Identification and characterization of novel proteins associated with CHD4

CHD (chromodomain helicase DNA binding protein) family is composed of nine members of chromatin remodeling factors that regulate chromatin structure in an ATP-dependent manner. Among them, CHD4 contributes to many basic cellular functions during development mainly through multiple proteins interacting with CHD4 including NuRD (nucleosome remodeling and deacetylase activities) complex, which contains histone deacetylase HDAC1/2. However, functions of CHD4 that are not mediated by NuRD complexes have also been found, implying the existence of unknown proteins that are associated with CHD4. In the present study, we generated CHD4 FLAG-tag knock-in cells in HeLa-S3 and HEK293T cells and sought proteins bound to CHD4 with the use of immunoprecipitation and liquid chromatography and tandem MS (LC-MS/MS) analysis. We found that LCORL (ligand dependent nuclear receptor corepressor like) and NOL4L (nucleolar protein 4 like) were reproducibly identified as a novel CHD4 interactors. RNA-sequencing analysis of HEK293T cells depleted of CHD4, LCORL, and NOL4L revealed consistent upregulation of genes related to the Notch pathway. Our results thus suggest that both NOL4L and LCORL may cooperate with CHD4 to suppress the Notch pathway in mammalian cells. Transcriprional control regulated by CHD4, NOL4L, and LCORL

染色质域解旋酶DNA结合蛋白（CHD, chromodomain helicase DNA binding protein）家族由9个ATP依赖型染色质重塑因子成员组成，可调控染色质结构。其中，CHD4主要通过与自身结合的多种蛋白（包括包含组蛋白去乙酰化酶HDAC1/2的NuRD（nucleosome remodeling and deacetylase activities, 核小体重塑与去乙酰化酶复合物））参与发育过程中的诸多基础细胞功能。然而，研究也发现了不依赖NuRD复合物介导的CHD4功能，这暗示存在与CHD4结合的未知蛋白。本研究中，我们在HeLa-S3与HEK293T细胞中构建了携带FLAG标签的CHD4敲入细胞株，并通过免疫沉淀结合液相色谱-串联质谱（LC-MS/MS）分析筛选与CHD4结合的蛋白。我们发现，LCORL（ligand dependent nuclear receptor corepressor like, 配体依赖性核受体辅阻遏物样蛋白）与NOL4L（nucleolar protein 4 like, 核仁蛋白4样蛋白）可重复被鉴定为新型CHD4互作蛋白。对沉默CHD4、LCORL与NOL4L的HEK293T细胞进行RNA测序分析，结果显示Notch信号通路相关基因呈现一致的上调趋势。综上，我们的研究结果表明，NOL4L与LCORL可与CHD4协同作用，在哺乳动物细胞中抑制Notch信号通路。CHD4、NOL4L与LCORL所介导的转录调控