Unravelling the Molecular Mechanism of DNA Demethylation around CTCF Binding Sites in Mammalian Cells

Binding of transcription factors to appropriate loci directs the transcriptional program and safeguards the success of various biological events, such as the normal embryonic development and suitable cellular responses to external stimuli. CTCF is a highly-conserved sequence-specific transcription factor with eleven tandem zinc finger domains, playing an essential part in insulator function and organization of the high-order structure of the genome, both of which are key to orchestrating the transcriptional program. Two CTCF molecules can bind to chromatin DNA in convergent orientations that stop the extruding chromatin loops to wire the intra-chromosomal long-range interactions, guiding the proper pairing between distalcis-regulatory elements and promoters of targeted genes. For instance, appropriate binding of CTCF is critical for determining the pattern of combinatorial expression of clustered proto-cadherin genes, and the antibody class switching in B lymphocytes. In our preliminary work, we have developed a novel method termed G-MAT that traces the foot-prints of endogenous CTCF in living mammalian cells. We found that CTCF searched its binding sites through stochastic collision to chromatin. DNA with methyl-CpG is preferred by CTCF. However, it has been well appreciated that CTCF binding sites are depleted of CpG methylation, indicating a demethylation event provoked by the binding of CTCF. This is also supported by an independent study led by Prof. Dirk Schübeler, a world-renowned expert in this field, showing that introduction of a CTCF motif into a methylated construct sparked the spreading of local demethylation. Therefore, we are intrigued to address the molecular mechanism of the demethylation around CTCF binding sites. With a BioID assay, which was employed to identify proteins co-occupying CTCF binding sites on chromatin, we found two methyl-CpG binding domain proteins (MBD2 and MBD3) interacting with CTCF, prioritized as candidate players in mediating the demethylation process. MBD proteins were previously shown to be associated with CpG-demethylation, suggesting a hypothesis that binding of CTCF recruits MBDs to chromatin and consequently causes demethylation. The reshaping of the local epigenetic environment of chromatin is required for binding of other transcriptional regulators. To test the hypothesis, we propose three specific objectives: 1) determining whether CTCF is required for recruitment of MBD proteins to its binding sites; 2) examining whether MBD proteins are essential for demethylation of DNA around CTCF binding sites; 3) exploring the biological consequences of MBD-mediated cytosine demethylation around CTCF binding sites. Results fromthe proposed experiments will provide answers to these questions, which will lead us to better understanding of the highly regulated binding of CTCF, demarcating the regulatory factors and effector enzymes implicated, and lay a theoretical foundation for future studies on the role of hypomethylation around CTCF binding in cellular function and disease.