DNA methylation in normal versus malignant melanocytes

描述（由申请方提供）：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，在肿瘤进展期间很常见。例如，DNA甲基化导致的失活是肿瘤抑制因子、凋亡因子、DNA修复酶和参与恶性进展为黑色素瘤和其他癌症的粘附分子下调的原因。此外，由于CpG启动子岛的DNA去甲基化，称为癌症/睾丸抗原的一组黑素瘤抗原异常地重新表达。在这个项目中，正常的人类黑素细胞和原发性黑色素瘤细胞将被用来评估CpG甲基化模式的变化和它们以高通量的方式影响的基因。这将通过探测启动子芯片阵列和基因组平铺阵列，采用修饰的DNA和ChIP芯片程序来实现。这些信息将用于评估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： 目标1：建立一种基于启动子芯片和染色体拼接芯片的全基因组CpG岛甲基化分析方法，比较正常黑素细胞和原发性黑素细胞中CpG岛甲基化状态。目标二：采用染色质免疫沉淀法鉴定这些基因启动子区甲基化CpG结合域蛋白（MBP）的结合区域（Chip/chip）。目标3：通过亚硫酸氢盐修饰DNA的方法，结合特定DNA区域的测序和基因表达分析，验证这两种方法的结果。从这些实验中获得的信息将揭示其表达受CpG岛甲基化/去甲基化事件调节的大部分基因的身份，以及在黑色素细胞向黑色素瘤的早期进展中CpG岛甲基化的变化程度。这些数据将用于设计一种更简单、更便宜的CpG岛微阵列芯片，可以用来探测黑素细胞病变中DNA甲基化的状态，作为恶性肿瘤的标志。

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.