Role of methyl-cytosine binding proteins in the gain and loss of pluripotency

甲基胞嘧啶结合蛋白（methylcytosine binding protein, MBDs）在表观遗传效应中起着重要的介导作用。它们与甲基化的DNA结合并招募组蛋白去乙酰化酶（hdac），这可能导致染色质凝聚和转录沉默。我们之前的研究表明，MeCP2水平在分化过程中增加，导致大规模的基因组重组。因此，我们正在研究mbd在多能性获得和丧失中的作用。我们已经生成并鉴定了一组特异性抗体，现在将完成我们对MBD在多能、分化和重编程（iPS）细胞中的表达和定位的系统分析。我们将进一步确定MBDs在这些细胞中的动态，特别是在基因组甲基胞嘧啶羟基化增加的反应中，甲基胞嘧啶是一种可能与表观遗传重编程相关的新型DNA修饰。利用特定的突变体，我们将研究MBDs在控制染色质组织和多能性获得或丧失的动力学中的作用。在过去的几年里，我们已经将单分子显微镜应用于活细胞研究，现在将以高时间和空间分辨率研究染色质动力学。我们将特别研究MBD染色质在单分子水平上的相互作用，并将比较多能和分化细胞中的染色质动力学。这项研究将阐明mbd的作用，并将解决在多能性和重编程中高阶基因组组织和动力学的功能相关性。

Methyl cytosine binding proteins (MBDs) play a central role in the mediation of epigenetic effects. They bind to methylated DNA and recruit histone deacetylases (HDACs), which may then cause chromatin condensation and transcriptional silencing. We have previously shown that MeCP2 levels increase during differentiation causing large scale genome reorganization. We are, therefore, investigating the role of MBDs in the gain and loss of pluripotency. We have generated and characterized a set of specific antibodies and will now complete our systematic analysis of MBD expression and localization in pluripotent, differentiated and reprogrammed (iPS) cells. We will further determine the dynamics of MBDs in these cells, especially in response to increased hydroxylation of genomic methyl cytosine, a novel DNA modification possibly linked to epigenetic reprogramming. Using specific mutants we will investigate the role of MBDs in controlling chromatin organization and dynamics in the gain or loss of pluripotency. Over the past years we have adapted single molecule microscopy to live cell studies and will now investigate chromatin dynamics with high temporal and spatial resolution. We will in particular study MBD chromatin interactions at the single molecule level and will compare chromatin dynamics in pluripotent versus differentiated cells. This study should clarify the role of MBDs and will address the functional relevance of higher order genome organization and dynamics in pluripotency and reprogramming.