Sites of Developmental & Tissue-specific DNA Methylation

发育部位

• 批准号: 6891365
• 负责人: WILLIAM A HELD
• 金额: $ 30.43万
• 依托单位: ROSWELL PARK CANCER INSTITUTE CORP
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6891365
• 关键词: CpG islands; DNA methylation; cell differentiation; chromatin; embryogenesis; flow cytometry; gene expression; genetically modified animals; genome; green fluorescent proteins; immunomagnetic separation; laboratory mouse; microarray technology; polymerase chain reaction

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.

DNA甲基化与染色质结构和基因表达有着内在的联系。发育过程中DNA甲基化的变化表明它可能在发育和组织特异性分化中发挥作用。限制性里程碑基因组扫描（RLGS）将用于鉴定DNA甲基化的发育和组织特异性差异位点。RLGS是一种末端标记DNA限制性内切片段的二维显示方法。RLGS以Notl作为限制性标记，针对基因丰富的CpG岛区，检测DNA甲基化差异，因为Notl的切割对甲基化敏感。RLGS将对不同年龄的C57BL6/J小鼠的不同组织进行采集。绿色荧光蛋白（GFP）转基因小鼠结合荧光激活细胞分选将用于标记和纯化组织中用于RLGS分析的选定细胞群。磁细胞分选和离心洗脱方法将用于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