TARGETS AND REQUIREMENTS OF METHYLATION IN HUMAN CELLS

据报道，哺乳动物细胞在胚胎发生、癌发生和外源DNA整合过程中发生从头甲基化。 虽然许多从头甲基化基因的甲基化模式已被广泛研究，从头甲基化的顺式和反式决定簇是未知的，并且支配从头甲基化的规则完全不清楚。 我们建议使用两个实验系统，一个基于oriP的稳定的附加型系统和一个flp重组介导的染色体整合的靶系统，来回答一些关于从头甲基化的基本问题。 在初步研究中，我们已经发现，EBNA1编码序列表现出可重复的从头甲基化，无论是在附加体和染色体上的从头甲基化时，从头甲基转移酶3a或3b存在。 这是一个有用的起点，以确定这两个甲基转移酶的要求和目标。 除了EBNA 1编码序列，我们还研究了内皮素受体B基因上游的CpG岛。 该CpG岛的染色体拷贝在前列腺癌细胞系中被甲基化。 然而，在该前列腺癌细胞系或表达任一种从头甲基转移酶的细胞中，其在稳定附加体上不甲基化。 利用flp重组的染色体整合靶系统将使我们能够剖析癌细胞中CpG岛甲基化在其自然位置的需求。 本提案中描述的实验旨在回答以下问题：1）已知的从头甲基转移酶Dnmt3a和Dnmt3b如何靶向DNA进行甲基化？ 2)这两种甲基转移酶对从头甲基化的要求是什么？ 3)是否有边界元件或因子可以保护区域免于从头甲基化？ 4)Dnmt3a和Dnmt3b的作用靶点是什么？5)CpG岛如何靶向癌细胞中的甲基化？

De novo methylation has been reported to occur in mammalian cells during embryogenesis, carcinogenesis, and foreign DNA integration. Although the methylation patterns of many de novo methylated genes have been studied extensively, the cis and trans determinants of de novo methylation are unknown and the rules that govern de novo methylation are entirely unclear. We propose to use two experimental systems, an oriP- based stable episomal system and a flp-recombination mediated chromosomally-integrated target system, to answer some of the essential questions regarding de novo methylation. In preliminary studies, we have found that the EBNA1 coding sequence exhibits reproducible de novo methylation both on the episome and in the chromosome when the de novo methyltransferase 3a or 3b is present. This is a useful starting point to define the requirements and targets of these two methyltransferases. In addition to the EBNA1 coding sequence, we have also examined the CpG island upstream of the endothelin receptor B gene. The chromosomal copies of this CpG island are methylated in a prostate cancer cell line. However, it is not methylated on the stable episome in this prostate cancer cell line or in cells expressing either of the de novo methyltransferases. A chromosomally-integrated target system utilizing flp-recombination will allow us to dissect the requirement for CpG island methylation in cancer cells at its natural location. Experiments described in this proposal are designed to answer the following questions: 1) How do the known de novo methyltransferases, Dnmt3a and Dnmt3b, target DNA for methylation? 2) What are the requirements for de novo methylation by these two methyltransferases? 3) Are there boundary elements or factors that can protect regions from de novo methylation? 4) What are the targets of Dnmt3a and Dnmt3b? 5) How is a CpG island targeted for methylation in cancer cells?