EPIGENETIC REGULATION IN A CANCER ASSOCIATED REGION

虽然突变通常在特定肿瘤的特定基因中被发现，但其他机制显然与肿瘤的发生有关。肿瘤中由启动子甲基化引起的基因失活是一种常见的发现，其他表观遗传学变化，如印迹丢失(LOI)，在几个例子中与疾病表型相关。印记控制区(ICR)存在于KCNQ1(KvLQT1)基因座上，被认为调节印记基因的表达，涉及散发性肿瘤和过度生长、癌症易感性和Beckwith-Wiedemannn综合征(BS)。在KCNQ1基因中发现了一个保守的CpG岛(KvDMR1)，它在体细胞和生殖系组织中都表现出不同的甲基化。与已报道的ICRs类似，KvDMR1具有直接重复结构，并与以印迹方式表达的反义非编码RNA相关。在BWS和一些癌症中已经检测到KvDMR1的遗传和表观遗传学变化。我们对KvDMR1是一种ICR的假设进行了验证，方法是定向缺失7号染色体远端的小鼠。对15.5-16.5天的胎儿的分析表明，KvDMR1缺失的父系遗传与生长迟缓和基因组印记的AQ中断有关。我们还证明了KvDMR1在增强子阻断实验中作为染色质绝缘体的功能，并且可能包含绝缘体相关蛋白CTCF的结合位点。建议开展以下研究：(1)对KvDMR1功能缺失所致的发育和分子异常进行全面的检测。(2)定位足以阻断增强子活性的最小DNA序列。(3)根据甲基化敏感性和对结合至关重要的残基的鉴定，准确定位和鉴定KvDMR1内的蛋白质结合位点。(4)通过靶标替换，在绝缘体活性所需的蛋白质结合部位引入失活突变，以证明这些部位在体内的重要性，并确定相同的突变是否影响适当印迹甲基化的建立和维持。本申请中提出的研究将把KvDMR1定义为印记控制区(ICR)，并阐明小鼠末端7/人11p15.5印迹结构域的调控机制，包括异常的表观遗传修饰如何导致肿瘤发生中的LOI。此外，印迹的破坏可能成为肿瘤细胞更具全局性表观遗传变化的范例。由于表观遗传现象往往是可逆的，对印迹的更好理解将有助于旨在恢复肿瘤适当印迹的药物设计。

Although mutations are commonly found in particular genes in specific tumors, other mechanisms are clearly involved in tumorigenesis. Gene inactivation in tumors by promoter methylation is a frequent finding and other epigenetic changes such as loss of imprinting (LOI) are associated with disease phenotypes in several examples. An imprinting control region (ICR), proposed to exist within the KCNQ1 (KvLQT1) locus, is thought to regulate the expression of imprinted genes involved in sporadic tumors and the overgrowth, cancer predisposition condition, Beckwith-Wiedemannn syndrome (BS). A conserved CpG-island (KvDMR1) has been identified within the KCNQ1 gene which exhibits differential methylation in both somatic and germline tissues. Similar to documented ICRs, KvDMR1 has direct repeat structures and is associated with an antisense non-coding RNA that is expressed in an imprinted fashion. Both genetic and epigenetic changes in KvDMR1 have been detected in BWS and some cancers. We tested the hypothesis that KvDMR1 is an ICR by generated a targeted deletion of the mouse locus on distal chromosome 7. The analysis of 15.5-16.5 day old fetuses demonstrated that paternally-inheritance of the KvDMR1 deletion is associated with growth retardation and aq disruption of genomic imprinting. We have also shown that KvDMR1 functions as a chromatin insulator in an enhancer-blocking assay and is likely to contain binding sites for the insulator associated protein CTCF. Studies are proposed to: (1) Carry out a comprehensive examination of KvDMR1 deficient mice for developmental and molecular abnormalities resulting from a lack of KvDMR1 function. (2) Locate the minimal DNA sequences sufficient for enhancer-blocking activity. (3) Precisely locate and characterize protein-binding sites within KvDMR1 with respect to methylation-sensitivity and identification of residues critical to binding. (4) Introduce into mice, by target replacement, an inactivating mutation in the protein binding sites required for insulator activity to demonstrate the significance of these sites in vivo, and to determine whether the same mutation affects the establishment and maintenance of proper imprinted methylation. Studies proposed in this application will define KvDMR1 as an imprinting control region (ICR) and shed light on the mechanisms by which the mouse distal 7/human 11p15.5 imprinted domain is regulated including how aberrant epigenetic modification could lead to LOI in tumorigenesis. Furthermore, disruption of imprinting may serve as a paradigm for more global epigenetic changes in tumor cells. Since epigenetic phenomena are often reversible, a better understanding of imprinting will facilitate drug design aimed at restoring proper imprinting in tumors.