Analysis of Imprinting on Mouse Distal Chromosome 7

至少有6个印迹基因（p57Kip2、Kvlqt1、Mash2、Ins2、Igf2和H19）被定位到小鼠7号染色体远端一个1兆碱基簇上。这些基因的印记表达和物理组织在人类中是保守的，它们映射到染色体11p15.5。这些基因正常印迹表达的破坏与小鼠的产前死亡率、贝克威斯-维德曼综合征（BWS）和人类的几种肿瘤有关。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这一区域。最后，组织培养研究强烈提示在该区域存在一种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的分子和遗传机制，识别该区域的新印迹基因，并开发与这些印迹基因错误表达相关的人类疾病的小鼠模型。我们使用分子方法鉴定了P1和细菌人工染色体（BAC）克隆，它们跨越小鼠远端大约1兆碱基印迹区。利用外显子陷阱和直接测序，我们已经确定了几个潜在的基因。其中一个基因是小鼠的Kvlqt1基因，该基因先前在人类基因研究中被确定为导致大多数长QT综合征的基因。人类KVLQT1的母系相关易位与BWS有关。我们已经完成了对小鼠Kvlqt1表达模式的广泛分析，表明它以一种发育调节的方式印记：早期胚胎中的表达来自母体染色体，但在新生幼鼠中是双等位基因。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）和BWS（显示完全亲本偏倚）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位点上产生了零突变和点突变，并使用囊胚注射产生了携带这些突变的小鼠。Kvlqt1零等位基因纯合的小鼠表现为双侧耳聋和严重的平衡障碍。突变小鼠内耳发育的组织学特征目前正在进行中，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分析来表征这些小鼠的心脏生理。初步分析表明，Kvlqt1基因活性的缺失导致小鼠QT间期延长。压力加剧了这种延伸，有趣的是，它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迹的机制，集中在H19和Igf2基因的协调调节上。H19仅在母体染色体上表达，而Igf2仅在父系染色体上表达。使用转基因小鼠，我们已经确定了沉默H19基因父本拷贝所需的元件。这些小鼠确定了两个关键元素：一个位于-0.7千碱基上游，另一个位于h19的外显子1。我们培育出携带突变的小鼠，使我们能够以一种暂时可控的方式分别删除这些区域。我们已经证明Igf2和H19共享一个共同的印迹控制区。也就是说，H19启动子上游的元件是沉默父本H19和母本Igf2等位基因所必需的。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，这种顺式作用元件必须存在以建立印迹的时间对两个基因来说是不同的。此外，我们的研究表明，印迹元件起到了边界的作用，限制了增强子元件进入边界另一侧的启动子。我们目前正在用转基因老鼠测试这个模型。

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.