Epigenetic Regulation in a Cancer Associated Region

描述(由申请人提供)： 项目摘要/摘要大多数人类癌症表现出全基因组表观遗传损伤的证据。一种常见的表观遗传学改变是印迹丢失(LOI)，它被定义为正常单等位基因的双等位表达或沉默。最近，一些研究提供了令人信服的证据，证明LOI在细胞转化中具有因果作用。Beckwith-Wiedemann综合征(BWS)是一种印迹障碍和癌症易感性疾病，很大比例的患者在人类染色体11p15.5上被称为KvDMR1的差异甲基化区域表现出甲基化缺失(LOM)。在几种成人癌症中也观察到这种突变，这种突变与肿瘤抑制基因CDKN1C的LOI(即沉默)有关。在过去19个月的授权期内，我们已经完成的研究表明，KvDMR1印记控制区(ICR)既包含Kcnq1ot1非编码RNA(NcRNA)的启动子，也包含在增强子阻断分析中具有抑制活性的序列。此外，我们还证明了表观遗传调节蛋白CTCF以等位基因特异性的方式与最小抑制序列结合。我们还完成了对我们的Kcnq1ot1截断小鼠模型的研究，该模型不仅证明了ncRNA在调节印迹表达中的关键作用，而且还揭示了肿瘤抑制基因CDKn1c特有的第二种沉默机制。结合CDKN1c零等位基因，这种截断小鼠在产生一种新的小鼠模型方面发挥了重要作用，该模型概括了BWS的几个方面。Kcnqot1截断小鼠也使我们能够证明，虽然ncRNA的表达是建立KvDMR1印记结构域的不等价核定位所必需的，但它在以后的发育中是必不可少的。目前的建议包括两个具体目的，旨在加深我们对KvDMR1功能机制(S)的理解：(1)KvDMR1中的CTCF结合位点将单独或与Kcnq1ot1截断一起删除。这些研究将确定CTCF结合在该基因座上的作用；(2)全长Kcnq1ot1的表达将在受精时受到条件调控，Kcnq1ot1启动子将在与父亲基因沉默发生的不同发育阶段相对应的时间点被删除；这些分析将确定ncRNA在配子发生过程中是否需要表达，以及一旦建立，ncRNA是否对于维持印记表达是必要的。由于包含生长调节基因的其他印迹结构域可能以类似于KvDMR1亚结构域的方式受到控制，而且很可能在哺乳动物基因组中存在许多其他CTCF介导的染色质绝缘子和ncRNA，因此这些研究获得的信息将与癌症的表观遗传学具有广泛的相关性。与公共卫生相关：项目叙述与人类的大多数基因不同，印记基因只表达于从父母那里继承的两条染色体中的一条。印迹基因通常是非常重要的生长调节基因，它们的异常表达可能导致发育障碍和癌症。该项目建议继续研究被称为KvDMR1的基因组区域，该区域调节一组印记基因的表达，包括肿瘤抑制基因。这一调控区域在被称为Beckwith-Wiedemann综合征(BWS)的过度生长和癌症易感性条件下以及在几种散发性癌症中被破坏。我们正在提议建立小鼠模型，以研究KvDMR1是如何工作的，以及它的放松调控如何导致疾病。由于人类基因组中可能存在许多与KvDMR1类似的其他区域，我们的研究将帮助我们了解这些区域是如何控制基因组的，并最终帮助开发治疗儿童印记障碍和癌症的新方法。

DESCRIPTION (provided by applicant): Project Summary/Abstract Most human cancers show evidence of genome-wide epigenetic lesions. One common epigenetic alteration is loss of imprinting (LOI) which is defined as either biallelic expression or silencing of normally monoallelically expressed genes. Recently, several studies have provided compelling evidence that LOI has a causal role in cellular transformation. A large proportion of patients with Beckwith-Wiedemann syndrome (BWS), an imprinting disorder and cancer predisposition condition, exhibit loss of methylation (LOM) at the differentially methylated region in human chromosome 11p15.5 known as KvDMR1. This epimutation, which is also observed in several adult cancers, is associated with LOI (i.e., silencing) of the tumor suppressor gene CDKN1C. During the last 19 month grant period, we have completed studies that show that the KvDMR1 imprinting control region (ICR) contains both the promoter for the Kcnq1ot1 noncoding RNA (ncRNA) and sequences possessing repressive activity in enhancer-blocking assays. Moreover, we have shown that the epigenetic regulatory protein CTCF binds to the minimal repressive sequence in an allele-specific manner. We have also completed studies on our Kcnq1ot1 truncation mouse model which has not only demonstrated a critical role for the ncRNA in regulating imprinted expression, but has uncovered a second silencing mechanism specific for the tumor suppressor gene Cdkn1c. In combination with a Cdkn1c null allele, this truncation mouse has been instrumental in generating a novel mouse model that recapitulates several aspects of BWS. The Kcnqot1 truncation mouse has also allowed us to demonstrate that, while expression of the ncRNA is required for the establishment of nonequivalent nuclear localization of the KvDMR1 imprinted domain, it is dispensable later in development. The current proposal consists of 2 specific aims intended to further our understanding of the mechanism(s) of KvDMR1 function: (1) The CTCF binding sites in KvDMR1 will be deleted either alone or in combination with the Kcnq1ot1 truncation. These studies will determine the role for CTCF binding at this locus; (2) Expression of full length Kcnq1ot1 will be conditionally regulated at fertilization, and the Kcnq1ot1 promoter will be deleted at time points corresponding to the different developmental stages where paternal gene silencing takes place; these analyses will determine whether expression of the ncRNA is required during gametogenesis, and whether the ncRNA is necessary for the maintenance of imprinted expression once it is established. Since other imprinted domains containing growth regulating genes may be controlled in a similar fashion as the KvDMR1 subdomain, and it is likely that there are many other CTCF mediated chromatin insulators and ncRNAs in the mammalian genome, information derived from these studies will have widespread relevance to the epigenetics of cancer. PUBLIC HEALTH RELEVANCE: Project Narrative Unlike most genes in human, imprinted genes are expressed from only one of the two chromosomes inherited from their parents. Imprinted genes are often very important regulators of growth, and their abnormal expression can lead to developmental disorders and cancer. This project proposes to continue investigation of a genomic region known as KvDMR1 that regulates the expression of a cluster of imprinted genes, including a tumor suppressor gene. This regulatory region is disrupted in the overgrowth and cancer predisposition condition known as Beckwith-Wiedemann syndrome (BWS), as well as in several sporadic cancers. We are proposing to generate mouse models to study how KvDMR1 works and how its deregulation can lead to disease. Since many other regions similar to KvDMR1 probably exist in the human genome, our studies will help us understand how these regions control the genome and eventually aid in developing new treatments to childhood imprinting disorders and cancer.