Identification and Characterization of Novel Cis-Regulatory Elements Controlling PTEN Expression

摘要 人们越来越认识到，非编码基因组可以塑造基因表达，因此， 癌症等疾病。然而，除了几个精选的例子外，我们对具体的CI- 影响许多关键癌基因和肿瘤抑制因子的调控机制。虽然不同的方法可以 在全基因组范围内确定候选顺式调控元件(CcRE)已成功提名 大集合假定的监管区域，许多这样的方法依赖于间接证据或从外部收集的证据 单元格的本地上下文。此外，将给定CCRE的调控活性与其基因伙伴配对 事实证明这很难。为了解决这一问题，我们的团队创造了第一批从功能上定义监管机构的技术 特定基因的电路，基于CRISPR-Cas9的表观基因组调控元件筛选(CERES)。这 平台利用核酸酶失活(D)Cas9偶联到表观基因组激活子(P300)和抑制子(Krab) 构造。这些构建物与针对以下区域的慢病毒短指南RNA(SgRNA)文库配对 目标基因周围可接近的染色质。因此，我们能够映射激活和激活的影响 抑制候选基因对靶基因的表达，从而定义 那个基因。在这种独特的内部技术方法的支持下，我们现在有兴趣定义 控制重要癌症基因表达的内源性机制。 在人类癌症中最常见的肿瘤抑制基因(TSG)改变之一是磷酸酶和 张力蛋白同源物(PTEN)，它编码一种脂类和蛋白质磷酸酶，负调控PI3K-AKT 路径，以及其他。在许多肿瘤中，PTEN在基因水平上发生突变或缺失。然而，一个很大的 部分患者在没有这些相关基因改变的情况下表现出PTEN表达缺失，这表明 控制PTEN表达的非编码改变的潜在作用。另外，我们也知道， PTEN的过度表达足以抑制癌细胞的增殖，促进细胞凋亡，并刺激免疫 监视系统。因此，针对内源性PTEN过度表达的技术，例如通过 操纵其顺式作用调节元件(CRE)，可能代表着一种有前途的治疗策略。 在这个方案中，我们将系统地定义控制PTEN表达的关键CRES。在此过程中，我们 将为理解非编码突变作为人类PTEN缺失的驱动因素的作用提供基础 肿瘤和生殖系肿瘤综合征。此外，这项工作将为确定 转录因子和信号通路通过这些关键的CRE调节PTEN的表达 这可能使最终设计针对其基因调控的疗法成为可能。

Abstract It is increasingly appreciated that the non-coding genome can shape gene expression and, consequently, diseases such as cancer. Beyond a few select examples, however, we know remarkably little about the specific cis- regulatory mechanisms influencing many key oncogenes and tumor suppressors. While various approaches to identify candidate cis-regulatory elements (ccREs) on a genome-wide scale have been successful in nominating large sets putative regulatory regions, many such methods rely on indirect evidence or evidence gleaned outside of the native context of the cell. Further, pairing the regulatory activity of a given ccRE with its gene partners has proven difficult. To address this, our groups created the first technologies for functionally defining the regulatory circuitry of specific genes, CRISPR-Cas9-Based Epigenomic Regulatory Element Screening (CERES). This platform utilizes nuclease-deactivated (d)Cas9 coupled to epigenomic activator (p300) and repressor (KRAB) constructs. These constructs are paired with lentiviral short guide RNA (sgRNA) libraries targeting areas of accessible chromatin surrounding a gene of interest. Thus, we are able to map the effect of both activation and repression of candidate loci on the expression of a target gene, and thereby define the regulatory elements for that gene. Powered by this unique in-house technological approach, we are now interested in defining the endogenous mechanisms controlling expression of important cancer genes. One of the most frequently altered tumor suppressor genes (TSGs) in human cancers is phosphatase and tensin homolog (PTEN), which encodes a lipid and protein phosphatase that negatively regulates the PI3K-AKT pathway, among others. PTEN is mutated or deleted at the genetic level in many tumors. However, a large fraction of patients exhibit loss of PTEN expression without these associated genetic alterations, suggesting a potential role for non-coding alterations controlling PTEN expression. Separately, it is also known that overexpression of PTEN is sufficient to inhibit cancer cell proliferation, drive apoptosis, and stimulate immune surveillance. Therefore, techniques which target endogenous PTEN for overexpression, for instance through the manipulation of its cis-acting regulatory elements (cREs), could represent a promising therapeutic strategy. In this proposal, we will systematically define the key cREs controlling PTEN expression. In so doing, we will provide a foundation for understanding the role of non-coding mutations as drivers of PTEN loss in human tumors and germline tumor syndromes. Further, this work will establish a foundation for identifying the transcription factors and signaling pathways that regulate PTEN expression through these critical cREs, work which could enable the eventual design of therapeutics that target its genetic regulation.