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: 7208927
• 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/7208927
• 关键词: DNA methylation; Wilms' tumor; animal genetic material tag; animal population genetics; catecholamines; chromatin; chromosome disorders; chromosomes; cytogenetics; developmental genetics; electrocardiography; epinephrine; functional /structural genomics; gene expression; gene mutation; genetic regulation; genetic transcription; genetically modified animals; genomic imprinting; heart electrical activity; laboratory mouse; long QT syndrome; norepinephrine; nucleic acid sequence; nucleic acid structure

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 Section 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 methylation-sensitive transcriptional insulator. Upon paternal inheritance, the DMR is methylated and the insulator is thereby inactivated, thus permitting expression of the Igf2 gene. Upon maternal inheritance, the unmethylated insulator is active and Igf2 transciption is blocked. In contrast, the methylated paternal 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. These experiments have led to the surprising discovery that DNA methylation, although crucial for correct transcriptonal regulation, is not the primary gametic imprint. We are using biochemical and molecular analyses to understand the epigentic marks that regulate imprinting and are devising strategies to alter these marks experimentally. 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 (LQTS) 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 ffor 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 beta-adrenergic stimulation is the primary extracardiac signal and the molecular basis for this effect is being dissected. To address the role of beta-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 beta-adrenergic synthesizing cells and all their descendants so that the fate of these cells can be assayed. These experiments demonstrate the major source of epinephrine (and norepinephrine) in the developing embryo is actually the heart. Thus the heart supplies the catecholamines to the midgestation embryo, the only developmental timepoint when these hormones are absolutely essential for life. We have generated knockin mice where the catecholamine synthesizing cardiac cells are marked for easy purification.

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良好模型。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。我们的部门正在研究小鼠 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 含有甲基化敏感的转录绝缘子。父系遗传后，DMR 被甲基化，绝缘体因此失活，从而允许 Igf2 基因表达。母系遗传后，未甲基化的绝缘体处于活跃状态，并且 Igf2 转录被阻断。相比之下，甲基化父本 H19DMR 通过直接干扰转录激活的 H19 启动子的表观遗传修饰来沉默 H19 基因。基于这些遗传学研究，我们设计了模型系统，在其中印记通常为非印记基因座（例如 Afp），以便更精确地定义印记和单等位基因表达的分子基础。这些实验令人惊讶地发现，DNA 甲基化虽然对于正确的转录调控至关重要，但并不是主要的配子印记。我们正在使用生化和分子分析来了解调节印记的表观遗传标记，并正在设计通过实验改变这些标记的策略。 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）病因学中重要基因的小组独立鉴定，以及在长 QT 综合征 (LQTS) 映射到 11p15.5（一种没有亲本遗传模式的疾病）中重要的基因的小组独立鉴定。亲本效应。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。这些小鼠的体内心电图显示异常的 T 波和 P 波形态以及 QT 和 JT 间期延长。然而，离体心脏的心电图是正常的。这些变化表明依赖于某些心外信号的心脏复极缺陷。进一步的研究表明，β-肾上腺素能刺激是主要的心外信号，并且正在剖析这种效应的分子基础。为了更广泛地阐明 β-肾上腺素能刺激在 LQTS 以及心脏发育和功能中的作用，我们开发了一种小鼠模型，其中表达 cre 重组酶代替 Pnmt 基因。 Pnmt 编码将去甲肾上腺素转化为肾上腺素的酶。因此，该等位基因纯合的小鼠不能产生任何肾上腺素，从而为鉴定该激素的特定作用提供了良好的遗传系统。此外，在Pnmt启动子控制下表达的cre重组酶将在适当的遗传背景下标记β-肾上腺素能合成细胞及其所有后代，以便可以测定这些细胞的命运。这些实验表明，发育中的胚胎中肾上腺素（和去甲肾上腺素）的主要来源实际上是心脏。因此，心脏向妊娠中期胚胎提供儿茶酚胺，这是这些激素对生命绝对必需的唯一发育时间点。我们已经产生了敲入小鼠，其中儿茶酚胺合成心脏细胞被标记以便于纯化。