Molecular Analysis of Transcriptional Enhancers in Hematopoiesis

项目摘要 阐明调节红细胞生成中基因表达的机制是研究红细胞生成的基础。 了解细胞分化，并开发红细胞疾病的新疗法。转录 增强子通过指导时空基因表达来确定细胞身份。最近，我们和其他人 通过染色质特征的基因组规模分析鉴定红细胞特异性增强子元件。进一步 分析揭示了GATA 1相互作用的共激活因子及其组合作为候选驱动因子， 增强子功能这些研究建立了一个全面的红细胞增强子元件目录， 绝大多数这些增强剂的分子组成和体内功能仍然未知。鉴于 增强子经常被疾病相关的遗传变异所靶向，因此必须填补这一空白， 知识本项目的目的是确定蛋白质复合物和长距离DNA 在红细胞生成过程中负责增强子组装和原位功能的相互作用。中央 假设是红系增强子是通过特定的谱系调节因子组合来组装的， 转录因子如GATA 1募集转录辅激活因子并启动长距离染色质 基因转录的相互作用。这一假设是在我们初步研究的基础上提出的 由三个单独的增强子组成的红细胞特异性超级增强子，其调节SLC 25 A37 基因，一种对铁代谢和血红素合成至关重要的线粒体铁转运蛋白，以及一种创新的 一种使用核酸内切酶缺陷的方法来鉴定与单个基因组位点相关的大分子， CRISPR系统的Cas9（dCas 9）和单向导RNA（sgRNA）组分。时遵循这些 初步数据，这一假设将通过追求三个具体目标进行测试：1）识别和表征 通过dCas 9亲和纯化原位检测SLC 25 A37增强子相关蛋白，随后进行定量分析。 蛋白质组学分析和验证研究。2)确定SLC 25 A37增强子的长程DNA相互作用 使用通过配对末端测序（dCas 9-ChIA-PET）的dCas 9介导的染色质相互作用分析。通过 比较单个SLC 25 A37增强子和 它们在红细胞生成过程中的时间变化，这些分析将建立染色质之间的功能联系， 循环和增强器功能。3)明确41例红系疾病相关的功能需求， 在dCas 9中使用工程化的dCas 9-LSD 1阻遏物复合物的进化上保守的超级增强子 敲入小鼠模型。这些研究将不仅阐明遗传控制的机制， 红细胞铁代谢的主要调节因子，而且还为原位分析增强子提供了新的工具， 调节组件。这些结果有望促进我们对组成的理解， 增强子在协调红系基因表达中的功能。最终，这些知识有可能 为红细胞疾病中靶向非编码调控元件的治疗策略的设计提供信息。

PROJECT SUMMARY Elucidating mechanisms that regulate gene expression in red blood cell production is fundamental to understanding cellular differentiation, and to developing new therapies for red cell disorders. Transcriptional enhancers determine cell identity by directing spatiotemporal gene expression. Recently, we and others have identified erythroid-specific enhancer elements through genome-scale profiling of chromatin features. Further analysis has uncovered GATA1-interacting coactivators and their combinations as candidate drivers of enhancer function. These studies established a comprehensive catalog of erythroid enhancer elements, yet the molecular composition and in vivo function of the vast majority of these enhancers remain unknown. Given that enhancers are frequently targeted by disease-associated genetic variations, it is imperative to fill this gap in knowledge. The objective of this project is to determine the protein complexes and long-range DNA interactions responsible for enhancer assembly and function in situ during erythropoiesis. The central hypothesis is that erythroid enhancers are assembled by specific combinations of lineage-regulating transcription factors such as GATA1 to recruit transcriptional coactivators and to initiate long-range chromatin interactions for gene transcription. This hypothesis has been formulated on the basis of our preliminary studies of an erythroid-specific super-enhancer comprised of three individual enhancers that regulates the SLC25A37 gene, a mitochondrial iron transporter essential for iron metabolism and heme synthesis, and an innovative approach to identify macromolecules associated with a single genomic locus using the endonuclease-deficient Cas9 (dCas9) and single guide RNA (sgRNA) components of the CRISPR system. Guided by these preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Identify and characterize SLC25A37 enhancer-associated proteins in situ by dCas9 affinity purification, followed by quantitative proteomic analysis and validation studies. 2) Determine SLC25A37 enhancer long-range DNA interactions using dCas9-mediated chromatin interaction analysis by paired-end sequencing (dCas9-ChIA-PET). By comparing the frequency and location of chromatin interactions between individual SLC25A37 enhancers and their temporal changes during erythropoiesis, these analyses will establish functional links between chromatin looping and enhancer function. 3) Define the functional requirement of 41 erythroid disease-associated, evolutionarily conserved super-enhancers using an engineered dCas9-LSD1 repressor complex in a dCas9 knockin mouse model. Together these studies will not only elucidate the mechanisms for the genetic control of a principal regulator of erythroid iron metabolism, but also provide new tools for in situ analysis of enhancer- regulating components. Such results are expected to advance our understanding of the composition and function of enhancers in coordinating erythroid gene expression. Ultimately, such knowledge has the potential to inform the design of therapeutic strategies to target non-coding regulatory elements in red cell disorders.