Epigenetic Regulation in a Cancer Associated Region

描述（由申请人提供）： 大多数人类癌症显示出全基因组表观遗传病变的证据。一种常见的表观遗传学改变是印迹缺失（LOI），其被定义为双等位基因表达或通常单等位基因表达的基因的沉默。最近，一些研究提供了令人信服的证据，LOI在细胞转化中具有因果作用。大部分患有Beckwith-Wiedemann综合征（BWS）（一种印记疾病和癌症易感性疾病）的患者在人类染色体11p15.5中的差异甲基化区域（称为KvDMR 1）表现出甲基化缺失（LOM）。这种表位突变，也在几种成人癌症中观察到，与LOI相关（即，沉默）的肿瘤抑制基因CDKN 1C。在过去19个月的资助期间，我们完成了研究，表明KvDMR 1印迹控制区（ICR）包含Kcnq 1 ot 1非编码RNA（ncRNA）的启动子和在增强子阻断试验中具有抑制活性的序列。此外，我们已经表明，表观遗传调节蛋白CTCF结合到最小的抑制序列在等位基因特异性的方式。我们还完成了对Kcnq 1 ot 1截短小鼠模型的研究，该模型不仅证明了ncRNA在调节印迹表达中的关键作用，而且还发现了肿瘤抑制基因Cdkn 1c特异性的第二种沉默机制。结合Cdkn 1c无效等位基因，这种截短小鼠有助于产生一种新的小鼠模型，概括了BWS的几个方面。Kcnqot 1截短小鼠也使我们能够证明，虽然ncRNA的表达是建立KvDMR 1印迹结构域的非等效核定位所必需的，但它在发育中是较晚的。目前的建议包括2个具体目标，旨在进一步了解KvDMR 1功能的机制：（1）KvDMR 1中的CTCF结合位点将单独或与Kcnq 1 ot 1截短组合删除。（2）Kcnq 1 ot 1全长基因的表达在受精过程中受到条件性调控，Kcnq 1 ot 1启动子在不同发育阶段被删除，此时父本基因沉默发生;这些分析将确定在配子发生期间是否需要ncRNA的表达，以及一旦建立，ncRNA是否是维持印记表达所必需的。由于其他包含生长调节基因的印记结构域可能以与KvDMR 1亚结构域类似的方式受到控制，并且哺乳动物基因组中可能存在许多其他CTCF介导的染色质绝缘子和ncRNA，因此来自这些研究的信息将与癌症的表观遗传学具有广泛的相关性。公共卫生相关性：与人类的大多数基因不同，印记基因只从父母遗传的两条染色体中的一条表达。印记基因通常是非常重要的生长调节因子，其异常表达可导致发育障碍和癌症。该项目建议继续研究被称为KvDMR 1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。这种调控区域在被称为Beckwith-Wiedemann综合征（BWS）的过度生长和癌症易感性条件以及几种散发性癌症中被破坏。我们建议建立小鼠模型来研究KvDMR 1是如何工作的，以及它的失调如何导致疾病。由于人类基因组中可能存在许多类似于KvDMR 1的其他区域，我们的研究将帮助我们了解这些区域如何控制基因组，并最终帮助开发儿童印记疾病和癌症的新疗法。

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.