DNA METHYLATION IDENTIFIES AND REGULATES IMPRINTS

DNA 甲基化识别和调节印记

• 批准号: 6018374
• 负责人: WILLIAM M RIDEOUT
• 金额: $ 4万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6018374
• 关键词: DNA methylation; genomic imprinting; laboratory mouse

Imprinting is necessary for proper mammalian development and is defined as the epigenetic modification of chromosomes during gametogenesis, which limits expression of imprinted loci to either the maternal or paternal allele. Paternal-specific loss of imprinting (LOI) and loss of heterozygosity (LOH) are common occurrences in several types of childhood cancer (e.g. Wilms' tumor, retinoblastoma, rhabdomyosarcoma, neuroblastoma, and acute myelogenous leukemia) and in several genetic disorders (e.g. Prader-Willi and Beckwith-Wiedermann syndromes). Sixteen, of an estimated 100, imprinted genes have been identified in mice and regions of parental-specific DNA methylation have been identified at many of these loci. Inactivation of the DNA methyltransferase (Dnmt) disrupts imprinted gene regulations and re-introduction of the Dnmt cDNA into these ES cells (rescued cells) fails to restore parental-specific methylation. This suggests a great opportunity to search for new imprinted loci by screening DNA for methylation differences between normal and rescued ES cells. A more focused screen for parental-specific methylation on mouse chromosome 12, which has been identified as an imprinted region, will also be done using DNA from flow-sorted metaphase mouse chromosomes t(4:12), either paternally or maternally inherited. These DNAs will be digested and fractionated on a methyl-DNA binding domain column (MBD) to isolated highly methylated fragments which will then use used in PCR-select based subtraction protocols to yield imprint-associated fragments. Clones of interest will be systematically examined for sequence homologies, expression, and imprinting. Ultimately, identified loci will be tested for developmental function by homologous recombinant knockouts and gain of function insertions in transgenic mice. In addition, a reversibly repressible allele of Dnmt will be made and used to study the role of Dnmt in gene imprinting during gametogenesis.

印记是哺乳动物正常发育所必需的，其定义为： 配子发生过程中染色体的表观遗传修饰， 将印记基因座的表达限制在母本或父本 等位基因父母特异性印记丧失（LOI）和 杂合性（洛合性缺失）在几种类型儿童中是常见的 癌症（例如Wilms肿瘤，视网膜母细胞瘤，横纹肌肉瘤， 神经母细胞瘤和急性髓性白血病）和几种遗传性白血病中， 疾病（例如Prader-Willi和Beckwith-Wiedermann综合征）。十六岁， 在估计的100种基因中，已经在老鼠身上发现了印记基因， 父母特异性DNA甲基化的区域已经在许多 这些地方。DNA甲基转移酶（Dnmt）的失活破坏了 印迹基因调控和重新引入Dnmt cDNA到这些 ES细胞（拯救的细胞）不能恢复亲本特异性甲基化。 这意味着一个很好的机会，寻找新的印记基因座， 筛选正常ES和挽救ES之间的甲基化差异的DNA 细胞对小鼠进行更集中的亲本特异性甲基化筛选 12号染色体，已被确定为印记区域，也将 使用来自流式分选的中期小鼠染色体t（4：12）的DNA进行， 要么是父亲遗传的，要么是母亲遗传的。这些DNA会被消化， 在甲基-DNA结合结构域柱（MBD）上分级以分离 高度甲基化的片段，然后将用于基于PCR选择的 减法方案以产生印迹相关片段。无性系 将系统地检查感兴趣序列同源性， 表达和印记。最终，将对确定的基因座进行检测， 通过同源重组敲除和获得 转基因小鼠中的功能插入。此外，可逆 将制备Dnmt的抑制性等位基因并用于研究Dnmt的作用 在配子形成过程中的基因印记。