GENOMIC IMPRINTING ON DISTAL MOUSE CHROMOSOME 7

研究的目的是为了了解分子机制 一组印迹基因在远端的调控表达 至少5个印记基因(p57Kip2、Mash2、Igf2、 INS-2和H19)已被映射到鼠标中的该集群和 染色体11p15.5上的人类共线区域。印记中的中断 这些基因的表达与先天死亡有关 小鼠和Beckwith-Wiedemann综合征以及在 人类。此外，主要的遗传缺陷也在增加 对长QT综合征的易感性图在这里。我们的目标是 了解等位基因受限的遗传和分子机制 这些基因的表达，以确定新的印记基因在 地区，并开发人类疾病的小鼠模型。当前 研究集中在三个主要研究领域。首先，我们希望 分离H19基因调控表达所需的遗传元件 完全从母体染色体转录而来的。使用转基因技术 小鼠我们已经确定了正常所需的顺式作用序列 发育特异的表达模式。我们还确认了 限制母体H19表达所需的要素 等位基因。这一规定依赖于拷贝数，这表明 目前还没有涉及母体特异性表达的其他元素 已被确认身份。因此我们创造了新的转基因系 细菌人工染色体克隆来寻找这些元件。我们的 第二个目标是了解基因在细胞内的协调调节 远端7簇生。对基因中断的分析表明H19 和Igf2基因共享增强子元件。奇怪的是，这两个基因 相反的印记，即Igf2只从父亲那里表达 染色体。H19基因的破坏，包括上游印记 控制因素，导致母体表达不当 IGF2。母体免疫球蛋白2的激活可能是由于 正常活跃的母体H19启动子因此缓解了竞争 在Igf2和H19启动子之间，H19启动子通常获胜。 或者，母体免疫球蛋白2的激活可能是机械性的 与H19的失活无关，但由于删除了 上游印记监管元素，我们在我们的 转基因实验。我们正在产生条件性删除突变 特定于该H19压印控制元件。这些突变，即 保持H19启动子和编码序列不变，将分析 H19基因表达与母体免疫球蛋白2沉默的关系 染色体。具体地说，我们希望我们的突变能诱导 H19在父本染色体中的不适当表达。我们的钥匙 那么，问题是，这种父性H19基因的表达会产生什么影响 在Igf2基因座上。抑制正常活跃的父亲免疫球蛋白Igf2 等位基因将支持H19和Igf2的竞争 发起人负责相互印记图案 两个基因。Igf2的双等位基因表达将支持一种模型 共同调控元件控制H19和Igf2的表达。在……里面 在后面的案例中，我们的努力将集中在了解这一案例是如何 元素特异性地沉默父亲染色体上的H19，但沉默 IGF2特异地位于母体染色体上。最后，我们寻求 识别和描述该区域的新基因。我们已经确定了 BAC和P1克隆，从p57Kip2上游到 在H19的下游。利用这些作为探测器，我们已经开始搜索 利用直接测序、外显子陷阱和 基因的选择方法。我们已经确定了人类的老鼠同源物 KVLQT1。KVLQT1和水貂基因的产物一起形成 功能性钾通道。这个渠道中的突变是 约占所有长QT间期的60%。人类KVLQT1仅表达 从母体染色体中，至少在胎儿组织中。我们有 在物理和遗传上将小鼠Vlqt1定位到远端7簇。 我们已经注意到发育调节基因的高水平表达 肺、肾、肠道、胎盘以及心脏。印记的印记 Kvlqt1处于严格的发育调控之下。而最早的 胚胎的表达是母亲特有的，父亲的等位基因变成了 变得越来越活跃，直到基因本质上是一个双等位基因 出生后一周。如果适用于人类，这些结果表明 Kvlqt1印迹的发育缺失可以解释缺乏 长QT间期遗传的亲本偏向。

Research is directed at understanding the molecular mechanisms for regulated expression of a cluster of imprinted genes on the distal end of mouse chromosome 7. At least five imprinted genes (p57Kip2, Mash2, Igf2, Ins-2, and H19) have been mapped to this cluster in mouse and to the human syntenic region on chromosome 11p15.5. Disruption in imprinted expression of these genes has been associated with prenatal lethality in mice and with Beckwith-Wiedemann Syndrome and with a number of tumors in humans. In addition, the principle genetic defect increasing susceptibility to long QT syndrome maps here. Our goals are to understand the genetic and molecular mechanisms for allele restricted expression of these genes, to identify novel imprinted genes in the region, and to develop mouse models for the human diseases. Current studies are focused on three major research areas. First we wish to isolate genetic elements required for regulated expression of H19, a gene transcribed exclusively from the maternal chromosome. Using transgenic mice we have identified cis acting sequences required for normal developmental-specific expression patterns. We have also identified elements required for restriction of H19 expression to the maternal allele. This regulation is copy-number dependent, suggesting that additional elements involved in maternal-specific expression have not yet been identified. We have therefore generated new transgenic lines using Bacterial Artificial Chromosome clones to search for these elements. Our second aim is to understand the coordinate regulation of genes in the distal 7 cluster. Analysis of gene disruptions indicates that the H19 and Igf2 genes share enhancer elements. Curiously, the two genes are oppositely imprinted, i.e. Igf2 is expressed only from the paternal chromosome. Disruption of the H19 gene, including upstream imprinting control elements, results in the inappropriate expression of maternal Igf2. The activation of maternal Igf2 may be due to the deletion of the normally active maternal H19 promoter thus relieving a competition between Igf2 and H19 promoters which the H19 promoter normally wins. Alternately, the activation of maternal Igf2 may be mechanistically independent of inactivation of H19 but due to the deletion of the upstream imprinting regulatory element that we identified in our transgenic experiments. We are generating conditional deletion mutations specific to this H19 imprinting control element. These mutations, which leave the H19 promoter and coding sequences intact, will assay the relationship between H19 expression and Igf2 silencing on the maternal chromosome. Specifically, we expect our mutation to induce inappropriate expression of H19 from the paternal chromosome. Our key question, then, is what the effect of this paternal H19 expression will be at the Igf2 locus. Silencing of the normally active paternal Igf2 allele will support the notion that competition of the H19 and Igf2 promoters is responsible for the reciprocal imprinting patterns of the two genes. Biallelic expression of Igf2 will support a model that a common regulatory element controls expression of both H19 and Igf2. In this later case, our efforts will focus on understanding how this one element silences H19 specifically on the paternal chromosome but silences Igf2 specifically on the maternal chromosome. Finally, we seek to identify and characterize novel genes in the region. We have identified BAC and P1 clones that span the region from upstream of p57Kip2 to downstream of H19. Using these as probes, we have begun a search for novel transcripts in the region using direct sequencing, exon trap, and cDNA selection approaches. We have identified the mouse homolog of human KVLQT1. The products of KVLQT1 and of the minK gene together form a functional potassium channel. Mutations in this channel are responsible for about 60% of all long QT cases. The human KVLQT1 is expressed only from the maternal chromosome, at least in fetal tissue. We have physically and genetically mapped mouse vlqt1 to the distal 7 cluster. We have noted high levels of developmentally regulated expression in lung, kidney, gut, and placenta as well as in heart. The imprinting of Kvlqt1 is under strict developmental regulation. While the earliest embryonic expression is maternal specific, the paternal allele becomes increasingly active until the gene is essentially biallelic within one week after birth. If applicable to humans, these results demonstrating developmental loss of imprinting of Kvlqt1 can explain the lack of parental bias in inheritance of long QT.