Sequences controlling H19 Gene Imprinting

描述(申请人提供)：哺乳动物的一个基因子集由基因组印记调节，这一过程导致某些基因的母系和父系等位基因表达不平等。因此，印记基因单一表达等位基因的有害突变或缺失将导致功能基因产品的缺失。在人类中，印记和印记基因的中断是人类遗传病Beckwith-Wiedemann综合征、Prader-Willi综合征和Angelman综合征、许多Silver-Russell综合征以及Wilms瘤等癌症的原因。这项建议的目的是调查印记基因亲本同一性建立和维持的机制。这项研究将使用H19基因，它是从老鼠和人类的母源等位基因中表达出来的。H19和连接的和相反印记的Igf2基因的印记至少部分是通过位于H19转录起始上游2kb的印记控制区(ICR)介导的。ICR也被称为差异甲基化结构域(DMD)，它在父系被抑制的等位基因上被高甲基化，并通过与母系等位基因上的CTCF结合而起到甲基化敏感的绝缘体的作用。本建议将通过以下实验来研究H19/Igf2基因座的印记机制：(1)确定除CTCF结合位点外的ICR/DMD序列和CTCF结合位点的适当间距是否对H19/Igf2印迹至关重要；(2)确定CTCF结合位点外的CpG突变是否会破坏印迹；(3)检测生殖细胞和胚胎细胞中H19基因座的染色质结构，并评估这些表观遗传修饰在印迹中的作用。除了阐明发育过程中该基因座印记的建立和保持机制外，这些实验还将对新发现的Beckwith-Wiedemann综合征和Silver-Russell综合征患者的突变进行建模，从而更好地了解疾病的病因。

DESCRIPTION (provided by applicant): A subset of genes in mammals is regulated by genomic imprinting, a process that results in unequal expression of the maternal and paternal alleles of certain genes. As a consequence, deleterious mutations or deletions in the single expressed allele of an imprinted gene will result in the absence of a functional gene product. In humans, disruptions in imprinting and imprinted genes account for the human genetic diseases Beckwith-Wiedemann Syndrome, Prader-Willi Syndrome and Angelman Syndrome, a number of cases of Silver-Russell Syndrome and for cancers such as Wilms' tumor. The objective of this proposal is to investigate the mechanism by which parental identity of imprinted genes is established and maintained. The studies will employ the H19 gene, which is expressed from the maternally-derived allele in mice and humans. The imprinting of H19 and the linked and oppositely imprinted Igf2 gene, is mediated, at least in part, through the 2 kb imprinting control region (ICR) that is located 2 kb upstream from the start of H19 transcription. The ICR, which is also designated the differentially methylated domain (DMD), is hypermethylated on the repressed paternal allele and acts as a methylation-sensitive insulator through CTCF-binding on the maternal allele. This proposal will investigate the mechanism of imprinting at the H19/Igf2 locus through the following experiments: (1) to determine whether ICR/DMD sequence in addition to CTCF binding sites and the proper spacing of the CTCF binding sites are critical for H19/Igf2 imprinting; (2) to determine if CpG mutations outside of the CTCF-binding sites disrupt imprinting; (3) To examine the chromatin structure of the H19 locus in germ cells and embryonic cells and assess the role of these epigenetic modifications in imprinting. In addition to elucidating the mechanism of imprint establishment and maintenance at this locus during development, these experiments will model newly identified mutations in individuals with Beckwith-Wiedemann Syndrome and Silver-Russell Syndrome, providing a better understanding of the etiology of the disease.