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。哺乳动物遗传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亲和父亲的等位基因中平等表达。然而，印记基因仅从一条染色体以亲本来源依赖的方式表达。因为沉默和活性启动子存在于单个核中，所以活性的差异不能用转录因子丰度来解释。因此，印迹基因的转录代表了一个明确的情况下，表观遗传机制限制基因表达。因此，印迹基因是理解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，由于亲本来源的限制性表达，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，而且容易受到控制调控的表观遗传程序的破坏。因此，印记基因经常与人类疾病有关，包括影响细胞生长、发育和行为的疾病。我们的单位正在研究小鼠7号染色体远端的一组基因。人类染色体11p15.5上的同线区域在基因组组织和单等位基因表达模式中是保守的。具体而言，我们正在解剖H19基因的母体特异性表达和Igf 2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Beckwith Wiedemann综合征（BWS）和Wilms肿瘤相关。我们已经证明，序列上游的H19启动子所需的印迹表达的H19转基因。这些序列被称为H19 DMR（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。我们已经从内源性基因座中删除了该区域，并表明父系遗传该突变的小鼠显示H19的双等位基因表达，而通过母系种系遗传该突变的小鼠显示正常沉默的Igf 2等位基因的抑制丧失。因此，H19 DMR是亲本来源特异性沉默子。通过构建等位基因，我们可以在特定的细胞和特定的发育时间点删除这个元件，我们能够证明DMR通过不同的机制沉默H19和Igf 2。具体来说，我们证明DMR包含一个转录绝缘子，在母系遗传后失活，这种活性是负责Igf 2的单等位基因表达。相反，H19 DMR通过指导H19启动子的表观遗传修饰使H19基因沉默，所述表观遗传修饰本身直接干扰转录激活。 我们研究的第二个重点是揭示KCNQ 1基因的生物学功能，也在这个位点。该基因已由寻找BWS病因学中重要基因的小组独立鉴定，BWS是一种具有起源遗传模式的疾病，以及在长QT综合征中重要的基因，映射到11 p15.5，一种没有起源的疾病。-起源的影响。我们已经阐明了这个基因的印迹复杂的发育调控，以解决这个明显的矛盾。最近，我们通过产生KCNQ 1缺陷的小鼠开发了遗传性LQTS模型。这些小鼠的ECG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。