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KvDMRI）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，与潜在肿瘤抑制基因CDKN1C的LOI（即沉默）相关。在最后一次授权期间，我们已经通过小鼠中第7号染色体远端的正向区域的靶向突变表明，KvDMRI是一个印记控制区（ICR），其缺失导致至少8个父系抑制基因的双等位基因表达。遗传分析表明，这种突变体中表现出的胎盘和胚胎生长缺陷可以归因于不同基因的过表达。我们已经表明，该基因座在细胞培养中充当增强子阻断剂，绝缘子相关蛋白CTCF以等位基因特异性方式在体内结合KvDMRI，并且CTCF结合位点的突变显著地消除增强子阻断活性。此外，我们已经确定了启动子和增强子样活动在这个位点是物理上分离的绝缘子的属性。目前的建议包括4个具体目标，旨在进一步我们对KvDMRI功能的机制的理解：（1）KvDMRI基因座将通过综合诱变和体内足迹法进行评估，以确定调节KvDMRI功能的蛋白质的额外DNA结合位点;（2）与这些位点结合的蛋白质将通过基于推定结合位点的序列的"候选"方法来鉴定，或通过常规的蛋白质纯化技术和质谱;（3）我们建议评估3个假定的调控元件中的每一个通过构建一系列条件突变小鼠，在KvDMRI内鉴定了它们对体内基因沉默的贡献的（绝缘子、启动子、增强子）;（4）非编码RNA转录本Kcnq1ot1的潜在作用将通过其提前终止和截短来测试。由于含有生长调节基因的其他印迹结构域可以以与KvDMRI亚结构域类似的方式控制，并且哺乳动物基因组中可能存在许多CTCF介导的染色质绝缘子，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。

DESCRIPTION (provided by applicant): 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), a cancer predisposition condition, exhibit loss of methylation (LOM) at the differentially methylated region in human chromosome 11p15.5 known as KvDMRI. This epimutation, which is also observed in several adult cancers, is associated with LOI (i.e. silencing) of the potential tumor suppressor gene CDKN1C. During the last grant period, we have shown by targeted mutation of the orthologous region in the mouse, distal chromosome 7, that KvDMRI is an imprinting control region (ICR) with its deletion resulting in biallelic expression of a least 8 paternally repressed genes. Genetic analysis has shown that the placental and embryonic growth deficiency exhibited in this mutant can be attributed to the overexpression of different genes. We have shown that this locus acts as an enhancer-blocker in cell culture, that the insulator- associated protein CTCF binds to KvDMRI in vivo in an allele-specific manner, and that mutation of the CTCF binding sites significantly abrogates enhancer-blocking activity. In addition, we have identified both promoter and enhancer-like activities at this locus which are physically separable from the insulator properties. The current proposal consists of 4 specific aims intended to further our understanding of the mechanism(s) of KvDMRI function: (1) The KvDMRI locus will be evaluated by comprehensive mutagenesis and in vivo footprinting to identify additional DNA binding sites for proteins regulating the function of KvDMRI; (2) The proteins binding to these sites will be identified by either a "candidate" approach based on the sequence of the putative binding site, or by conventional protein purification techniques and mass spectrometry; (3) We propose to assess each of the 3 putative regulatory elements (insulator, promoter, enhancer) identified within KvDMRI for their contribution to gene silencing in vivo by constructing a series of conditionally mutant mice; (4) The potential role of the noncoding RNA transcript Kcnq1ot1 will be tested by its premature termination and truncation. Since other imprinted domains containing growth regulating genes may be controlled in a similar fashion as the KvDMRI subdomain, and it is likely that there are many CTCF mediated chromatin insulators in the mammalian genome, information derived from these studies will have widespread relevance to the epigenetics of cancer.