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

项目摘要 目前迫切需要了解控制ACE 2表达的调节机制， 新型冠状病毒SARS-CoV-2的细胞受体，在生理和病理条件下的人体细胞中 条件ACE 2与SARS-CoV-2刺突蛋白的结合，沿着蛋白水解切割， ACE 2促进冠状病毒进入靶细胞、病毒复制和传播。因此，ACE 2 已被认为是病毒感染性和疾病病理学的关键因素。虽然ACE 2被劫持 SARS-CoV-2在COVID-19的发病机制中，其主要生理作用是抵消组织损伤， 炎症作为肾素-血管紧张素系统（RAS）的一部分。ACE2的表达仅限于特定的 组织驻留细胞类型，发育调节，对生理（如性别）高度反应 激素）和病理（例如炎症或病毒感染）信号。通过调查表观遗传 在人类ACE2基因座的主要ACE2表达细胞类型的景观，我们确定了一组基因- 远端顺式调节元件（CREs）显示组织特异性活性，与假定的细胞类型一致， ACE 2的特异性增强子元件。组织和发育阶段特异性 ACE2的表达是未知的，并且控制ACE2表达的调节机制响应于 生理和病理信号仍未得到探索，突出了知识上的重大差距。没有 深入了解控制ACE2表达的分子和细胞途径，基因或 ACE2的药理学调节作为COVID-19的治疗方法可能仍然无法实现。 这个项目的目标是缩小我们对转录机制理解的关键差距 在正常和病理条件下控制ACE2表达。核心假设是ACE 2 表达受组织特异性CREs的组合控制，以募集细胞类型特异性和/或信号转导。 依赖性转录因子这一假设是根据组织特异性 ACE 2在人细胞中的表达谱，多个基因远端CREs的存在，由活性CREs区分， ACE 2表达细胞中的染色质特征，以及ACE 2表达的差异效应。 生理或病理刺激。在这些发现的指导下，这一假设将由两个 具体目标：1）确定组织特异性顺式调节元件在控制ACE2中的功能作用 表达及其对刺激的反应; 2）鉴定和表征染色质调节复合物 通过CRISPR/dCas9介导的亲和纯化负责ACE2表达。通过共同努力， 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学的关键方面，而且还建立了急需的资源 的候选顺式和反式调节因子所需的ACE 2在人类细胞中的表达，从而促进 持续开发创新策略，以利用ACE2作为COVID-19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.