Direct association between PU.1 and MeCP2 that recruits mSin3A-HDAC complex for PU.1-mediated transcriptional repression
Direct association between PU.1 and MeCP2 that recruits mSin3A-HDAC complex for PU.1-mediated transcriptional repression
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DOI：
10.1038/sj.onc.1207182
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发表时间：
2003-11
期刊：
影响因子：
8
通讯作者：
Mitsuhiro Suzuki;Toshiyuki Yamada;F. Kihara-Negishi;T. Sakurai;T. Oikawa
中科院分区：
文献类型：
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作者：
Mitsuhiro Suzuki;Toshiyuki Yamada;F. Kihara-Negishi;T. Sakurai;T. Oikawa
聚氨酯。 1 是转录因子 Ets 家族的成员，通过与其他转录因子和辅因子的相互作用参与造血细胞分化。鉴定与 PU 结合的新蛋白质。如图 1 所示，我们使用结合 GST-PU 的谷胱甘肽-琼脂糖珠柱进行亲和纯化。 1 融合蛋白并使用鼠红白血病 (MEL) 细胞提取物分离出几种单独的蛋白。对这些蛋白质的序列分析表明，其中一个是 MeCP2，一种甲基 CpG 结合蛋白。 GST-pull-down 测定和免疫沉淀测定表明 PU。 1 通过其 Ets 结构域直接与 MeCP2 结合，并且 MeCP2 与 PU 结合。 1 通过其氨基末端结构域或反式抑制结构域。 MeCP2 抑制 PU 的转录活性。 1 在具有三聚 PU 的报告构建体上。 1个结合位点。这种下调在组蛋白脱乙酰酶抑制剂曲古抑菌素 A (TSA) 存在下得以恢复。 MeCP2被集成到PU中。 1-mSin3A-HDAC 复合体，但不在 PU 中。 1-CBP复合物。染色质免疫沉淀 (ChIP) 检测表明 PU。 1和MeCP2在PU处并置。报告构建体和 PU 上有 1 个结合位点。 β-珠蛋白基因内含子中插入序列 2 (IVS2) 区域的 1 个结合位点，已被认为在未分化的 MEL 细胞中调节该基因的表达。在 MEL 细胞的红系分化过程中，该复合物从该区域消失。我们的结果表明 MeCP2 充当 PU 的辅阻遏物。 1 可能是由于促进了 mSin3A 和 HDAC 的复合物形成。
PU. 1, a member of the Ets family of transcription factors, is implicated in hematopoietic cell differentiation through its interactions with other transcriptional factors and cofactors. To identify a novel protein (s) binding to PU. 1, we carried out affinity purification using a column of Glutathione-Sepharose beads bound to GST-PU. 1 fusion protein and isolated several individual proteins using murine erythroleukemia (MEL) cell extracts. Sequence analysis of these proteins revealed that one was MeCP2 a methyl CpG binding protein. GST-pull-down assay and immunoprecipitation assay showed that PU. 1 bound directly to MeCP2 via its Ets domain and MeCP2 bound to PU. 1 via either its amino terminal domain or trans-repression domain. MeCP2 repressed transcriptional activity of PU. 1 on a reporter construct with trimerized PU. 1 binding sites. This downregulation was recovered in the presence of histone deacetylase inhibitor, trichostatin A (TSA). MeCP2 was integrated in PU. 1-mSin3A-HDAC complex but not in PU. 1-CBP complex. Chromatin immunoprecipitation (ChIP) assays showed that PU. 1 and MeCP2 were collocated at the PU. 1 binding site on the reporter construct and the PU. 1 binding site of the intervening sequence 2 (IVS2) region in the intron of the β-globin gene, which has been proposed to regulate expression of the gene, in undifferentiated MEL cells. The complex disappeared from the region during the course of erythroid differentiation of MEL cells. Our results suggest that MeCP2 acts as a corepressor of PU. 1 probably due to facilitating complex formation with mSin3A and HDACs.