EPIGENETIC REGULATION IN A CANCER ASSOCIATED REGION

虽然突变通常在特定肿瘤的特定基因中发现，但肿瘤发生显然涉及其他机制。在肿瘤中，启动子甲基化导致基因失活是一个常见的发现，在一些例子中，其他表观遗传变化，如印迹丧失（LOI）与疾病表型相关。印迹控制区（ICR）被认为存在于KCNQ1 （KvLQT1）基因座中，它被认为调控了与散发性肿瘤和过度生长、癌症易感状态beckwithwithwiedemann综合征（BS）相关的印迹基因的表达。在KCNQ1基因中发现了一个保守的cpg岛（KvDMR1），它在体细胞和种系组织中都表现出不同的甲基化。与文献记载的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。KvDMR1的遗传和表观遗传变化已在BWS和一些癌症中发现。我们验证了KvDMR1是一种ICR的假设，通过在小鼠远端7号染色体上产生靶向缺失。对15.5-16.5日龄胎儿的分析表明，KvDMR1缺失的父系遗传与生长迟缓和基因组印迹的破坏有关。我们还在增强子阻断实验中发现KvDMR1作为染色质绝缘体起作用，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缺陷小鼠进行全面检查，发现KvDMR1功能缺失导致的发育和分子异常。(2)找到足以抑制增强子活性的最小DNA序列。(3)精确定位和表征KvDMR1蛋白结合位点的甲基化敏感性和鉴定对结合至关重要的残基。(4)将绝缘子活性所需蛋白结合位点的失活突变通过靶置换引入小鼠，以证明这些位点在体内的重要性，并确定同一突变是否影响适当印迹甲基化的建立和维持。本应用程序提出的研究将定义KvDMR1为印迹控制区域（ICR），并阐明小鼠远端7/人11p15.5印迹结构域的调控机制，包括异常表观遗传修饰如何导致肿瘤发生中的LOI。此外，印迹的破坏可以作为肿瘤细胞中更全面的表观遗传变化的范例。由于表观遗传现象通常是可逆的，因此更好地理解印迹将有助于设计旨在恢复肿瘤中适当印迹的药物。

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.