Determining cis- and trans- regulatory mechanisms of epigenetic bivalency

项目总结/摘要 在多细胞生物中，生殖细胞提供产生后代所需的所有物质，包括 在DNA中编码的遗传指令和指导发育基因表达的调节信息。 重要的是，生殖细胞必须保持建立全能性的潜力，同时也作为终末细胞发挥作用。 分化细胞表观遗传修饰是编码生殖细胞信息的一种机制- 特定的调节程序，同时也允许保留发育可塑性。一种特殊的表观遗传 一种称为二价的状态存在于生殖细胞和胚胎干细胞（ESCs）中，可能有助于平衡 细胞命运限制和可塑性的竞争需求。在二价结构域，两个矛盾的组蛋白 在转录沉默基因的启动子中，修饰占据了相同的核小体：赖氨酸的三甲基化 4对组蛋白3（H3 K4 me 3），促进转录激活，和H3 K27 me 3，促进 转录抑制发育基因的启动子区域建立了二价， 以在体细胞谱系特化过程中“平衡”这些基因的条件表达。然而，尽管其 潜在的重要性，在调节早期发展，目前有一个差距，我们的理解， 调节二价的分子机制及其对生殖细胞生物学、胚胎发育和生殖系统的作用 可塑性和发展。本项目的目标是发现顺式和反式调节机制 导致了二价。具体来说，我们将利用转基因小鼠胚胎干细胞来测试 假设不同的序列元件负责建立二价，并且存在蛋白质 在二价环境中特异性地维持组蛋白修饰。目标1中的实验将检验 特异性序列元件对使用候选物和 无偏见的方法。首先，我们将评估一个假定的CCCTC结合因子（CTCF）结合位点的作用， 在特定的测试位点Traf 6调节二价。其次，我们将系统地询问序列元素 在Traf 6启动子中使用成簇的规则间隔的短回文重复序列（CRISPR）技术， 系统地切除启动子的短片段，并确定哪些序列基序是必需的， 建立二价。目标2将通过使用基因组识别反式作用的二价染色质的新调节子， 在三个小鼠ESC报告细胞系中进行宽CRISPR筛选。总之，这些实验将确定两个位点- 对于定义和维持二价启动子重要的特异性和全局机制。这些数据将 推进我们对二价顺式和反式调控的理解，并提供对 这种染色质状态在发育中的作用。我们的研究结果将对生殖细胞功能和生育能力产生影响， 表观遗传和胚胎发育和分化。

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.