Determining cis- and trans- regulatory mechanisms of epigenetic bivalency

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PROJECT SUMMARY/ ABSTRACT In multicellular organisms, germ cells provide all the material necessary to generate offspring, including both genetic instructions encoded in DNA and regulatory information that guides developmental gene expression. Importantly, germ cells must retain the potential to establish totipotency while also functioning as terminally differentiated cells. Epigenetic modifications are one mechanism that encodes information about germ cell- specific regulatory programs while also permitting retention of developmental plasticity. A specialized epigenetic state called bivalency exists in germ cells and embryonic stem cells (ESCs), and may help to balance the competing requirements for cell fate restriction and plasticity. At bivalent domains, two contradictory histone modifications occupy the same nucleosome in promoters of transcriptionally silent genes: trimethylation of lysine 4 on histone 3 (H3K4me3), which promotes transcriptional activation, and H3K27me3, which promotes transcriptional repression. Bivalency is established in promoter regions of developmental genes and is thought to ‘poise’ these genes for conditional expression during somatic lineage specification. However, despite its potential importance in regulating early development, there is currently a gap in our understanding of the molecular machinery that regulates bivalency and its functional contributions to germ cell biology, embryo plasticity, and development. The goal of this project is to discover cis- and trans- regulatory mechanisms that contribute to bivalency. Specifically, we will utilize transgenic mouse embryonic stem cells to test the hypothesis that distinct sequence elements are responsible for establishing bivalency and that there are proteins maintaining histone modifications specifically in a bivalent context. Experiments in Aim 1 will test the contribution of specific sequence elements to establishment and maintenance of bivalency using both candidate and unbiased approaches. First, we will evaluate the role of a putative CCCTC-binding factor (CTCF) binding site in regulating bivalency at a specific test locus, Traf6. Second, we will systematically interrogate sequence elements in the Traf6 promoter using clustered regularly interspaced short palindromic repeats (CRISPR) technology to systematically ablate short pieces of the promoter and determine which sequence motifs are necessary to establish bivalency. Aim 2 will identify trans-acting novel regulators of bivalent chromatin by using a genome- wide CRISPR screen in three mouse ESC reporter lines. Together, these experiments will identify both locus- specific and global mechanisms important for defining and maintaining bivalent promoters. These data will advance our understanding of the cis- and trans- regulatory control of bivalency and provide insight into the function of this chromatin state in development. Our results will have implications in germ cell function and fertility, epigenetic inheritance, and embryonic development and differentiation.