Elucidating the transcriptional mechanisms that control the expression of the SARS-CoV-2 receptor ACE2

项目总结 迫切需要了解控制ACE2表达的调控机制，即 新型冠状病毒SARS-CoV-2在生理和病理条件下的细胞受体 条件。ACE2与SARS-CoV-2刺突蛋白的结合及其蛋白水解性切割 ACE2，促进冠状病毒进入靶细胞、病毒复制和传播。因此，ACE2 已被认为是病毒传染性和疾病病理学的关键因素。尽管ACE2被劫持了 SARS-CoV-2在新冠肺炎的发病机制中，其主要生理作用是中和组织损伤和 炎症是肾素血管紧张素系统(RAS)的一部分。ACE2的表达被限制在特定的 组织驻留细胞类型，发育受调节，对生理(如性别)高度敏感 激素)和病理信号(如炎症或病毒感染)。通过调查表观遗传学 在人类ACE2基因座上主要表达ACE2的细胞类型中，我们鉴定了一组基因- 远端顺式调节元件(CRE)显示组织特异性活性，与推测的细胞类型一致- ACE2的特定增强子元素。组织和发育阶段特异性的分子基础 ACE2的表达尚不清楚，控制ACE2表达的调控机制 生理和病理信号仍未被探索，突显了知识中的关键差距。如果没有一个 深入了解控制ACE2表达的分子和细胞途径、遗传或 血管紧张素转换酶2作为新冠肺炎的一种治疗方法的药物调节可能仍然无法实现。 这个项目的目标是弥合我们对转录机制的理解上的一个关键差距。 在正常和病理条件下控制ACE2的表达。中心假设是ACE2 表达受组织特异性CRE的组合控制，以招募特定细胞类型和/或信号- 依赖转录因子。这一假设是在组织特异性的基础上提出的 ACE2在人细胞中的表达谱，多个基因的存在--以Active为界的远端Cres ACE2表达细胞的染色质特征及其对ACE2表达的差异性影响 生理或病理刺激。在这些发现的指导下，这一假设将由两个人进行检验 具体目标：1)确定组织特异性顺式调控元件在控制ACE2中的功能作用 表达及其对刺激的反应；2)鉴定和鉴定染色质调节复合体 通过CRISPR/dCas9介导的亲和纯化，负责ACE2的表达。一起，通过专注于 经过验证的SARS-CoV-2进入受体，我们的研究不仅将促进我们对SARS-CoV-2的机制的理解 病毒易感性和感染后病理的关键方面，但也建立了急需的资源 在人类细胞中表达ACE2所需的候选顺式和反式调控因子，从而促进 正在制定创新战略，以利用ACE2作为针对新冠肺炎的靶向治疗。

PROJECT SUMMARY There is a pressing urgency to understand the regulatory mechanisms that control the expression of ACE2, the cellular receptor for the new coronavirus SARS-CoV-2, in human cells under physiological and pathological conditions. The binding between ACE2 and the SARS-CoV-2 spike protein, along with proteolytic cleavage of ACE2, facilitates entry of the coronavirus into target cells, viral replication, and transmission. As such, ACE2 has been proposed as a key factor in virus infectivity and disease pathology. Although ACE2 is hijacked by SARS-CoV-2 in the pathogenesis of COVID-19, its primary physiological role is to counteract tissue injury and inflammation as part of the renin-angiotensin system (RAS). The expression of ACE2 is restricted to specific tissue-resident cell types, developmentally regulated, and highly responsive to physiological (e.g. sex hormones) and pathological (e.g. inflammation or virus infection) signals. By surveying the epigenetic landscapes at the human ACE2 locus in the major ACE2-expressing cell types, we identified a set of gene- distal cis-regulatory elements (CREs) displaying tissue-specific activity, consistent with putative cell type- specific enhancer elements for ACE2. The molecular basis for the tissue- and developmental stage-specific expression of ACE2 is unknown, and the regulatory mechanisms controlling ACE2 expression in response to physiological and pathological signals remain unexplored, highlighting a critical gap in knowledge. Without an in-depth understanding of the molecular and cellular pathways that control ACE2 expression, the genetic or pharmacological modulation of ACE2 as an approach to therapy for COVID-19 will likely remain unreachable. The objective of this project is to close a critical gap in our understanding of the transcriptional mechanisms controlling ACE2 expression under normal and pathological conditions. The central hypothesis is that ACE2 expression is controlled by combinations of tissue-specific CREs to recruit cell type-specific and/or signal- dependent transcription factors. This hypothesis has been formulated on the basis of the tissue-specific expression profiles of ACE2 in human cells, the presence of multiple gene-distal CREs demarcated by active chromatin signatures in ACE2-expressing cells, and the differential effects on ACE2 expression upon physiological or pathological stimulations. Guided by these findings, this hypothesis will be tested by two specific aims: 1) Determine the functional roles of tissue-specific cis-regulatory elements in controlling ACE2 expression and its responses to stimuli; 2) Identify and characterize chromatin regulatory complexes responsible for ACE2 expression by CRISPR/dCas9-mediated affinity purification. Together, by focusing on the validated entry receptor for SARS-CoV-2, our studies will not only advance our mechanistic understanding of critical aspects of viral susceptibility and post-infection pathology, but also establish a much-needed resource of candidate cis- and trans-regulatory factors required for ACE2 expression in human cells, thus facilitating ongoing development of innovative strategies to leverage ACE2 as targeted therapies against COVID-19.