Mechanism of Chromatin Insulator Imprinting

染色质绝缘体印迹机制

• 批准号: 6852647
• 负责人: Piroska Edit Szabó
• 金额: $ 36.75万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6852647
• 关键词: DNA binding protein; DNA footprinting; DNA methylation; Prader Willi syndrome; alleles; binding sites; chromatin; chromosomes; developmental genetics; flow cytometry; gel mobility shift assay; gene expression; genetic mapping; genetic regulatory element; genetically modified animals; genomic imprinting; germ cells; hydatidiform mole; insulinlike growth factor; laboratory mouse; nucleic acid sequence; polymerase chain reaction; protein binding; site directed mutagenesis; teratoma

DESCRIPTION (provided by applicant): The broad long term objective is to determine the mechanism and role in development of the epigenetic -system of genomic imprinting. Imprinting of the H19 and insulin-like growth factor 2 (Igf2) genes (resulting in their monoallelic expression) is achieved through paternal-specific methylation of a 2.4 kb imprinting control region (ICR) as inherited from the male germ line. The specific aims are designed to further understanding of (1) how male germ cell-specific methylation of the ICR is achieved-this would be the imprinting mechanism, and (2) how the maternal ICR functions as a chromatin insulator in somatic cells. The specific aims are (1) in somatic cells, to map the ICR for sites of parent-of-origin allele-specific protein binding, and similarly, in germ cells, to map the ICR for sites of sex-specific protein binding. Sites characterized in this way will be candidates for mediating imprinting or male germ cell-specific ICR methylation. (2) Mutate these candidate sites in mice to test their role in mediating imprinting, and (3) identify the proteins binding to these sites. The hypothesis being tested by these aims is that there is a "primary protein difference" between the two germ lines which determines imprinting. These studies are related to health in that genomic imprinting underlies a number of human diseases, e.g., hydatidiform mole, ovarian teratoma, and the Prader-Willi and Beckwith-Wiedemann syndromes. Further understanding of the genetic basis of genomic imprinting will therefore shed light on the genetic basis of these diseases. The research design and methods for achieving this goal will involve (1) in vivo footprinting of the ICR to visualize protein binding sites, (2) targeted mutagenesis of ICR sites in mice and assessment of effects on imprinting by allele-specific expression and methylation analysis of H19 and Igf2, and (3) competitive and supershift electrophoretic mobility shift assays to identify proteins binding to ICR sites. Further assessment of protein identity by examining mouse mutants for candidate proteins.

描述（由申请人提供）：长期目标是 确定表观遗传系统发展的机制和作用 基因组印记。 H19和胰岛素样生长因子2的印记2 （IGF2）基因（导致其单相表达）通过 2.4 kb印迹控制区（ICR）的父亲特异性甲基化AS 从雄性生殖系继承。具体目标旨在进一步 了解（1）ICR的男性生殖细胞特异性甲基化如何 实现的 - 这将是烙印机制，以及（2）母体ICR如何 在体细胞中充当染色质绝缘子。具体目的是（1） 在体细胞中，绘制ICR的原始等位基因特异性位点 蛋白质结合，类似地，在生殖细胞中，以绘制ICR的位点 性别特异性蛋白质结合。以这种方式描述的网站将是 用于介导印迹或男性生殖细胞特异性ICR甲基化的候选者。 （2）在小鼠中突变这些候选部位以测试其在中介中的作用 印刷和（3）识别与这些位点结合的蛋白质。这 这些目标测试的假设是有一个“主要蛋白质” 两种决定印迹的细菌线之间的差异。 研究与健康有关，因为基因组烙印是许多基础 人类疾病，例如氢化巨摩尔，卵巢畸胎瘤和prader-willi 和贝克维斯·威德曼综合症。进一步理解的遗传基础 因此，基因组印记将阐明这些遗传基础 疾病。实现此目标的研究设计和方法将涉及 （1）ICR的体内足迹可视化蛋白质结合位点，（2） 小鼠ICR位点的靶向诱变以及对影响的影响 通过等位基因特异性表达和H19和甲基化分析的印迹 IGF2和（3）竞争和超弹性电泳移动偏移测定法 鉴定蛋白质与ICR位点结合。进一步评估蛋白质 通过检查小鼠突变体的候选蛋白质身份。