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: 6813784
• 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/6813784
• 关键词: DNA methylation; animal genetic material tag; animal population genetics; catecholamines; chromatin; cytogenetics; epinephrine; functional /structural genomics; gene expression; genetic regulation; genetically modified animals; genomic imprinting; 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. 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 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 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 B-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 transgenic mice where the catecholamine synthesizing cardiac cells are marked for easy purification.

烙印代表了正常孟德尔遗传学的奇怪蔑视。哺乳动物继承了两组完整的染色体，一组来自母亲，一组来自父亲，大多数常染色体基因将同样地从母亲和父亲等位基因中表达。然而，印迹基因仅从一个依赖性的父母的染色体中表达。由于单个核中存在沉默和活跃的启动子，因此活性的差异无法通过转录因子丰度来解释。因此，印迹基因的转录代表了一种明显的情况，在这种情况下，表观遗传机制限制了基因表达。因此，印迹基因是理解DNA修饰和染色质结构在维持基因表达模式中的作用的良好模型。此外，由于原始限制的表达，由印迹基因确定的表型不仅容易受到基因本身的突变的影响，而且也会在控制调节的表观遗传程序中破坏。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。我们的部分正在研究小鼠染色体染色体远端的一组基因。人类在11p15.5上的人类的同义区域在基因组组织和单相表达模式中保守。具体而言，我们正在解剖H19基因的母体特异性表达和IGF2基因的父亲特异性表达。这两个基因中的烙印突变的丧失与贝克维斯·威德曼综合征（BWS）和威尔姆斯的肿瘤有关。我们已经证明，H19启动子上游的序列是H19转基因表达所必需的。这些序列称为H19DMR（对于差异甲基化的区域），因为它们仅在父亲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，并表明遗传了这种突变的小鼠遗传性的H19的双重表达，而通过母体种系遗传突变的小鼠显示出正常静音IGF2等位基因的抑制丧失。因此，H19DMR是原始特异性消音器。通过构造等位基因，我们可以在特定细胞中删除该元素，在特定的发育时间点，我们能够通过不同的机制证明DMR沉默H19和IGF2。具体而言，我们证明了DMR包含甲基化敏感的转录绝缘子。父亲遗传后，DMR被甲基化，因此被灭活，从而允许IGF2基因的表达。母体遗传后，未甲基化的绝缘子具有活性，而IGF2的转移被阻塞。相反，甲基化的父亲H19DMR通过指导直接干扰转录激活的H19启动子的表观遗传修饰来使H19基因沉默。基于这些遗传研究，我们设计了模型系统，在该模型系统中，我们正通常非标记基因座（例如AFP），以便更精确地定义了印迹和单相表达的分子基础。这些实验导致了一个令人惊讶的发现，即DNA甲基化虽然对于正确的转录调节至关重要，但并不是主要的配子烙印。 我们研究的第二个重点是在该基因座中发现KCNQ1基因的生物学功能。该基因已通过寻找BWS病因的基因，一种具有原始遗传遗传模式的疾病的基因独立鉴定，对于长QT综合征（LQTS），映射到11P15.5的基因，一种没有父母的疾病。我们已经阐明了该基因印迹的复杂发展调节，以解决这种明显的悖论。最近，我们通过生成缺乏KCNQ1的小鼠来开发了一个用于遗传的LQT的模型。这些小鼠的体内ECG显示出异常的T波和P波形态以及QT和JT间隔的延长。但是，孤立心脏的心电图是正常的。这些变化表明心脏复极化缺陷取决于某些心外信号。进一步的研究表明，β-肾上腺素能刺激是原发性信号，并且正在解剖这种作用的分子基础。为了解决β-肾上腺素能刺激在LQT中的作用以及更普遍的心脏发育和功能，我们开发了一种小鼠模型，其中CRE重组酶代替PNMT基因表达了CRE重组酶。 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，因此为识别这种激素的特定作用提供了良好的遗传系统。此外，在适当的遗传背景下，在PNMT启动子控制下表达的CRE重组酶将标记B-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。这些实验证明了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，心脏将儿茶酚胺提供给中间植物的胚胎，这是这些激素绝对对生命至关重要的唯一发育时点。我们已经产生了转基因小鼠，其中有纯净的儿茶酚胺合成心脏细胞的标记。