Molecular Analysis of Transcriptional Enhancers in Hematopoiesis

项目总结 破译在造血过程中控制谱系特异性基因转录的机制对 确定细胞分化的基本原理和血液疾病的新疗法。增强剂 是非编码顺式作用DNA序列，通过指导时空基因表达来控制细胞命运。 尽管假定的增强子的全基因组图谱的可用性越来越高，但仍然很难 阐明在谱系分化过程中控制增强子功能的分子过程，突出了一个 主要障碍是理解正常和病理性造血的主要决定因素。这个 我们现在面临的挑战是确定谱系定义增强剂的监管组件的特征 生理相关的背景，并在血统指定期间询问它们在体内的功能。 应对这些挑战需要从增强子图谱研究转向对 它们在细胞分化过程中天然染色质中的分子组成。这个项目的目标是 原位确定控制增强子激活的蛋白质和RNA复合体，并建立体内 谱系特异性增强子在红系细胞分化和造血中的作用。中环 假设增强剂是由组织特异性转录因子、染色质组合而成 调节因子和RNA复合体，指导谱系特异基因的长程染色质相互作用 抄写。这一假说的提出是基于 该项目之前的资助期，包括开发基于CRISPR/dCas9的亲和力捕获 基因位点特异性染色质相互作用和增强子靶向性表观遗传扰动系统的体内研究 增强剂功能的询问。使用这些方法，我们揭示了组织原则 控制红系特异的超级增强子并建立新的分子链接 3D染色质结构和体内增强子功能的分级组织。在这些指导下 初步数据，我们的假设将通过三个具体目标进行验证：1)确定增强子调节 基于dCas9的邻近标记研究细胞分化中的染色质复合体。2)识别和 描述控制增强子结构和功能的染色质相关RNA。3)阐明体内的 “Hub”增强子在单细胞CRISPR表观遗传学扰动中的造血功能。把这些放在一起 研究不仅将阐明细胞主要决定因素的转录控制机制 但也为在原位和体内分析增强子的结构和功能建立了新的工具。 这样的结果有望推动我们从机械上理解 造血过程中增强剂的组成和体内功能。最终，这些发现将有助于 开发通过遗传或药物调节增强子活性的创新方法 操作，导致血液病的基于机制的治疗。

PROJECT SUMMARY Deciphering mechanisms that control lineage-specific gene transcription during hematopoiesis is critical for identifying fundamental principles of cellular differentiation and new therapies for blood disorders. Enhancers are noncoding cis-acting DNA sequences that control cell fate by directing spatiotemporal gene expression. Despite the increasing availability of genome-wide profiles of putative enhancers, it remains difficult to elucidate the molecular processes controlling enhancer function during lineage differentiation, highlighting a major impediment for understanding the major determinants of normal and pathological hematopoiesis. The challenges we are now facing are to characterize the regulatory components of lineage-defining enhancers in physiologically relevant contexts and to interrogate their in vivo function during lineage specification. Addressing these challenges requires a shift from enhancer mapping studies to detailed characterization of their molecular composition in native chromatin during cellular differentiation. The objectives of this project are to determine the protein and RNA complexes controlling enhancer activation in situ, and to establish the in vivo function of lineage-specifying enhancers during erythroid cell differentiation and hematopoiesis. The central hypothesis is that enhancers are assembled by combinations of tissue-specific transcription factors, chromatin regulators, and RNA complexes to direct long-range chromatin interactions for lineage-specific gene transcription. This hypothesis has been formulated on the basis of the substantial progresses made during the prior funding period of this project, including the development of CRISPR/dCas9-based affinity capture of locus-specific chromatin interactions and enhancer-targeting epigenetic perturbation systems for in vivo interrogation of enhancer function. Using these approaches, we uncovered the organizational principles controlling erythroid lineage-specific super-enhancers and established new molecular links between hierarchical organization of 3D chromatin structures and in vivo enhancer function. Guided by these preliminary data, our hypothesis will be tested by three specific aims: 1) Determine enhancer-regulating chromatin complexes during cellular differentiation by dCas9-based proximity labeling. 2) Identify and characterize chromatin-associated RNAs that control enhancer structure and function. 3) Elucidate the in vivo function of “hub” enhancers in hematopoiesis by single-cell CRISPR epigenetic perturbation. Together these studies will not only elucidate mechanisms for the transcriptional control of principal determinants of cellular differentiation, but also establish new tools for the analysis of enhancer structure-function in situ and in vivo. Such results are expected to advance our mechanistic understanding of the causal relationships between enhancer composition and in vivo function during hematopoiesis. Ultimately, these findings will facilitate the development of innovative approaches to modulate enhancer activity through genetic or pharmacological manipulations, resulting in mechanism-based therapies for hematologic disorders.