Defining the Function of Age-Related Epimutation in Intestinal Tumorigenesis

项目总结 与DNA甲基化的表观遗传学改变相关的有丝分裂稳定的基因沉默 现在被认为是人类癌症的共同特征。事实上，最近的表观基因组学研究表明 几乎所有的肿瘤类型都含有数百个异常高甲基化和沉默的启动子CpG岛- 相关(CGI相关)基因，突出了识别司机表型突变的必要性。在这方面，我们 开发了一种新的肿瘤抑制基因p16(也称为细胞周期蛋白)突变的小鼠模型。 依赖的激酶抑制物2A)。我们发现，基因工程的启动子甲基化导致p16基因加速 衰老过程中小鼠体组织的突变。我们证明了p16突变使小鼠易患上 到自发的肿瘤发展。此外，我们的初步工作表明，p16突变体可以 与突变的APC协同使用ApcMin/小鼠加速肠道肿瘤的形成。重要的是，我们 发现p16突变的功能超出了经典已知的细胞周期控制，可以诱导肿瘤发生 肠道肿瘤的转化。因此，根据我们强劲的初步数据，我们假设 P16突变，通常在人类散发性癌中观察到，是年龄相关表观遗传学改变的一部分。 与基因改变合作，推动肠癌的发生和发展。我们将对此进行测试1) 确定p16突变如何促进肠道肿瘤的发生。我们将评估生物 P16突变与WNT-APC-β两条关键信号通路相互作用的后果 连环蛋白和MAPK-RAS-RAF；2)决定p16表观遗传缺陷的逆转是否抑制肿瘤 成长。我们将通过应用基于CRISPR的方法来评估肿瘤对p16突变逆转的反应。 靶向p16启动子去甲基化；以及3)确定转录因子是否作为阅读器 DNA甲基化介导了p16基因突变的功能。我们将研究分子事件， P16表观遗传沉默是DNA甲基化依赖的反式作用蛋白结合的结果 随后招募染色质抑制复合体。这些研究将阐明异常的DNA是如何 甲基化，一种重要的表观遗传机制，调节肿瘤抑制基因的表达 结直肠肿瘤的发生。从这项建议中得出的结果可能会对设计产生独特的影响 结肠癌的表观遗传疗法。

PROJECT SUMMARY Epimutation – mitotically stable gene silencing associated with epigenetic alteration in DNA methylation – is now recognized as a common feature of human cancer. Indeed, recent epigenomic studies revealed that nearly all tumor types harbor hundreds of aberrantly hypermethylated and silenced promoter CpG island- associated (CGI-associated) genes, highlighting the need to identify driver epimutations. In this regard, we developed a novel mouse model of epimutation in a tumor suppressor gene p16 (also known as cyclin- dependent kinase inhibitor 2A). We found that engineered promoter methylation leads to accelerated p16 epimutation in mouse somatic tissues during aging. We demonstrated that p16 epimutation predisposes mice to spontaneous tumor development. In addition, our preliminary work shown that p16 epimutation can cooperate with mutational Apc to accelerate intestinal tumorigenesis using the ApcMin/+ mice. Importantly, we found that p16 epimutation functions beyond the classically known cell-cycle control to induce malignant transformation of intestinal neoplasms. Therefore, based on our strong preliminary data, we hypothesize that p16 epimutation, commonly observed in human sporadic CRCs as part of the age-related epigenetic alteration, cooperates with genetic alterations to drive intestinal cancer initiation and progression. We will test this by 1) Determine how p16 epimutation promotes intestinal tumorigenesis. We will assess the biological consequences of p16 epimutation through its interactions with two key signaling pathways: WNT-APC-β- catenin and MAPK-RAS-RAF; 2) Determine whether reversal of epigenetic defects in p16 suppresses tumor growth. We will evaluate tumor responses to the reversal of p16 epimutation by applying a CRISPR-based targeted p16 promoter demethylation; and 3) Determine whether transcription factors as a reader of methylated DNA mediate the function of p16 epimutation. We will investigate the molecular events by which p16 epigenetic silencing is a result from DNA methylation dependent trans-acting protein binding which subsequently recruits chromatin repressive complexes. These studies will elucidate how aberrant DNA methylation, an important epigenetic mechanism regulating the expression of tumor suppressor genes, drives colorectal tumorigenesis. The results derived from this proposal could have a unique impact in the design of colon cancer epigenetic therapies.