Mechanism of Chromatin Insulator Imprinting

描述（由申请人提供）：广泛的长期目标是 确定的机制和作用，在发展中的表观遗传系统， 基因组印记H19和胰岛素样生长因子2的印迹 （Igf 2）基因（导致其单等位基因表达）通过以下途径实现： 2.4 kb印迹控制区（ICR）的父系特异性甲基化， 遗传自男性生殖细胞具体目标旨在进一步 理解（1）男性生殖细胞特异性ICR甲基化是如何 这将是印迹机制，以及（2）如何母ICR 在体细胞中起染色质绝缘体的作用。具体目标是：（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，以及类似地，在生殖细胞中，绘制ICR的位点， 性别特异性蛋白结合。以这种方式描述的网站将 介导印迹或雄性生殖细胞特异性ICR甲基化的候选者。 (2)在小鼠中突变这些候选位点，以测试它们在介导 印迹，和（3）鉴定与这些位点结合的蛋白质。的 这些目标正在验证的假设是，存在一种“初级蛋白质 这是两个生殖系之间的“差异”，决定了印记。这些 研究与健康有关，因为基因组印记是许多 人类疾病，例如，葡萄胎，卵巢畸胎瘤，普拉德-威利 和Beckwith-Wiedemann综合征。进一步了解遗传基础 因此，基因组印记将揭示这些基因的遗传基础。 疾病实现这一目标的研究设计和方法将涉及 (1)ICR的体内足迹法以可视化蛋白结合位点，（2） 小鼠ICR位点的靶向诱变和对 通过H19的等位基因特异性表达和甲基化分析进行印迹， igf 2和（3）竞争性和超迁移电泳迁移率变动分析 以鉴定与ICR位点结合的蛋白质。蛋白质的进一步评估 通过检查小鼠突变体的候选蛋白质来鉴定。

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.