TARGETS AND REQUIREMENTS OF METHYLATION IN HUMAN CELLS

据报道，新生甲基化发生在哺乳动物细胞的胚胎发生、癌变和外源DNA整合过程中。尽管许多新生甲基化基因的甲基化模式已被广泛研究，但新生甲基化的顺式和反式决定因素尚不清楚，控制新生甲基化的规则也完全不清楚。我们建议使用两个实验系统，一个基于oriP的稳定episomal系统和一个flip -recombination介导的染色体整合靶系统，来回答一些关于从头甲基化的基本问题。在初步研究中，我们发现当存在新生甲基转移酶3a或3b时，EBNA1编码序列在小体和染色体上都表现出可重复的新生甲基化。这是定义这两种甲基转移酶的要求和目标的一个有用的起点。除了EBNA1编码序列外，我们还研究了内皮素受体B基因上游的CpG岛。在前列腺癌细胞系中，这个CpG岛的染色体拷贝被甲基化。然而，在这种前列腺癌细胞系或表达任何一种新生甲基转移酶的细胞中，它在稳定的片段上都没有甲基化。利用flip -重组的染色体整合靶系统将使我们能够在癌细胞的自然位置剖析CpG岛甲基化的需求。本提案中描述的实验旨在回答以下问题：1)已知的新生甲基转移酶Dnmt3a和Dnmt3b如何靶向DNA进行甲基化？2)这两种甲基转移酶的从头甲基化要求是什么？3)是否有边界元素或因素可以保护区域免于重新甲基化？4) Dnmt3a和Dnmt3b的靶点是什么？5)癌细胞中CpG岛是如何靶向甲基化的？

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?