Sequences controlling H19 Gene Imprinting

描述（由申请人提供）：哺乳动物中的一个基因子集受基因组印记调节，这是一种导致某些基因的母本和父本等位基因不平等表达的过程。因此，印记基因的单个表达等位基因中的有害突变或缺失将导致功能性基因产物的缺失。在人类中，印记和印记基因的破坏导致了人类遗传疾病Beckwith-Wiedemann综合征、Prader-Willi综合征和Angelman综合征，一些Silver-Russell综合征病例以及诸如Wilms肿瘤的癌症。这个建议的目的是调查的机制，父母的身份印记基因的建立和维护。这些研究将使用H19基因，该基因在小鼠和人类中表达自母体来源的等位基因。H19和连接的且相反印迹的Igf 2基因的印迹至少部分地通过位于H19转录起始点上游2kb的2kb印迹控制区（ICR）介导。ICR也被称为差异甲基化结构域（DMD），其在受抑制的父本等位基因上被高甲基化，并且通过与母本等位基因上的CTCF结合而充当甲基化敏感的绝缘体。本研究拟通过以下实验探讨H19/Igf 2位点的印迹机制：（1）确定除了CTCF结合位点之外的ICR/DMD序列和CTCF结合位点的适当间隔是否对H19/Igf 2印迹至关重要;（2）确定CTCF结合位点之外的CpG突变是否破坏印迹;（3）检测H19基因座在生殖细胞和胚胎细胞中的染色质结构，并评估这些表观遗传修饰在印迹中的作用。除了阐明在发育过程中在该位点建立和维持印记的机制外，这些实验还将模拟Beckwith-Wiedemann综合征和Silver-Russell综合征患者中新发现的突变，从而更好地了解该疾病的病因。

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.