Establishment and maintenance of epigenetic states that govern and stabilize cell fate upon differentiation are crucial for the development of multicellular organisms. DNA methylation, which is mitotically heritable, is an important component of mammalian epigenetic gene regulation. Over the past decade, the Human Epigenome Projects have comprehensively profiled tissue- and cell-type-specific DNA methylation and identified dynamic methylation differences. The timing of developmentally programmed DNA methylation and associated mechanisms of transcriptional regulation during early cell-lineage specification, however, remain poorly understood. This proposal builds upon our recent discovery that, in addition to canonical transcriptional repression by DNA methylation at promoter CpG islands (CGIs), transcriptional activation of a group of developmental genes is related to gene body CGI methylation during human embryonic stem cell (hESC) differentiation. One particular gene of interest is Hypermethylated in cancer 1 (HIC1) gene, which is a tumor suppressor and a candidate gene for a developmental disorder Miller-Dieker syndrome. We found that CGI methylation at the 3' end of HIC1 (3' CGI methylation) is highly conserved in human and mouse, and may specify mesenchymal Hic1-expressing during fetal development. More importantly, our preliminary studies suggest that 3' CGI methylation regulates Hic1 transcription via a CCCTC-binding factor (CTCF)-dependent mechanism. Based on these findings, the proposed research uses mouse models to investigate whether and how the 3' CGI methylation controls the temporal and spatial expression of Hic1 in developing embryos. Specifically, we will: 1 ? Investigate the mechanism by which 3' CGI methylation regulates Hic1 gene activation. Using Hic1-citrine (a yellow fluorescent protein) reporter mice, we will perform chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) to systemically map CTCF-bindings at the Hic1 locus in tissues known to express Hic1 in mouse embryos. 2 ? Investigate the function of Hic1 3' CGI methylation during mouse development. Using a novel mouse model to enable CRISPR-based targeted DNA demethylation, we will determine whether Hic1 3' CGI demethylation affects transcriptional regulation of Hic1 from embryonic development into birth, and whether defective epigenetic regulation leads to developmental defects. Altogether, this exploratory project will establish a combination of novel technologies for in vivo studies to dissect epigenetic transcriptional regulation. The successful completion of these studies will yield important insights into the function role of DNA methylation for mammalian development.
(Relevance Statement) The precise regulation of gene expression is essential for development, and defective transcriptional regulation can lead to developmental diseases. Employing novel mouse models, our research is aimed at understanding the functional role of epigenetic gene regulation by DNA methylation and whether these epigenetics can impact normal development. Elucidating these fundamental epigenetic mechanisms may eventually inform the design of effective interventions to prevent human developmental disorders.
