Sequences controlling H19 Gene Imprinting

描述（由申请人提供）：哺乳动物的基因子集受基因组印记调节，这一过程导致某些基因的母系和父系等位基因的不平等表达。因此，印迹基因的单个表达等位基因的有害突变或缺失将导致缺乏功能性基因产物。在人类中，印迹和印迹基因的破坏是人类遗传疾病贝克威斯-魏德曼综合征、普瑞德-威利综合征和安吉尔曼综合征的原因，也是银罗素综合征和威尔姆斯肿瘤等癌症的原因。本研究的目的是探讨印迹基因的亲本身份是如何建立和维持的。这些研究将使用H19基因，该基因是由小鼠和人类的母系等位基因表达的。H19和Igf2基因的印迹至少部分是通过位于H19转录开始上游2kb的2kb印迹控制区（ICR）介导的。ICR，也被称为差异甲基化结构域（DMD），在受抑制的父本等位基因上被超甲基化，并通过ctcf结合在母本等位基因上作为甲基化敏感绝缘子。本课题将通过以下实验探讨H19/Igf2基因位点印迹的机制：(1)确定ICR/DMD序列以及CTCF结合位点的合适间距是否对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)研究生殖细胞和胚胎细胞中H19位点的染色质结构，并评估这些表观遗传修饰在印迹中的作用。除了阐明该基因座在发育过程中印记建立和维持的机制外，这些实验还将对Beckwith-Wiedemann综合征和Silver-Russell综合征患者中新发现的突变进行建模，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.