邻近依赖性生物素识别 (BioID) 揭示了胚胎干细胞分化过程中的动态 LSD1-CoREST 相互作用组,Molecular Omics

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邻近依赖性生物素识别 (BioID) 揭示了胚胎干细胞分化过程中的动态 LSD1-CoREST 相互作用组
Molecular Omics ( IF 3.0 ) Pub Date : 2021-10-14 , DOI: 10.1039/d1mo00236h
Claire E Barnes ₁ , David M English ₁ , Megan Broderick ₁ , Mark O Collins _{2,

3} , Shaun M Cowley ₁

Affiliation

赖氨酸特异性脱甲基酶 1 (LSD1) 作为 CoREST 复合物的一部分，与 REST (CoREST) 和组蛋白脱乙酰酶 1 (HDAC1) 的共阻遏物一起调节基因表达。CoREST 通过核心成分以及与染色质相关因子和转录因子的大量短暂相互作用被招募到特定的基因组位点。我们假设许多较弱和短暂的关联可能很难使用传统的免疫共沉淀方法来识别。因此，我们在三种不同的细胞类型中对 CoREST 复合物的四个不同成员进行了邻近依赖性生物素识别 (BioID)，以鉴定 LSD1/CoREST 相关蛋白的综合网络。在 HEK293T 细胞中，我们鉴定了 302 个 CoREST 相关蛋白。该组中有 18 个已知 CoREST 组件中的 16 个以及许多新颖的关联，包括组蛋白甲基化的读取器（CHD3、4、6、7 和 8）、写入器（KMT2B 和 KMT2D）和擦除器（KDM2B）。然而，其他含有复合物的HDAC1（例如Sin3）的成分基本上不存在。为了检查原代细胞类型中 CoREST 相互作用组的动态性质，我们在胚胎干 (ES) 细胞中用 BirA*-LSD1 替换内源性 LSD1，并在多能、早期和晚期分化环境中进行 BioID。我们鉴定了 156 个 LSD1 相关蛋白，其中 67 个在所有三个时间点 (43%) 上都是组成型相关的，包括与 MMB 和 ChAHP 复合物的新关联，这意味着大多数相互作用因子都是动态的且依赖于细胞类型。总共，我们在三种不同的细胞类型中对 LSD1 进行了 16 次独立的 BioID 实验，产生了 LSD1 相关蛋白的明确网络，这应该为该领域提供主要资源。

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Proximity-dependent biotin identification (BioID) reveals a dynamic LSD1–CoREST interactome during embryonic stem cell differentiation

Lysine specific demethylase 1 (LSD1) regulates gene expression as part of the CoREST complex, along with co-repressor of REST (CoREST) and histone deacetylase 1 (HDAC1). CoREST is recruited to specific genomic loci by core components and numerous transient interactions with chromatin-associated factors and transcription factors. We hypothesise that many of these weaker and transient associations may be difficult to identify using traditional co-immunoprecipitation methods. We have therefore employed proximity-dependent biotin-identification (BioID) with four different members of the CoREST complex, in three different cell types, to identify a comprehensive network of LSD1/CoREST associated proteins. In HEK293T cells, we identified 302 CoREST-associated proteins. Among this group were 16 of 18 known CoREST components and numerous novel associations, including readers (CHD3, 4, 6, 7 and 8), writers (KMT2B and KMT2D) and erasers (KDM2B) of histone methylation. However, components of other HDAC1 containing complexes (e.g. Sin3) were largely absent. To examine the dynamic nature of the CoREST interactome in a primary cell type, we replaced endogenous LSD1 with BirA*-LSD1 in embryonic stem (ES) cells and performed BioID in pluripotent, early- and late-differentiating environments. We identified 156 LSD1-associated proteins of which 67 were constitutively associated across all three time-points (43%), including novel associations with the MMB and ChAHP complexes, implying that the majority of interactors are both dynamic and cell type dependent. In total, we have performed 16 independent BioID experiments for LSD1 in three different cell types, producing a definitive network of LSD1-assoicated proteins that should provide a major resource for the field.

更新日期：2021-12-09

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