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

基本信息

批准号：
10708831
负责人：
Kira Marshall
金额：
$ 3.26万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10708831
关键词：
Ablation Affect Binding Sites CCCTC-binding factor CRISPR screen CRISPR/Cas technology Cell Differentiation process Cell Separation Cell physiology Cells Cellular biology Chromatin Conserved Sequence CpG Islands DNA Data Development Developmental Gene Disease ES Cell Line 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 candidate identification design developmental plasticity embryonic stem cell experimental study gene repression genome-wide histone modification insight male next generation novel offspring programs promoter recruit

项目摘要

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.

项目概要/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 DNA 中编码的遗传指令和指导发育基因表达的调控信息。重要的是，生殖细胞必须保留建立全能性的潜力，同时也发挥终末细胞的作用。分化细胞的表观遗传修饰是编码生殖细胞信息的一种机制。特定的监管计划，同时还允许保留发育可塑性。生殖细胞和胚胎干细胞 (ESC) 中存在称为二价的状态，可能有助于平衡在二价域，两个相互矛盾的组蛋白竞争细胞命运限制和可塑性。修饰在转录沉默基因的启动子中占据相同的核小体：赖氨酸的三甲基化组蛋白 3 (H3K4me3) 上的 4 促进转录激活，以及 H3K27me3 促进转录激活转录抑制是在发育基因的启动子区域建立的，并且被认为是二价性。然而，尽管如此，在体细胞谱系规范期间“平衡”这些基因的条件表达。尽管在调节早期发育方面具有潜在的重要性，但目前我们对调节二价的分子机制及其对生殖细胞生物学、胚胎的功能贡献该项目的目标是发现顺式和反式调节机制。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目标 1 中的实验将测试其贡献。使用候选和维持二价的特定序列元件首先，我们将评估假定的 CCCTC 结合因子 (CTCF) 结合位点的作用。在特定测试位点 Traf6 上调节二价其次，我们将系统地询问序列元件。在 Traf6 启动子中使用成簇规则间隔短回文重复序列 (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要的目标 2 将通过使用基因组来识别二价染色质的反式作用新型调节因子。对三个小鼠 ESC 报告基因系进行广泛的 CRISPR 筛选，这些实验将共同鉴定这两个基因座。这些数据对于定义和维持二价启动子非常重要。我们对二价的顺式和反式监管控制的理解，并提供了对这种染色质状态在发育中的功能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。