Determining cis- and trans- regulatory mechanisms of epigenetic bivalency

确定表观遗传二价的顺式和反式调控机制

• 批准号: 10534801
• 负责人: Kira Marshall
• 金额: $ 4.68万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2025-09-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10534801
• 关键词: Address; Affect; Binding Sites; CCCTC-binding factor; CRISPR screen; CRISPR/Cas technology; Cell Differentiation process; Cell physiology; Cells; Cellular biology; Chromatin; Common Core; Conserved Sequence; CpG Islands; DNA; Data; Development; Developmental Gene; Disease; Elements; Embryo; Embryonic Development; Epigenetic Process; Equilibrium; Exhibits; Fertility; Fertilization; Flow Cytometry; Foundations; Future; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Transcription; Genome; Germ Cells; Germ Lines; Goals; Histones; Instruction; Knowledge; Lysine; Maintenance; Mammals; Methylation; Modeling; Modification; Molecular; Mus; Nature; Nucleosomes; Organism; Phase; Process; Promoter Regions; Proteins; Reading; Reporter; Research; Role; Specific qualifier value; Sperm Maturation; Spermatogenesis; Testing; Totipotency; Transcriptional Activation; Transgenic Mice; Work; Writing; base; design; developmental plasticity; embryonic stem cell; experimental study; gene repression; genome-wide; histone modification; insight; male; next generation; novel; offspring; programs; promoter; recruit

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.

项目摘要/摘要 在多细胞生物体中，生殖细胞提供产生后代所需的所有物质，包括 编码在DNA中的遗传指令和指导发育基因表达的调控信息。 重要的是，生殖细胞必须保持建立全能性的潜力，同时还必须发挥终末功能 分化的细胞。表观遗传修饰是对生殖细胞信息进行编码的一种机制- 特定的调控计划，同时也允许保留发育可塑性。一个专门的表观遗传学家 生殖细胞和胚胎干细胞(ESCs)中存在一种称为二价体的状态，这种状态可能有助于平衡 对细胞命运限制和可塑性的相互竞争的要求。在二价区，两个相互矛盾的组蛋白 修饰在转录沉默基因的启动子中占据相同的核小体：赖氨酸的三甲基化 4在促进转录激活的组蛋白3(H3K4me3)和促进转录激活的H3K27me3上 转录抑制。二价体建立在发育基因的启动子区域，并被认为 使这些基因在体细胞谱系指定过程中有条件地表达。然而，尽管它的 在调节早期发育方面的潜在重要性，目前我们对 调节二价体及其对生殖细胞生物学、胚胎的功能贡献的分子机制 可塑性和发展性。这个项目的目标是发现顺式和跨式调节机制。 这导致了二价体。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体，并且存在蛋白质 特别是在二价环境中维持组蛋白修饰。目标1中的实验将测试其贡献 特定序列元件的使用来建立和维持二价体 不带偏见的方法。首先，我们将评估假定的CCCTC结合因子(CTCF)结合位点在 调节一个特定测试位点的二价性，Traf6。第二，我们将系统地询问序列元素 在Traf6启动子中，使用簇状规则间隔短回文重复(CRISPR)技术 系统地消融启动子的短片段，并确定哪些序列基序是必需的 建立二价体。AIM 2将通过使用基因组来鉴定二价染色质的反式作用新调节子- CRISPR宽屏有三条鼠标ESC报告线。总之，这些实验将识别这两个基因座- 对定义和维持二价启动子很重要的具体和全球机制。这些数据将 增进我们对二价体顺式和跨调节控制的理解，并提供对 这种染色质状态在发育中的作用。我们的结果将对生殖细胞功能和生育能力产生影响， 表观遗传，胚胎发育和分化。