Characterization of MBD3L1 and MBD3L2

描述（由申请人提供）：哺乳动物基因组编码几种蛋白质，其具有可以结合在CpG二核苷酸处含有5-甲基胞嘧啶的DNA的结构域（甲基-CpG结合结构域或MBD;存在于例如蛋白质MeCP 2、MBD 1、MBD 2、MBD 3和MBD 4中）。这些蛋白质中的两种，MBD 2和MBD 3，是密切相关的，并且MBD 2是已知的转录阻遏物。最近，我们已经鉴定并克隆了MBD 2和MBD 3的两个新的同源物，我们将其命名为MBD 3L 1和MBD 3L 2（“MBD 3样1和2”）。MBD 3L 1和MBD 3L 2都缺乏MBD结构域。MBD 3L 1是一种睾丸特异性转录抑制因子。在本申请中，我们建议详细分析这些新的MBD 3 - 1样蛋白。我们的假设是，MBD 3L 1和MBD 3L 2是转录调节因子，与MBD 2和/或MBD 3一起发挥作用以调节基因活性。我们提出四个具体目标：1）表征MBD 3L 1和MBD 3L 2的转录抑制功能及其对CpG甲基化的依赖性，2）鉴定和表征MBD 3L 1和MBD 3L 2的体内结合配偶体（初步数据表明这些蛋白质可以与MBD 2和MBD 3形成异二聚体），3）确定MBD 3L 1和MBD 3L 2是否是体内更大复合物的组分（例如，Mi-2/NuRD复合物），和4）获得Mbd 311和Mbd 312敲除小鼠。我们将检验Mbd 311敲除小鼠将具有雄性不育表型的假设。由于这两种蛋白质都可能作为转录调节因子发挥作用，因此确定Mbd 311和Mbd 312（-/-）细胞中的基因表达模式是否发生变化将是重要的。

DESCRIPTION (provided by applicant): Mammalian genomes encode several proteins, which have domains that can bind to DNA containing 5- methylcytosines at CpG dinucleotides (methyI-CpG binding domains or MBDs; present for example in the proteins MeCP2, MBD1, MBD2, MBD3, and MBD4). Two of these proteins, MBD2 and MBD3, are closely related and MBD2 is a known transcriptional repressor. Recently, we have identified and cloned two new homologues of MBD2 and MBD3, which we have named MBD3L1 and MBD3L2 (for "MBD3-1ike 1 and 2"). Both MBD3L1 and MBD3L2 lack the MBD domains. MBD3L1 has so far been characterized as a testisspecific transcriptional repressor. In this application, we propose to analyze these new MBD3-1ike proteins in detail. Our hypothesis is that MBD3L1 and MBD3L2 are transcriptional regulators that function in conjunction with MBD2 and/or MBD3 to modulate gene activity. We propose four Specific Aims: 1) to characterize the transcriptional repression functions of MBD3L1 and MBD3L2 and its dependence on CpG methylation, 2) to identify and characterize the in vivo binding partners of MBD3L1 and MBD3L2 (preliminary data suggest that these proteins can form heterodimers with MBD2 and MBD3), 3) to determine if MBD3L1 and MBD3L2 are components of a larger complex in vivo (e.g., the Mi-2/NuRD complex), and 4) to derive knockout mice for Mbd311 and Mbd312. We will test the hypothesis that Mbd311 knockout mice will have a male sterility phenotype. Since both proteins are likely functioning as transcriptional regulators, it will be important to determine if there are changes in gene expression patterns in Mbd311 and Mbd312 (-/-) cells.