Two highly related p66 proteins comprise a new family of potent transcriptional repressors interacting with MBD2 and MBD3
Two highly related p66 proteins comprise a new family of potent transcriptional repressors interacting with MBD2 and MBD3
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DOI：
10.1074/jbc.m207467200
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发表时间：
2002-10-25
影响因子：
4.8
通讯作者：
Renkawitz, R
中科院分区：
文献类型：
--
作者：
Brackertz, M;Boeke, J;Renkawitz, R
甲基化CpG结合域蛋白（MBD）介导甲基化DNA的功能反应。MBD 2和MBD 3是MeCP 1蛋白复合物的组分，其含有Mi-2/NuRD复合物，并包括66和68-kDa多肽。在这里，我们确定了两个高度相关的66 kDa的蛋白在酵母双杂交筛选MBD 2b。基于与先前鉴定的Mi-2/NuRD复合物的爪蟾p66亚基的高度序列保守性，我们将这些蛋白质命名为hp 66 α和hp 66 β。hp 66 alpha是非洲爪蟾p66的人类直向同源物，而hp 66 beta，先前被鉴定为人类MeCP 1复合物的组分，是p66基因家族的第二个成员。hp 66 α和MBD 2的共沉淀证明了它们在体内的关联。此外，共聚焦显微镜显示hp 66 α与hp 66 β和MBD 2在斑点图案中的核共定位。hp 66 α是一种有效的转录抑制因子，可使基因活性降低约100倍，并在细胞系和胎儿及成人组织中与hp 66 β广泛共表达。我们证明了p66家族成员与MBD 2和MBD 3的直接结合。有趣的是，hp 66 α，结合的亲和力比hp 66 β高，通过两个相互作用域相互作用，而不是hp 66 β中存在的单个相互作用域。这些结果表明，两个高度相关的哺乳动物p66蛋白显示重叠的功能，并参与甲基化依赖的转录抑制。
Methyl-CpG-binding domain proteins (MBD) mediate functional responses of methylated DNA. MBD2 and MBD3 are components of the MeCP1 protein complex, which contains the Mi-2/NuRD complex and includes 66 and 68-kDa polypeptides. Here we identified two highly related 66-kDa proteins in a yeast two-hybrid screen with MBD2b. Based on the high degree of sequence conservation to the previously identified Xenopus p66 subunit of the Mi-2/NuRD complex, we termed these proteins hp66alpha and hp66beta. hp66alpha is the human orthologue of Xenopus p66, whereas hp66beta, previously identified as a component of the human MeCP1 complex, is a second member of a p66 gene family. Coprecipitation of hp66alpha and MBD2 demonstrates their in vivo association. Furthermore, confocal microscopy shows a nuclear colocalization of hp66alpha with hp66beta and MBD2 in a speckled pattern. hp66alpha is a potent transcriptional repressor reducing gene activity about 100-fold and is ubiquitously coexpressed with hp66beta in cell lines and in fetal and adult tissues. We demonstrate direct binding of both p66 family members to MBD2 as well as MBD3. Interestingly, hp66alpha, which binds with a higher affinity than hp66beta, interacts via two interaction domains in contrast to a single interaction domain present in hp66beta. These results demonstrate that two highly related mammalian p66 proteins display overlapping functions and are involved in methylation dependent transcriptional repression.