Epigenetic Regulation in a Cancer Associated Region

描述(申请人提供)：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。很大比例的Beckwith-Wiedemann综合征(BWS)患者表现出人类染色体11p15.5差异甲基化区域(称为KvDMRI)的甲基化缺失(LOM)。在几种成人癌症中也观察到这种突变，这种突变与潜在的肿瘤抑制基因CDKN1C的LOI(即沉默)有关。在上一次授予期间，我们通过对小鼠7号染色体远端的同源区域的定向突变表明，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，该突变体的胎盘和胚胎生长缺陷可归因于不同基因的过度表达。我们已经证明，该基因座在细胞培养中作为增强子阻滞剂，绝缘体相关蛋白CTCF在体内以等位基因特异性的方式与KvDMRI结合，CTCF结合位点的突变显著取消了增强子阻断剂的活性。此外，我们还鉴定了该基因的启动子和增强子样活性，它们在物理上与绝缘体的性质是分开的。目前的建议包括4个具体目标，旨在加深我们对KvDMRI功能机制(S)的理解：(1)KvDMRI基因座将通过全面的突变和体内足迹来评估，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测结合位点序列的“候选”方法或通过传统的蛋白质纯化技术和质谱学来鉴定；(3)我们建议通过构建一系列条件突变小鼠来评估KvDMRI中确定的3个假定调控元件(绝缘子、启动子、增强子)在体内对基因沉默的贡献；(4)非编码RNA转录物Kcnq1ot1的潜在作用将通过其提前终止和截断来测试。由于包含生长调节基因的其他印迹结构域可能以类似于KvDMRI亚结构域的方式受到控制，并且很可能在哺乳动物基因组中存在许多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.