EPIGENETIC REGULATION IN A CANCER ASSOCIATED REGION

点击翻译按钮获取中文摘要

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.