Identification and characterization of novel proteins associated with CHD4
Identification and characterization of novel proteins associated with CHD4
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与 CHD4 相关的新型蛋白质的鉴定和表征
DOI：
10.1111/gtc.12909
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发表时间：
2021
期刊：
影响因子：
2.1
通讯作者：
Nakayama Keiichi I.
中科院分区：
文献类型：
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作者：
Sakaguchi Chihiro;Ichihara Kazuya;Nita Akihiro;Katayama Yuta;Nakayama Keiichi I.
CHD（chromodomain helicase DNA binding protein，染色体结构域解旋酶DNA结合蛋白）家族由9种染色质重塑因子组成，它们以ATP依赖性方式改变染色质结构。CHD4通过与多种蛋白质相互作用，包括形成NuRD（核小体重塑和脱乙酰酶活性）复合物，有助于调节各种细胞活动和过程，包括发育。CHD4的功能似乎不是由NuRD复合物或其他已知的相互作用物介导的，然而，这表明存在未识别的蛋白质也与CHD4相关。我们在这里产生了具有FLAG表位标记的CHD4的敲入等位基因的HeLa‐S3和HEK 293 T细胞，并使用这些细胞通过免疫沉淀，然后通过液相色谱和串联质谱来鉴定与CHD4结合的蛋白质。LCORL（配体依赖性核受体辅阻遏物样）和NOL4L（核仁蛋白4样）被可重复地鉴定为新的CHD4相互作用物。此外，去除CHD4、LCORL或NOL4L的HEK293T细胞的RNA测序分析显示与Notch信号通路相关的基因一致上调。因此，我们的研究结果表明，LCORL和NOL4L可能与CHD4合作，以抑制哺乳动物细胞中的Notch通路。
The CHD (chromodomain helicase DNA binding protein) family consists of nine chromatin remodeling factors that alter chromatin structure in an ATP‐dependent manner. CHD4 contributes to the regulation of various cellular activities and processes including development through interaction with multiple proteins including formation of the NuRD (nucleosome remodeling and deacetylase activity) complex. Functions of CHD4 that appear not to be mediated by the NuRD complex or other known interactors have also been identified, however, suggesting the existence of unrecognized proteins that also associate with CHD4. We here generated HeLa‐S3 and HEK293T cells with a knock‐in allele for FLAG epitope‐tagged CHD4 and used these cells to identify proteins that bind to CHD4 with the use of immunoprecipitation followed by liquid chromatography and tandem mass spectrometry. LCORL (ligand‐dependent nuclear receptor corepressor like) and NOL4L (nucleolar protein 4 like) were reproducibly identified as novel CHD4 interactors. Furthermore, RNA‐sequencing analysis of HEK293T cells depleted of CHD4, LCORL, or NOL4L revealed consistent up‐regulation of genes related to the Notch signaling pathway. Our results thus suggest that both LCORL and NOL4L may cooperate with CHD4 to suppress the Notch pathway in mammalian cells.