PROJECT 3 ROLE OF CTCF IN CHROMATIN AND NUCLEAR ORGANIZATION AT THE FSHD 4qD4Z4 LOCUS The chromatin insulator protein, CTCF, has been shown to protect genes from epigenetic silencing. CTCF binding is generally inhibited by DNA methylation and CTCF prevents spreading of DNA methylation. Recent evidence suggests that CTCF can also act to bring spatially separated chromatin domains in close proximity, both in cis and in trans. In collaboration with Dr. van der Maarel, we have mapped CTCF binding sites in the D4Z4 unit and demonstrated both enhanced binding of CTCF to the partially deleted pathogenic D4Z4 allele in FSHD cells and decreased DNA methylation at these CTCF binding sites. Moreover, enhanced CTCF binding to D4Z4 was also observed in undifferentiated ES cells. These findings lead to the hypothesis that inappropriate CTCF binding in the D4Z4 units in FSHD might interfere with the establishment or maintenance of developmentally regulated epigenetic repression and result in inappropriate transcription or nuclear localization. Therefore, our long-term goal is to determine whether CTCF binding on the disease associated allele regulates regional chromatin structure and/or RNA transcription. In our Aim1, we will determine whether the CTCF sites in the D4Z4 units function as insulators, transcriptional regulators, or chromatin boundary elements, and whether CTCF is necessary for regulating D4Z4 chromatin structure and transcription. These studies will be done using siRNA-mediated knock-down of CTCF in control and FSHD human muscle cells and D4Z4 mouse models.
In Aim 2, we will detennine whether human IPS cells generated from control and FSHD fibroblasts accurately recapitulate the chromatin structure of D4Z4 in ES cells before and during in vitro differentiation, and whether CTCF binding and/or deletion of D4Z4 subunits prevent a developmentally regulated chromatin-mediated repression of D4Z4 transcripts. Finally, in Aim 3, we will use chromatin conformation capture in combination with chromatin immunoprecipitation techniques to determine whether CTCF, chromatin context, and/or developmental state mediate intra- or inter-chromosomal interactions at the 4q D4Z4 locus and whether these interactions are altered in FSHD cells.
The significance of this proposal is that characterization of the chromatin structure and long range chromatin interactions specific to the disease associated D4Z4 repeat and their developmental regulation will contribute to our understanding of the epigenetic mechanisms underlying FSHD. Moreover, the identified mechanisms will be important for the development of methods for re-establishing repressive chromatin structure on the pathogenic alleles as new therapeutic strategies for FSHD.
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