Cardoso

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

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.