Cells utilize epigenetic programs to regulate gene expression and cell fate. As multiple cancer types have defects in epigenetic pathways, understanding the basic principles underlying epigenetic mechanisms is paramount to our ability to diagnose, treat and potentially cure various cancer types. This proposal aims to use X chromosome inactivation (XCI) to elucidate mechanisms of epigenetic gene regulation. Mammalian females silence one of their two X-chromosomes using epigenetic programs. Little is known as to how a cell chooses an active or inactive X chromosome (Xa or Xi, respectively). Furthermore, the genomic sequences utilized to compact the Xi into a transcriptionally silent structure are equally elusive. Imprinted XCI in the developing mouse embryo serves as an appropriate model system for this analysis as evidence of choice and the initial events of Xi compaction both occur during early development. This proposal has two specific aims. The first addresses the role of transcription in the choice between the Xa and Xi. The second develops a novel strategy to identify the genomic sequences used to compact the Xi. Embryo culturing methods will be used to manipulate the transcriptional activity of the maternal and paternal pronuclei during early mouse development and analysis of X-chromosome activity will be carried out by allele-specific RT-PCR, FISH, reporter gene expression and immunofluorescence. Secondly, chromosomal conformational capture, chromatin immunoprecipitation and Solexa sequencing methodologies will be combined to identify X-linked sequences associated with chromatin modifications known to be required for the compaction of the Xi. Overall structure of the inactive X-chromosome will then be inferred and de novo motif analysis employed to identify sequences required for each chromatin modification. Health Relevance: Defects in X-chromosome inactivation during adulthood may be linked to a predisposition to cancer and improper regulation of genes on the X-chromosome are implicated in both breast and ovarian cancers, Furthermore, functional defects in genetic programs required for X-chromosome inactivation are also implicated in general cancer progression. By studying how mammalian females silence of their two X chromosomes, we can address both specific and general genetic mechanisms that play a role in cancer progression.
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