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

摘要 越来越多的人认识到，非编码基因组可以塑造基因表达，因此， 疾病，如癌症。然而，除了一些精选的例子之外，我们对具体的顺式- 影响许多关键癌基因和肿瘤抑制因子的调节机制。虽然各种方法， 在全基因组范围内鉴定候选顺式调节元件（ccRE）已经成功地提名 大集合假定的调控区域，许多这样的方法依赖于间接证据或证据收集外部 细胞的原生环境。此外，将给定ccRE的调节活性与其基因伴侣配对， 证明困难。为了解决这个问题，我们的团队创造了第一个技术，用于功能性地定义监管 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的（d）与表观基因组激活因子（p300）和阻遏因子（KRAB）偶联的Cas9 结构。这些构建体与靶向靶向肿瘤区域的慢病毒短向导RNA（sgRNA）文库配对。 可接近的染色质围绕感兴趣的基因。因此，我们能够映射激活和 抑制候选基因座对靶基因表达的抑制，从而定义用于 这个基因。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。 人类癌症中最常改变的肿瘤抑制基因（TSG）之一是磷酸酶， 张力蛋白同源物（PTEN），其编码负调节PI 3 K-AKT的脂质和蛋白磷酸酶 路，除此之外。在许多肿瘤中，PTEN在基因水平上突变或缺失。然而，一个大的 一部分患者表现出PTEN表达的缺失，而没有这些相关的遗传改变，这表明 非编码改变控制PTEN表达的潜在作用。另外，还已知， PTEN的过表达足以抑制癌细胞增殖，驱动凋亡，并刺激免疫应答。 监视因此，靶向内源性PTEN过表达的技术，例如通过靶向内源性PTEN过表达的技术， 操纵其顺式作用调控元件（克雷斯），可以代表一种有前途的治疗策略。 在这个提议中，我们将系统地定义控制PTEN表达的关键克雷斯。我们这样做 这将为理解非编码突变作为人类PTEN丢失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 通过这些关键的克雷斯调节PTEN表达的转录因子和信号通路起作用， 这可能使最终设计出针对其遗传调控的治疗方法成为可能。

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.