Eto2/MTG16 and MTGR1 are heteromeric corepressors of the TAL1/SCL transcription factor in murine erythroid progenitors.
Eto2/MTG16 and MTGR1 are heteromeric corepressors of the TAL1/SCL transcription factor in murine erythroid progenitors.
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DOI：
10.1016/j.bbrc.2009.09.111
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发表时间：
2009-12-11
影响因子：
3.1
通讯作者：
Brandt SJ
中科院分区：
文献类型：
--
作者：
Cai Y;Xu Z;Xie J;Ham AJ;Koury MJ;Hiebert SW;Brandt SJ
TAL1（或 SCL）基因最初是通过 T 细胞急性淋巴细胞白血病的染色体易位而发现的，它编码对造血和血管发育至关重要的基本螺旋-环-螺旋（bHLH）转录因子。为了鉴定其相互作用伙伴，我们在小鼠红白血病 (MEL) 细胞中表达了串联表位标记蛋白，并通过液相色谱-串联质谱分析对亲和纯化的含 Tal1 复合物进行了表征。除了已知的相互作用蛋白外，还从分析的肽片段中鉴定出与八二十一 (ETO) 辅阻遏物相关的两种蛋白 Eto2/Mtg16 和 Mtgr1。 Tal1 与 Eto2 和 Mtgr1 的相互作用通过 Tal1、Eto2 和 Mtgr1 转染的 COS-7 细胞、表达 V5 表位标记的 Tal1 蛋白的 MEL 细胞和未转染的 MEL 细胞中的共免疫沉淀分析进行验证。 Gal4 融合蛋白的作图分析表明，Tal1 的 bHLH 结构域和 Eto2 的 TAF110 结构域相互作用需要，瞬时转染和谷胱甘肽 S 转移酶下拉分析表明，Mtgr1 和 Eto2 增强了彼此与 Tal1 的关联。在分化的 MEL 细胞中强制表达 Eto2 会抑制蛋白 4.2 (P4.2) 基因的启动子，该基因是红系祖细胞中 TAL1 的直接靶标，并且 Eto2 和 Mtgr1 的转导增强了 Tal1 介导的基因抑制。最后，染色质免疫沉淀分析显示，MEL 细胞中 Eto2 对 P4.2 启动子的占据随着分化而减少，同时 Eto2 蛋白丰度也随之下降。这些结果将 Eto2 和 Mtgr1 确定为 Tal1 的真实相互作用伴侣，并表明它们在红系分化过程中充当该 bHLH 转录因子的异聚辅阻遏物。
The TAL1 (or SCL) gene, originally discovered through its involvement by a chromosomal translocation in T-cell acute lymphoblastic leukemia, encodes a basic helix-loop-helix (bHLH) transcription factor essential for hematopoietic and vascular development. To identify its interaction partners, we expressed a tandem epitope-tagged protein in murine erythroleukemia (MEL) cells and characterized affinity-purified Tal1-containing complexes by liquid chromatography-tandem mass spectrometry analysis. In addition to known interacting proteins, two proteins related to the Eight-Twenty-One (ETO) corepressor, Eto2/Mtg16 and Mtgr1, were identified from the peptide fragments analyzed. Tal1 interaction with Eto2 and Mtgr1 was verified by coimmunoprecipitation analysis in Tal1, Eto2-, and Mtgr1-transfected COS-7 cells, MEL cells expressing V5 epitope-tagged Tal1 protein, and non-transfected MEL cells. Mapping analysis with Gal4 fusion proteins demonstrated a requirement for the bHLH domain of Tal1 and TAF110 domain of Eto2 for their interaction, and transient transfection and glutathione S-transferase pull-down analysis showed that Mtgr1 and Eto2 enhanced the other’s association with Tal1. Enforced expression of Eto2 in differentiating MEL cells inhibited the promoter of the Protein 4.2 (P4.2) gene, a direct target of TAL1 in erythroid progenitors, and transduction of Eto2 and Mtgr1 augmented Tal1-mediated gene repression. Finally, chromatin immunoprecipitation analysis revealed that Eto2 occupancy of the P4.2 promoter in MEL cells decreased with differentiation, in parallel with a decline in Eto2 protein abundance. These results identify Eto2 and Mtgr1 as authentic interaction partners of Tal1 and suggest they act as heteromeric corepressors of this bHLH transcription factor during erythroid differentiation.
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