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
• 负责人: Karl Eric Pfeifer
• 金额: $ 92.42万
• 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/8553889
• 关键词: 11p15.5; Address; Adrenergic Agents; Affect; Age; Alleles; Animals; Area; Arrhythmia; Beckwith-Wiedemann Syndrome; Behavior; Biological Models; Biological Process; Calcium ion; Calsequestrin; Cardiac; Cardiac Myocytes; Cell Nucleus; Cell physiology; Chromatin Structure; Chromosomes; Chromosomes, Human, Pair 7; Clinical; Coupling; DNA Modification Process; Deletion Mutagenesis; Development; Developmental Process; Disease; Distal; Drug usage; Elements; Epigenetic Process; Fathers; Functional RNA; Gene Cluster; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Mutation; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genomics; Germ Cells; Goals; Growth and Development function; H19 gene; Heart; Human; Inherited; Insertional Mutagenesis; Intervention; Ion Channel; Late Effects; Mammals; Metabolism; Methylation; Modeling; Molecular; Mothers; Mus; Mutation; Nephroblastoma; Parents; Patients; Pattern; Phenotype; Pilot Projects; Proteins; Protocols documentation; Regulation; Research; Role; Sarcoplasmic Reticulum; Structure; Surface; Testing; Therapeutic Intervention; Tissues; Transcriptional Silencer Elements; adrenergic; base; cancer type; cell growth; cell type; coping mechanism; developmental disease; efficacy testing; gain of function; gene function; gene therapy; genetic analysis; human disease; imprint; mouse model; novel; premature; programs; promoter; research study; restoration; therapeutic target; transcription factor; voltage

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. Expression of both H19 and Igf2 is dependent upon a shared set of enhancer elements downstream of both genes. We have identified a 2.4 kb ICR (for Imprinting Control Region) upstream of the H19 promoter. Using conditional deletion and insertional mutagenesis we have identified three functions associated with this element. First, this element acts to distinguish the parental origin of any chromosome into which it is inserted. Specifically, the CpGs within this region become hypermethylated upon paternal inheritance. Second, this element functions as a CTCF-dependent, methylation-sensitive transcriptional insulator. By reorganizing the long-range interactions of nearby promoter and enhancer elements, this insulator is able to direct parental-specific activation of nearby genes. Finally, this ICR also acts as a developmentally regulated silencer element when paternally inherited. Specifically, the methylated ICR induces changes in chromatin structure of neighboring sequences that impacts gene expression. Our current goals are to identify and characterize the protein factors and non-coding RNAs that interact with the ICR and establish the chromatin structures associated with the maternal and paternal chromosomes. We are addressing these issues both in germ cells, where the imprints are established, and in somatic tissues where expression of Igf2 and H19 are most critical for normal, healthy cell function. A second focus of our research is to generate mouse models for cardiac arrhythmias. We first focused on uncovering the biological function of the imprinted Kcnq1 gene, located just upstream of Igf2. More recently, we have generated a mouse model for Calsequestrin2 deficiency. We demonstrate that calsequestrin2 is not essential for cardiac calcium ion storage, which can be maintained by an expansion of the sarcoplasmic reticulum (SR) volume and surface area. Rather, the primary function of calsequestrin appears to be the regulation of the SR calcium ion release channel during conditions of beta-adrenergic stimulation. The loss of calsequestrin2 thus results in premature calcium ion release from the SR, leading to voltage changes that result in premature contraction of cardiomyocytes and thus arrhythmia. The validity of this mouse model has been recently confirmed by demonstration that drugs that we used to successfully ameliorate the mouse arrhythmias were highly effective in pilot studies on human patients. In the past year, we have demonstrated that the arrhythmias associated with calsequestrin2-deficiency worsen signficantly with age. We have recently generated and are now analyzing conditional alleles of calsequestrin 2. Using these models we have analyzed the effect of late-onset loss of calsequestrin 2 gene function, thus modeling a common human condition. Our results indicate that the phenotypes associated with loss of gene function late in development are much more severe. Thus we we believe that the the developing heart has mechanisms for coping aberrant regulation of Ca++ metabolism that can permanently protect the heart. We are initiating genomic approaches that will identify these mechanism and then evaluate whether these mechanisms represent therapeutic targets. We are also now determining the effect of restoration of calsequestrin 2 gene function to animals that have developed in the absence of any active calsequestrin 2 gene. Together these experiments will help us understand how calsequestin 2 gene activity regulates sarcoplasmic reticulum structure and also help us develop novel therapies for human patients with both congenital and acquired deficiencies in Ca++ excitation-contraction coupling.

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在母亲和父亲的等位基因中平等表达。然而，印记基因仅从一条染色体以亲本来源依赖的方式表达。因为沉默和活性启动子存在于单个核中，所以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一个明确的情况下，表观遗传机制限制基因表达。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性表达，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，而且容易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。我们的部门正在研究小鼠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。具体而言，我们正在解剖H19基因的母体特异性表达和Igf 2基因的父体特异性表达的分子基础。这两个基因中的印记突变的缺失与Beckwith Wiedemann综合征（BWS）和Wilms肿瘤相关。H19和Igf 2的表达都依赖于两个基因下游的一组共享的增强子元件。 我们已经鉴定了H19启动子上游的2.4kb ICR（Imprinting Control Region）。 使用条件性缺失和插入突变，我们已经确定了三个功能与此元素。 首先，该元件的作用是区分它所插入的任何染色体的亲本来源。 具体而言，该区域内的CpG在父系遗传后变得高甲基化。 第二，该元件作为CTCF依赖性、甲基化敏感性转录绝缘子发挥功能。 通过重组附近的启动子和增强子元件的长程相互作用，这种绝缘子能够指导附近基因的亲本特异性激活。 最后，这ICR也作为一个发展调节沉默元件时，父系遗传。 具体来说，甲基化的ICR会诱导邻近序列的染色质结构发生变化，从而影响基因表达。 我们目前的目标是鉴定和表征与ICR相互作用的蛋白质因子和非编码RNA，并建立与母本和父本染色体相关的染色质结构。我们正在解决这些问题，无论是在生殖细胞，其中建立的印记，并在体细胞组织中的表达Igf 2和H19是最关键的正常，健康的细胞功能。 我们研究的第二个重点是产生心律失常的小鼠模型。 我们首先专注于揭示位于Igf 2上游的印迹Kcnq 1基因的生物学功能。 最近，我们已经产生了钙螯合蛋白2缺乏症的小鼠模型。 我们证明，钙螯合蛋白2是不是必不可少的心脏钙离子储存，这可以维持扩张的肌浆网（SR）的体积和表面积。 相反，钙螯合蛋白的主要功能似乎是在β-肾上腺素能刺激条件下调节SR钙离子释放通道。 因此，钙螯合蛋白2的丢失导致SR过早释放钙离子，导致电压变化，导致心肌细胞过早收缩，从而导致心律失常。 这种小鼠模型的有效性最近已经通过证明我们用于成功改善小鼠心律失常的药物在人类患者的初步研究中非常有效而得到证实。 在过去的一年中，我们已经证明了与钙螯合素2缺乏相关的心律失常随着年龄的增长而显著恶化。 我们最近已经产生并正在分析钙螯合蛋白2的条件等位基因。 使用这些模型，我们分析了迟发性钙螯合蛋白2基因功能丧失的影响，从而模拟了一种常见的人类疾病。 我们的研究结果表明，与发育后期基因功能丧失相关的表型更为严重。因此，我们相信发育中的心脏具有应对钙代谢异常调节的机制，可以永久保护心脏。 我们正在启动基因组方法，以确定这些机制，然后评估这些机制是否代表治疗靶点。我们现在也在确定恢复钙螯合蛋白2基因功能对在没有任何活性钙螯合蛋白2基因的情况下发育的动物的影响。 这些实验将帮助我们了解calsequestin 2基因活性如何调节肌浆网结构，并帮助我们为先天性和后天性Ca++兴奋-收缩偶联缺陷的人类患者开发新的治疗方法。