Analysis Of Imprinting On Mouse Distal Chromosome 7

小鼠远端染色体 7 上的印记分析

基本信息

项目摘要

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 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。我们研究组正在研究小鼠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含有甲基化敏感的转录绝缘子。在父系遗传时,DMR甲基化,绝缘体因此失活,从而允许Igf2基因的表达。在母系遗传时,未甲基化的绝缘体被激活,而Igf2转导被阻断。相反,父亲的H19DMR甲基化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。在这些遗传学研究的基础上,我们设计了正常印记非印记基因座(如AFP)的模型系统,以便更准确地定义印记和单等位基因表达的分子基础。这些实验导致了令人惊讶的发现,DNA甲基化虽然对正确的转录调节至关重要,但并不是主要的配子印记。 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。该基因已经被寻找在BWS的病因学中重要的基因和在长QT综合征(LQTS)中的重要基因独立地识别出来,BWS是一种具有亲本遗传模式的疾病,而长QT综合征(LQTS)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。这些小鼠的活体心电图显示异常的T波和P波形态,QT和JT间期延长。然而,孤立心脏的心电图是正常的。这些变化表明心脏复极缺陷依赖于某些心外信号。进一步的研究表明,β-肾上腺素能刺激是主要的心外信号,这种作用的分子基础正在被剖析。为了阐明β-肾上腺素能刺激在LQTS和心脏发育和功能中的作用,我们开发了一种小鼠模型,在该模型中,cre重组酶代替Pnmt基因表达。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,从而为识别这种荷尔蒙的特定作用提供了一个良好的遗传系统。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-肾上腺素合成细胞及其所有后代,从而可以检测这些细胞的命运。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,心脏为中期胚胎提供儿茶酚胺,这是唯一的发育时间点,当这些激素对生命绝对必要时。我们已经产生了敲击小鼠,在那里合成儿茶酚胺的心肌细胞被标记为易于纯化。

项目成果

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Karl Eric Pfeifer其他文献

Karl Eric Pfeifer的其他文献

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{{ truncateString('Karl Eric Pfeifer', 18)}}的其他基金

Analysis of Imprinting on Mouse Distal Chromosome 7
小鼠远端7号染色体印记分析
  • 批准号:
    6432581
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
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
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Epigenetic mechanisms regulating the Igf2/H19 and Kcnq1 locus
调节 Igf2/H19 和 Kcnq1 位点的表观遗传机制
  • 批准号:
    8351152
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Generating new mouse models
生成新的鼠标模型
  • 批准号:
    10908197
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
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
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Regulated expression and developmental functions of the H19 long noncoding RNA
H19长非编码RNA的调控表达和发育功能
  • 批准号:
    10685191
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Epigenetic mechanisms regulating the Igf2/H19 and Kcnq1 locus
调节 Igf2/H19 和 Kcnq1 位点的表观遗传机制
  • 批准号:
    10266483
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
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
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
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:
    7968609
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Epigenetic mechanisms regulating the Igf2/H19 and Kcnq1 locus
调节 Igf2/H19 和 Kcnq1 位点的表观遗传机制
  • 批准号:
    8553889
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
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