Regulation of hematopoiesis by epigenetic timing control

项目总结： 干细胞命运决定的时机对多细胞组织的大小和功能至关重要。在许多 发育过程中，干细胞只有在跨越数天和细胞的长时间延迟后才能分化 分裂，使得稀有的多能祖细胞种群呈指数级增长。分化中的变异 时机会导致组织大小和形态发生戏剧性的变化，并可能有助于人类的发育 和疾病。然而，分化过程中的时间控制的潜在机制仍然不清楚。 这项提议将使用造血模型系统来理解表观遗传调控 决定细胞命运的时间。最近的证据表明，表观遗传机制在 顺式病毒中的单个基因组基因座在数天和细胞分裂的过程中可能会缓慢变化，实现 发育基因激活的限速步骤。然而，目前还不清楚这是否是一种选择时机的范例 控制在细胞分化过程中被广泛使用。 该提案旨在确定表观遗传计时机制(目标1)的流行度，并发现 它们如何受到非编码DNA元件的调控(目标2)。表观遗传调控，不同于转录 在同一细胞(如X染色体)中，反式调节因子在每个基因拷贝上独立发挥作用 停用)。因此，为了研究表观遗传时序控制流行率和机制，这项提议将 使用可以单独监测单个基因拷贝活动的小鼠模型。在Aim 1中，一辆F1 具有频繁单核苷酸多态的杂交小鼠品系将被用于分析个体 用Cut&Tag分析全基因组等位基因。这种单细胞基因组学方法可以通过以下方式区分表观遗传状态 量化蛋白质-DNA相互作用，并将揭示表观遗传计时机制的共性 造血术。在目标2中，将使用两个副本的基因报告系统来提供高灵敏度的读数 造血祖细胞分化过程中的表观遗传调控。CRISPR/CAS9方法将是 用于有效识别功能性非编码DNA元件，并阐明它们对表观遗传学的影响 定时控制。 该研究金将为计算基因组学和CRISPR/CAS9提供培训机会 基因组和表观基因组编辑。这些领域的专家将担任技能培训和职业生涯的导师。 发展。这项培训计划包括量身定做的课程、研讨会和讲习班，以加强 培训环境，并促进向博士后研究奖学金的过渡。预期的结果 这项提议将为组织发育和动态平衡过程中的时间控制建立一个新的模型。会的 也为血液疾病的发病机制提供了新的见解，并启发了表观遗传学的新方法 在基于细胞的治疗中重新编程。

Project Summary: The timing of stem cell fate decisions is critical for multicellular tissue size and function. In many developmental processes, stem cells differentiate only after a long time-delay spanning multiple days and cell divisions, allowing rare populations of multipotent progenitors to expand exponentially. Variation in differentiation timing generates dramatic changes in tissue size and morphology and likely contributes to human development and disease. However, the mechanism underlying timing control during differentiation remains unclear. This proposal will use hematopoiesis a model system to understand how epigenetic regulation contributes to the timing of cell fate decisions. Recent evidence suggests that epigenetic mechanisms acting at individual genomic loci in cis can change slowly over the course of multiple days and cell divisions, implementing rate-limiting steps to developmental gene activation. However, it remains unclear if this is a paradigm for timing control broadly utilized during cell differentiation. This proposal aims to determine the prevalence of epigenetic timing mechanisms (Aim 1) and uncover how they can be regulated by non-coding DNA elements (Aim 2). Epigenetic regulation, unlike transcription factor regulation in trans, functions at each gene copy independently in the same cell (e.g. X-chromosome inactivation). Therefore, to investigate epigenetic timing control prevalence and mechanisms, this proposal will use mouse models in which the activity of individual gene copies can be monitored separately. In Aim 1, a F1 hybrid mouse strain that harbors frequent single nucleotide polymorphisms will be used to analyze individual alleles genome-wide by CUT&Tag. This single-cell genomics approach can distinguish epigenetic states by quantifying of protein-DNA interactions and will reveal the generality of epigenetic timing mechanisms during hematopoiesis. In Aim 2, a two-copy gene reporter system will be used to provide a highly sensitive readout for epigenetic regulation in hematopoietic progenitors undergoing differentiation. CRISPR/Cas9 approaches will be used to efficiently identify functional non-coding DNA elements and elucidate how they contribute to epigenetic timing control. This fellowship will provide an opportunity for training in computational genomics and CRISPR/Cas9 genome and epigenome editing. Experts in these fields will serve as mentors for skills training and career development. This training plan incorporates tailored courses, seminars and workshops that will enhance the training environment and facilitate the transition to a postdoctoral research fellowship. The expected outcome of this proposal will establish a new model for timing control during tissue development and homeostasis. It will also provide new insights into blood disease pathogenesis and inspire new approaches for epigenetic reprogramming in cell-based therapies.