decreased p300 assembly. Moreover, immunoprecipitation and purified protein pull down assay revealed that p300 physically interacted with CHD4 at DNA damage sites, and this interaction was dependent on the chromodomain and ATPase/helicase domain of CHD4 and the CH2, Bd and HAT domains of p300. These results indicate that p300 and CHD4 could function cooperatively at DSB sites and provide a new insight into the detailed crosstalk among the chromatin remodelling proteins.