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挑战。哺乳动物遗传了两组完整的染色体，一组来自母亲，另一组来自父亲，大多数常染色体基因将从母亲和父亲的等位基因中平等表达。然而，印记基因只在一条染色体上以亲本依赖的方式表达。因为沉默和活跃的启动子存在于单个细胞核中，所以活性的差异不能用转录因子的丰度来解释。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，印迹基因是一个模型，用于理解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。此外，由于亲本来源的限制表达，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，而且还会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。具体地说，我们正在剖析H19基因母系特异性表达和Igf2基因父系特异性表达的分子基础。这两个基因印记突变的丢失与Beckwith Wiedemann综合征(BWS)和Wilms‘s瘤有关。我们已经证明了H19启动子上游的序列是H19转基因印迹表达所必需的。这些序列被称为H19DMR(差异甲基化区域)，因为它们只在父亲的染色体上特异性地超甲基化。我们已经从内源基因座中删除了这个区域，并表明继承这种突变的小鼠在父系中表现出H19的双等位基因表达，而通过母系遗传突变的小鼠表现出正常沉默的Igf2等位基因的抑制丧失。因此，H19DMR是一种原产地特定的消声器。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。具体地说，我们证明了DMR包含一个转录绝缘子，该绝缘子在母系遗传时被灭活，并且这一活动与Igf2的单等位基因表达有关。相反，H19DMR通过引导H19启动子的表观遗传修饰直接干扰转录激活来沉默H19基因。我们研究的第二个重点是揭示KCNQ1基因的生物学功能，也是在这个基因座上。这个基因已经被寻找在BWS病因学中重要的基因和在长QT综合征中重要的基因定位到11p15.5的小组独立识别，BWS是一种具有亲本遗传模式的疾病，11p15.5是一种没有亲本效应的疾病。我们已经阐明了该基因印记的复杂发育规律，以解决这一明显的悖论。最近，我们通过产生KCNQ1缺陷的小鼠发展了一种遗传性LQTS的模型。这些小鼠的心电图显示异常的T波和P波形态以及QT和JT间期的延长。这些改变都预示着心脏复极缺陷。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。