Analysis Of Imprinting On Mouse Distal Chromosome 7

小鼠远端染色体 7 上的印记分析

• 批准号: 6541232
• 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/6541232
• 关键词: DNA methylation; animal genetic material tag; gene expression; genetic mapping; genetic regulation; genetically modified animals; genomic imprinting; laboratory mouse; long QT syndrome; nucleic acid sequence; structural genes

Imprinting represents a curious defiance of normal Mendelian genetics. Mammals inherit two complete sets of chromosomes, one from the mother and one from the father, and most autosomal genes will be expressed equally from maternal and paternal alleles. Imprinted genes, however, are expressed from only one chromosome in a parent-of-origin dependent manner. Because silent and active promoters are present in a single nucleus, the differences in activity cannot be explained by transcription factor abundance. Thus the transcriptional of imprinted genes represents a clear situation in which epigenetic mechanisms restrict gene expression. Therefore imprinted genes are a model for understanding the role of DNA modifications and chromatin structure in maintaining appropriate patterns of gene expression. Further, because of parent-of-origin restricted expression, phenotypes determined by imprinted genes are not only susceptible to mutations of the genes themselves but also to disruptions in the epigenetic programs controlling regulation. Thus imprinted genes are frequently associated with human diseases, including disorders affecting cell growth, development, and behavior. Our Unit is investigating a cluster of genes on the distal end of mouse chromosome 7. The syntenic region in humans on chromosome 11p15.5 is conserved in genomic organization and in monoallelic expression patterns. Specifically we are dissecting the molecular basis for the maternal specific expression of the H19 gene and the paternal specific expression of the Igf2 gene. Loss of imprinting mutations in these two genes is associated with Beckwith Wiedemann Syndrome (BWS) and with Wilms' tumor. We have demonstrated that sequences upstream of the H19 promoter are required for imprinted expression of H19 transgenes. These sequences are called the H19DMR (for differentially methylated region) because they are specifically hypermethylated only on the paternal chromosome. We have deleted this region from the endogenous locus and shown that mice inheriting this mutation paternally show biallelic expression of H19 while mice inheriting the mutation through the maternal germline show loss of repression of the normally silent Igf2 allele. Thus the H19DMR is a parent-of-origin specific silencer. By constructing alleles in which we could delete this element in specific cells and at specific developmental time points we were able to demonstrate that the DMR silences H19 and Igf2 by distinct mechanisms. Specifically, we demonstrate that the DMR contains a transcriptional insulator that is inactivated upon maternal inheritance and that this activity is responsible for monoallelic expression of Igf2. In contrast, the H19DMR silences the H19 gene by directing epigenetic modifications of the H19 promoter that themeselves directly interfere with transcriptional activation. A second focus of our research is to uncover the biological function of the KCNQ1 gene, also in this locus. This gene has been identified independently by groups looking for genes important in the etiology of BWS, a disease with parent-of-origin inheritance patterns, and for genes important in Long QT syndromes mapping to 11p15.5, a disease with no parent-of-origin effects. We have elucidated the complex developmental regulation of imprinting of this gene so to resolve this apparent paradox. Recently, we have developed a model for inherited LQTS by generating mice deficient in KCNQ1. ECGs from these mice show abnormal T-wave and P-wave morphologies and prolongation of the QT and JT intervals. These changes are both indicative of cardiac repolarization defects. In addition, these mice show profound bilateral deafness and balance disorders. Histological analyses demonstrate that mutant mice cannot secrete functional endolymph.

印记代表了对正常孟德尔遗传学的一种奇怪的蔑视。哺乳动物继承了两套完整的染色体，一套来自母亲，一套来自父亲，大多数常染色体基因将在母本和父本等位基因中同等表达。然而，印记基因仅从一条染色体以依赖于亲本的方式表达。由于沉默启动子和活性启动子存在于单个核中，因此活性差异不能用转录因子丰度来解释。因此，印记基因的转录代表了表观遗传机制限制基因表达的明显情况。因此，印记基因是了解 DNA 修饰和染色质结构在维持适当的基因表达模式中的作用的模型。此外，由于亲本表达受到限制，由印记基因决定的表型不仅容易受到基因本身突变的影响，而且还容易受到控制调节的表观遗传程序的破坏。因此，印记基因经常与人类疾病相关，包括影响细胞生长、发育和行为的疾病。我们的小组正在研究小鼠 7 号染色体远端的一组基因。人类 11p15.5 号染色体上的同线性区域在基因组组织和单等位基因表达模式中是保守的。具体来说，我们正在剖析 H19 基因的母体特异性表达和 Igf2 基因的父体特异性表达的分子基础。这两个基因中印记突变的缺失与贝克威斯·维德曼综合征 (BWS) 和维尔姆斯氏瘤有关。我们已经证明 H19 启动子上游的序列是 H19 转基因的印记表达所必需的。这些序列被称为 H19DMR（差异甲基化区域），因为它们仅在父本染色体上特异性高甲基化。我们从内源基因座中删除了该区域，并表明从父系继承该突变的小鼠表现出 H19 的双等位基因表达，而通过母系种系遗传该突变的小鼠则表现出对通常沉默的 Igf2 等位基因的抑制的丧失。因此，H19DMR 是亲本特异性沉默子。通过构建可以在特定细胞和特定发育时间点删除该元件的等位基因，我们能够证明 DMR 通过不同的机制沉默 H19 和 Igf2。具体来说，我们证明 DMR 含有一种转录绝缘子，该绝缘子在母系遗传时失活，并且这种活性负责 Igf2 的单等位基因表达。相比之下，H19DMR 通过指导 H19 启动子的表观遗传修饰来沉默 H19 基因，而 H19 启动子本身直接干扰转录激活。 我们研究的第二个重点是揭示 KCNQ1 基因（也在该位点）的生物学功能。该基因已由寻找 BWS（一种具有亲本遗传模式的疾病）病因学中重要基因以及映射到 11p15.5（一种无亲本疾病）的长 QT 综合征中重要基因的小组独立鉴定。 -起源效应。我们已经阐明了该基因印记的复杂发育调控，从而解决了这一明显的悖论。最近，我们通过生成 KCNQ1 缺陷的小鼠开发了遗传性 LQTS 模型。这些小鼠的心电图显示 T 波和 P 波形态异常以及 QT 和 JT 间期延长。这些变化都表明心脏复极缺陷。此外，这些小鼠表现出严重的双侧耳聋和平衡障碍。组织学分析表明突变小鼠不能分泌功能性内淋巴。