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: 6671892
• 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/6671892
• 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 good models 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 directly interfere with transcriptional activation. Based on these genetic studies, we have devised model systems where we imprint normally non-imprinted loci (e.g. Afp) in order to more precisely define the molecular basis for imprinting and monoallelic expression. 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. In vivo ECGs from these mice show abnormal T-wave and P-wave morphologies and prolongation of the QT and JT intervals. However, ECGs of isolated hearts are normal. These changes are indicative of cardiac repolarization defects that are dependent upon some extracardiac signal. Further studies demonstrate that B-adrenergic stimulation is the primary extracardiac signal and the molecular basis for this effect is being dissected. To address the role of B-adrenergic stimulation in LQTS and in cardiac development and function more generally, we have developed a mouse model in which the cre recombinase enzyme is expressed in place of the Pnmt gene. Pnmt encodes the enzyme converting norepinephrine to epinephrine. Thus mice homozygous for this allele cannot make any epinephrine and thus offer a good genetic system for identifying the specific role of this hormone. Moreover, the cre recombinase expressed under control of the Pnmt promoter will, in the appropriate genetic background, mark B-adrenergic synthesizing cells and all their descendants so that the fate of these cells can be assayed.

印记是对正常孟德尔遗传学的一种奇特的挑战。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从母亲和父亲的等位基因中平等地表达。然而，印迹基因仅在一条染色体上以依赖于亲本的方式表达。由于沉默启动子和活跃启动子存在于单个细胞核中，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表观遗传机制限制基因表达的明确情况。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良好模型。此外，由于亲本的限制性表达，由印迹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控的表观遗传程序的破坏。因此，印迹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。本小组正在研究小鼠7号染色体远端的一组基因。人类11p15.5染色体上的共链区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基因的父亲特异性表达的分子基础。这两个基因的印迹突变缺失与Beckwith Wiedemann综合征（BWS）和Wilms肿瘤有关。我们已经证明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），因为它们只在父系染色体上特异性高甲基化。我们从内源性基因座中删除了这一区域，结果表明，通过父系遗传该突变的小鼠表现出H19的双等位基因表达，而通过母系遗传该突变的小鼠表现出对通常沉默的Igf2等位基因的抑制缺失。因此，H19DMR是一个父源特定的消音器。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该元件，我们能够证明DMR通过不同的机制沉默H19和Igf2。具体来说，我们证明了DMR包含一个转录绝缘子，该绝缘子在母体遗传时失活，并且该活性负责Igf2的单等位基因表达。相反，H19DMR通过指导H19启动子的表观遗传修饰，直接干扰转录激活，从而使H19基因沉默。基于这些遗传研究，我们设计了模型系统，在其中我们印记通常非印记位点（例如Afp），以便更精确地定义印迹和单等位基因表达的分子基础。