Sites of Developmental & Tissue-specific DNA Methylation

DNA甲基化与染色质结构和基因表达有内在联系。DNA甲基化在发育过程中的变化表明，它可能在发育和组织特异性分化中发挥作用。限制性地标基因组扫描(RLGS)将被用于识别DNA甲基化的发育和组织特异性差异的位置。RLGS是一种末端标记DNA限制性片段的二维展示方法。使用Not1作为限制标志，RLGS针对基因丰富的CpG岛区并检测DNA甲基化的差异，因为Not1的切割是甲基化敏感的。RLGS将对不同年龄的C57BL6/J小鼠的不同组织进行收集。绿色荧光蛋白(GFP)转基因小鼠将与荧光激活的细胞分类相结合，用于标记和纯化组织中选定的细胞群，用于RLGS分析。磁性细胞分选和离心洗脱法将被用来纯化成年小鼠体内的“分化”细胞群，用于RLGS分析。“虚拟”RLGS软件开发的新进展 为快速克隆DNA甲基化/染色质结构具有组织特异性差异的基因组区域提供独特的机会。定量和半定量甲基化敏感的聚合酶链式反应，以及亚硫酸氢盐测序，将被用来划定区域甲基化的边界和密度。与该区域相关的基因的组织表达模式将使用现有的微阵列数据来访问，以评估染色质结构对基因表达可能产生的“远距离影响”。定量RT-PCR方法将用于评估具有明显CpG岛启动子DNA甲基化的基因的表达。越来越清楚的是，表观遗传改变在 衰老与癌症和其他一些疾病的发展。然而，我们对表观遗传变化在正常发育过程中的作用的理解还很少。在完成小鼠基因组序列方面的最新进展和现在可用的大量生物信息学工具使得现在是对基因组甲基化的发育和组织特异性差异进行系统的、全基因组范围的调查并评估其对基因表达和分化的影响的好时机。

DNA methylation is intrinsically linked to chromatin structure and gene expression. Changes in DNA methylation during development suggest that it may play a role in development and in tissue-specific differentiation. Restriction Landmark Genomic Scanning (RLGS) will be used to identify sites of developmental and tissue specific differences in DNA methylation. RLGS is a method for the two dimensional display of end-labeled DNA restriction fragments. Using Notl as the restriction landmark, RLGS targets gene rich CpG island regions and detects differences in DNA methylation since cleavage by Notl is methylation sensitive. RLGS will be performed on a collection of different tissues from C57BL6/J mice of different ages. Green Fluorescent Protein (GFP) transgenic mice in conjunction with fluorescence activated cell sorting will be used to mark and purify selected populations of cells within a tissue for RLGS analysis. Magnetic Cell Sorting and Centrifugal Elutriation methods will be used to purify "differentiating" cell populations within adult mice for RLGS analysis. New advances in development of "virtual" RLGS software provide unique opportunities to rapidly clone "in silico" the genomic regions with tissue specific differences in DNA methylation/chromatin structure. Quantitative and semi-quantitative methylation sensitive PCR, along with bisulfite sequencing, will be used to delineate the boundaries and density of methylation in the regions. The tissue expression patterns of genes associated with the region will be accessed using available microarray data to evaluate possible "long distance effects" of chromatin structure on gene expression. Quantitative RTPCR methods will be used to evaluate expression of genes with apparent CpG island promoter DNA methylation. It is becoming increasing clear that epigenetic alterations have a major role in aging and the development of Cancer and some other diseases. Yet our understanding of the role of epigenetic alterations during normal development is poorly understood. Recent advances in completing the sequence of the mouse genome and the vast array of bioinformatics tools that are now available makes this an opportune time to undertake a systematic, genome-wide investigation of developmental and tissue-specific differences in genome methylation and assess its impact on gene expression and differentiation.