Molecular Analysis of Transcriptional Enhancers in Hematopoiesis

项目摘要 在造血过程中控制谱系特异性基因转录的机制对于 确定细胞分化的基本原则和血液疾病的新疗法。增强剂 是通过指导时空基因表达来控制细胞命运的非编码顺式作用DNA序列。 尽管全基因组范围内的推定增强子谱的可用性越来越高，但仍然难以 阐明了在谱系分化过程中控制增强子功能的分子过程，强调了 这是理解正常和病理性造血的主要决定因素的主要障碍。的 我们现在面临的挑战是描述谱系定义增强子的调控成分， 生理学相关的背景，并询问他们在谱系特化过程中的体内功能。 解决这些挑战需要从增强子定位研究转向详细表征 它们在细胞分化期间在天然染色质中的分子组成。本项目的目标是 确定原位控制增强子激活的蛋白质和RNA复合物，并建立体内 谱系特异性增强子在红系细胞分化和造血过程中的功能。中央 假设增强子是由组织特异性转录因子、染色质 调节子和RNA复合物来指导谱系特异性基因的长距离染色质相互作用 转录。这一假设是根据2000年期间取得的重大进展提出的。 该项目的前期资助，包括开发基于CRISPR/dCas 9的亲和捕获技术， 基因座特异性染色质相互作用和增强子靶向表观遗传扰动系统 询问增强子功能。使用这些方法，我们发现了组织原则 控制红系特异性超级增强子，并在 3D染色质结构的分级组织和体内增强子功能。时遵循这些 根据初步数据，我们的假设将通过三个具体目标进行测试：1）确定增强子调节 通过基于dCas 9的邻近标记在细胞分化期间检测染色质复合物。2)识别和 表征控制增强子结构和功能的染色质相关RNA。3)阐明体内 通过单细胞CRISPR表观遗传扰动的“枢纽”增强子在造血中的功能。综合这些 这些研究不仅阐明了转录控制细胞内主要决定子的机制， 分化，而且还建立了原位和体内分析增强子结构-功能的新工具。 这样的结果有望促进我们对因果关系的机械理解， 增强剂组合物和造血过程中的体内功能。最终，这些发现将有助于 开发通过遗传或药理学调节增强子活性的创新方法 操作，导致基于机制的治疗血液病。

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.