DNA methylation in normal versus malignant melanocytes

描述（由申请人提供）：CpG二核苷酸岛的异常DNA甲基化是哺乳动物基因组的主要表观遗传修饰，在肿瘤进展过程中很常见。例如，DNA甲基化失活导致肿瘤抑制因子、凋亡因子、DNA修复酶和粘附分子的下调，这些分子参与恶性进展为黑色素瘤和其他癌症。此外，一组黑色素瘤抗原，被称为癌症/睾丸抗原，由于CpG启动子岛的DNA去甲基化而异常地重新表达。在这个项目中，正常的人类黑色素细胞和原发性黑色素瘤细胞将以高通量的方式评估CpG甲基化模式的变化及其影响的基因。这将通过探测启动子芯片阵列和采用改良DNA和芯片上芯片程序的基因组平铺阵列来完成。该信息将用于评估CpG岛甲基化作为早期黑素细胞病变肿瘤进展的标志物。具体目标是：

DESCRIPTION (provided by applicant): Aberrant DNA methylation at CpG dinucleotide islands, the major epigenetic modification of mammalian genomes, is frequent during tumor progression. For example, inactivation by DNA methylation is the cause for down-regulation of tumor suppressors, apoptotic factors, DNA repair enzymes, and adhesion molecules involved in malignant progression to melanoma and other cancers. In addition, a panel of melanoma antigens, known as cancer/testis-antigens is aberrantly re-expressed due to DNA demethylation of CpG promoter islands. In this project normal human melanocytes and primary melanoma cells will be employed to assess changes in CpG methylation patterns and the genes they affect in a high throughput manner. This will be accomplished by probing promoter chip arrays and genomic tiling arrays employing modified DNA and ChIP on chip procedures. The information will be used to assess CpG island methylation as a marker for tumor progression of early melanocytic lesion. The specific aims are: Aim 1: To establish a procedure to probe promoter chip and chromosome tiling arrays for analysis of the methylation status of CpG rich islands on a genome-wide basis, determine the methylation status of CpG island regions in normal melanocytes versus primary melanoma cells. Aim 2: To employ chromatin immunoprecipitation procedure to identify binding regions of methyl-CpG-binding domain proteins (MBP) in the promoter region of these genes (Chip/chip). Aim 3: To validate the results of these two procedures by the bisulfite DNA modification method combined with sequencing of specific DNA regions and gene expression analysis. The information derived from these experiments will reveal the identity of a large fraction of genes whose expression is modulated by CpG island methylation/demethylation events and the extent of changes in CpG island methylation in the early progression of melanocytes to melanomas. The data will then be used to design a simpler and cheaper CpG island microarray chip that can be sed to probe the status of DNA methylation in melanocytic lesions as markers for malignancy.