Analysis of Imprinting on Mouse Distal Chromosome 7

小鼠远端7号染色体印记分析

• 批准号: 6432581
• 负责人: Karl Eric Pfeifer
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432581
• 关键词: DNA methylation; animal genetic material tag; gene expression; genetic mapping; genetic regulation; genetically modified animals; genomic imprinting; laboratory mouse; nucleic acid sequence; structural genes

At least six imprinted genes (p57Kip2, Kvlqt1, Mash2, Ins2, Igf2, and H19) have been mapped to a one megabase cluster at the distal end of mouse chromosome 7. The imprinted expression and physical organization of these genes is conserved in humans where they map to chromosome 11p15.5. Disruption of the normal imprinted expression of these genes is associated with prenatal lethality in mice and with Beckwith-Wiedemann Syndrome (BWS) and with several tumors in humans. In addition, the most frequent genetic defect associated with long QT syndrome maps to this region in humans. Finally, tissue culture studies strongly suggest the existence of a novel gene with tumor suppressor activity in this region. Our goals are to understand the molecular and genetic mechanisms underlying the allele restricted expression of these genes, to identify novel imprinted genes in the region, and to develop mouse models for the human diseases associated with misexpression of these imprinted genes.We have used molecular approaches to identify P1 and Bacterial Artificial Chromosome (BAC) clones that span the approximately 1 megabase imprinted region of distal mouse. Using exon trap and direct sequencing we have identified several potential genes. One gene so identified is the mouse Kvlqt1 gene, previously identified in human genetic studies as the gene responsible for the majority of cases of long QT syndrome. Maternally associated translocations in human KVLQT1 are associated with BWS. We have completed extensive analysis of the expression pattern of mouse Kvlqt1 showing that it is imprinted in a developmentally regulated fashion: expression in the early embryo is from the maternal chromosome but is biallelic in new born pups. These findings help to explain the paradoxical association of the gene with both long QT syndrome, inherited as a dominant mutation with no parent-of-origin effect, and also with BWS which shows complete parent of origin bias. To further elucidate the genetics of these diseases we have generated null and point mutations at the Kvlqt1 locus in embryonic stem cells have used blastocyst injection to generate mice carrying these mutations. Mice homozygous for null alleles of Kvlqt1 show bilateral deafness and severe balance disorders. Histological characterization of inner ear development in mutant mice is currently underway to determine the basis for this phenotype. In addition we have begun characterization of the heart physiology in these mice by ECG analysis in vivo and ex vivo and by analysis of the electrophysiology of single cardiac myocytes. Preliminary analysis indicates that deletion of the Kvlqt1 gene activity results in extended QT intervals in mice. This extension is exacerbated by stress and, interestingly, shows a strong gender bias.We are using molecular genetic approaches to characterize the mechanisms of imprinting concentrating on coordinate regulation of the H19 and Igf2 genes. H19 is expressed only from the maternal chromosome while Igf2 is expressed only paternally. Using transgenic mice we have identified elements required for silencing of the paternal copies of H19 transgenes. These mice identify two crucial elements: one element upstream of -0.7 kilobases and a second within H19s exon 1. We have generated mice carrying mutations that allow us to delete these regions separately and in a temporally controlled manner. We have shown that both Igf2 and H19 share a common imprinting control region. That is, the element just upstream of the H19 promoter is required for silencing of the paternal H19 and of the maternal Igf2 alleles. However, the molecular mechanisms for silencing of these genes is distinct. That is, the times during development when this cis acting element must be present to establish imprinting are distinct for the two genes. Further, our studies suggest that the imprinting element acts as a boundary, restricting access of enhancer elements to promoters on the other side of the boundary. We are currently testing this model using transgenic mice.

至少有 6 个印记基因（p57Kip2、Kvlqt1、Mash2、Ins2、Igf2 和 H19）已被定位到小鼠 7 号染色体远端的一个兆碱基簇。这些基因的印记表达和物理组织在人类中是保守的，其中它们映射到染色体 11p15.5。这些基因正常印记表达的破坏与小鼠的产前致死率、贝克威斯-维德曼综合征 (BWS) 以及人类的多种肿瘤有关。此外，与长 QT 综合征相关的最常见的遗传缺陷也位于人类的该区域。最后，组织培养研究强烈表明该区域存在具有肿瘤抑制活性的新基因。我们的目标是了解这些基因的等位基因限制性表达背后的分子和遗传机制，识别该区域中的新印记基因，并开发与这些印记基因错误表达相关的人类疾病的小鼠模型。我们使用了分子方法鉴定跨越小鼠远端约 1 兆碱基印记区域的 P1 和细菌人工染色体 (BAC) 克隆。使用外显子捕获和直接测序，我们已经鉴定了几个潜在的基因。这样确定的一个基因是小鼠 Kvlqt1 基因，该基因先前在人类遗传学研究中被确定为导致大多数长 QT 综合征病例的基因。人类 KVLQT1 中与母体相关的易位与 BWS 相关。我们已经完成了对小鼠 Kvlqt1 表达模式的广泛分析，表明它以发育调节方式印记：早期胚胎中的表达来自母体染色体，但在新生幼崽中是双等位基因。这些发现有助于解释该基因与长QT综合征（作为显性突变遗传而无亲本效应）以及与BWS（显示出完全亲本偏差）之间的矛盾关联。为了进一步阐明这些疾病的遗传学，我们在胚胎干细胞的 Kvlqt1 基因座上产生了零突变和点突变，并使用囊胚注射来产生携带这些突变的小鼠。 Kvlqt1 无效等位基因纯合的小鼠表现出双侧耳聋和严重的平衡障碍。目前正在进行突变小鼠内耳发育的组织学表征，以确定这种表型的基础。此外，我们已经开始通过体内和离体心电图分析以及单个心肌细胞的电生理学分析来表征这些小鼠的心脏生理学。初步分析表明，Kvlqt1 基因活性的缺失会导致小鼠 QT 间期延长。这种延伸因压力而加剧，有趣的是，显示出强烈的性别偏见。我们正在使用分子遗传学方法来表征印记机制，重点是 H19 和 Igf2 基因的协调调节。 H19 仅在母本染色体上表达，而 Igf2 仅在父本染色体上表达。使用转基因小鼠，我们已经确定了沉默 H19 转基因父本拷贝所需的元件。这些小鼠识别出两个关键元素：一个元素位于 -0.7 kb 上游，第二个元素位于 H19 外显子 1 内。我们已经培育出携带突变的小鼠，这些突变使我们能够以时间控制的方式单独删除这些区域。我们已经证明 Igf2 和 H19 共享一个共同的印记控制区域。也就是说，父本 H19 和母本 Igf2 等位基因的沉默需要 H19 启动子上游的元件。然而，这些基因沉默的分子机制是不同的。也就是说，在发育过程中，必须存在该顺式作用元件以建立印记的时间对于这两个基因来说是不同的。此外，我们的研究表明，印记元件充当边界，限制增强子元件接触边界另一侧的启动子。我们目前正在使用转基因小鼠测试该模型。