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
• 负责人: Karl Eric Pfeifer
• 金额: $ 151.95万
• 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/10266483
• 关键词: 11p15.5; Ablation; Address; Adrenergic Agents; Adult; Affect; Age; Alleles; Animals; Arrhythmia; Beckwith-Wiedemann Syndrome; Behavior; Biological Models; Birth; CRISPR/Cas technology; Calcium ion; Calsequestrin; Cardiac; Cardiac Myocytes; Cardiac conduction system; Cell Aging; Cell Cycle Regulation; Cell Differentiation process; Cell Nucleus; Cell physiology; Cells; Chromatin Structure; Chromosome 7; Chromosomes; Complex; DNA Modification Process; Data; Defect; Deletion Mutagenesis; Development; Developmental Process; Disease; Disease Progression; Disease model; Distal; Drug usage; Elements; Endothelial Cells; Endothelium; Epigenetic Process; Fathers; Fibrosis; Gene Cluster; Gene Expression; Gene Expression Profile; Gene Mutation; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genomics; Germ Cells; Goals; Growth and Development function; H19 gene; Health; Heart; Heart Rate; Human; Hypertrophy; IGF2 gene; Inherited; Insertional Mutagenesis; MAP Kinase Gene; Mammals; Mesenchymal; Messenger RNA; Methylation; MicroRNAs; Modeling; Molecular; Mothers; Mouse Strains; Mus; Muscle Cells; Mutation; Myocardial dysfunction; Nephroblastoma; Parents; Pathologic; Pathway interactions; Patients; Pattern; Peptides; Phenocopy; Phenotype; Physiological; Pilot Projects; Proteins; Recording of previous events; Regulation; Research; Risk; Role; Skeletal Muscle; Small Interfering RNA; Stress; TP53 gene; Therapeutic Intervention; Time; Tissues; Transcriptional Silencer Elements; Translations; Untranslated RNA; Ventricular Tachycardia; age related; cancer type; cell growth; clinically significant; coronary fibrosis; developmental disease; epigenome; flexibility; gene function; genetic analysis; heart function; human disease; imprint; in vitro Model; mature animal; mouse model; muscle regeneration; mutant; novel; postnatal; premature; prevent; programs; promoter; response; senescence; 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 transcription 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. Especially, we are focusing on 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. We are also working to establish mouse models that mimic the Beckwith Wiedemann syndrome phenotypes associated with loss of imprinting at the Igf2/H19 locus in humans. Most recently we have demonstrated defects in muscle cell differentiation and in muscle regeneration in cells where Igf2/H19 imprinting is disrupted. We have demonstrated that even a <2-fold increase in Igf2 expression will result in large-scale disruption in cell cycle regulation by hyperactivation of the MAPK pathway. In addition, decreased expression of H19 disrupts normal regulation of p53 in muscle cells so that they can no longer respond to Wnt stimulation and therefore do no undergo normal hypertrophy. Thus, loss of imprinting of both H19 and Igf2 genes are relevant to overgrowth phenotypes in BWS We are now characterizing cardiac dysfunction phenotypes in these mutant animals. During early development, extra expression of Igf2 results in physiologic hypertrophy. However, hypertrophy diminishes after birth (when Igf2 expression stops) and there are no long-term health consequences. However, loss of the H19 lncRNA results in pathological hypertrophy and reduced cardiac function that progresses in the postnatal heart. Genetic analyses indicate that H19 prevents premature endothelial to mesenchymal transition. In the absence of H19, endothelial cells mis-express mesenchymal markers and adult mice show significant fibrosis. Using CRISPR-Cas9 technologies, we have generated novel mouse strains that carry mutations in specific H19 domains. These analyses demonstrate that H19 sequences that interact with let7 microRNAs are necessary to prevent cardiac fibrosis and functional defects. Finally, using in vitro models, we have learned that abrupt depletion of H19 by siRNA or by genetic ablation results in rapid onset of cellular senescence. H19 lncRNA interacts directly with p21 mRNA and both destabilizes the mRNA and inhibits protein translation. Upon loss of H19, p21 mRNA and peptide levels rise rapidly to induce senescence. A second research goal is to generate mouse models for cardiac arrhythmias. Most recently, we have generated mouse models for Calsequestrin2 deficiency. We demonstrated that calsequestrin2 is not essential for cardiac calcium ion storage. 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 two years, we have demonstrated that mouse arrhythmias associated with calsequestrin2-deficiency worsen significantly with age. This age-dependent increase in cardiac phenotypes had already been known to occur in humans. We are now completing genomic analyses to identify genes and pathways that are dysregulated specifically in older mice where arrhythmia phenotypes are strongest. We have recently completed analyses of conditional alleles of calsequestrin 2. Casq2-deficient mice closely phenocopy the human disease. That is mice show normal heart function (but reduced heart rate) under basal conditions but develop polymorphic ventricular tachycardia (CPVT) in response to stress. Phenotypic analyses of these mice show that the CPVT phenotype is independent of developmental history. That is, the presence of arrhythmia depends on the status of Casq2 at the time of analysis with minimal influence by the hearts developmental history in regard to Casq2 gene function. Moreover, our data indicate that CPVT phenotype is dependent upon concurrent loss of Casq2 peptide in both the cardiac conduction system (CCS) and in working cardiomyocytes. The practical significance of this finding is that therapies that rescue Casq2 only in the CCS may be sufficient to prevent CPVT. In contrast to the CPVT phenotype, heart rate phenotypes are dependent only on the loss of Casq2 in the CCS. More interestingly, heart rates are dependent upon CCS developmental history. That is, heart rates are determined by two factors: 1) the status of Casq2 at the time of analysis and 2) CCS developmental history in regard to Casq2 gene function. Altogether, our data indicate that the relationship between heart rate and CPVT is complex but support the idea that reduced basal heart rate is a central contributor to increased risk of stress induced arrhythmias in Calsequestrin-deficient hearts.

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保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。尤其是，我们专注于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）和威尔姆斯肿瘤有关。 H19和IGF2的表达都取决于两个基因下游的一组共享的增强子元素。我们已经确定了H19启动子上游的2.4 kb ICR（用于印记控制区域）。使用条件缺失和插入诱变，我们确定了与该元素相关的三个函数。首先，该元素的作用是区分其插入的任何染色体的父母来源。具体而言，该区域内的CP​​G在父亲遗传下变得高甲基化。其次，该元素充当CTCF依赖性的，甲基化敏感的转录绝缘子。通过重新组织附近启动子和增强子元素的远距离相互作用，该绝缘子能够引导附近基因的特定于父母的激活。最后，当遗传遗传时，该ICR也充当了发育调节的消音器元素。具体而言，甲基化的ICR诱导影响基因表达的相邻序列的染色质结构的变化。我们目前的目标是识别和表征与ICR相互作用的蛋白质因子和非编码RNA，并建立与母体和父亲染色体相关的染色质结构。我们都在生殖细胞（建立烙印）以及IGF2和H19表达对正常健康细胞功能最重要的体细胞组织中解决这些问题。 我们还在努力建立模仿Wiedemann综合征表型与人类IGF2/H19基因座的遗传相关的小鼠模型。最近，我们证明了肌肉细胞分化和肌肉再生的缺陷，其中IGF2/H19印迹破坏了。我们已经证明，即使IGF2表达的增加<2倍也将通过MAPK途径的过度激活而导致细胞周期调节的大规模破坏。此外，H19的表达降低会破坏肌肉细胞中p53的正常调节，因此它们无法再对Wnt刺激做出反应，因此不经历正常的肥大。因此，H19和IGF2基因的印迹丧失与BWS中的过度生长表型有关 我们现在正在表征这些突变动物中心脏功能障碍表型。在早期发育过程中，IGF2的额外表达会导致生理肥大。但是，肥大会在出生后（IGF2表达停止）减少，并且没有长期的健康后果。然而，H19 lncRNA的丧失导致病理肥大和心脏心脏发展的心脏功能降低。 遗传分析表明，H19防止了间充质转变过早内皮。在没有H19的情况下，内皮细胞错误表达的间充质标记和成年小鼠显示出明显的纤维化。 使用CRISPR-CAS9技术，我们产生了新型的小鼠菌株，这些小鼠菌株在特定的H19域中携带突变。 这些分析表明，与LET7 microRNA相互作用的H19序列对于预防心脏纤维化和功能缺陷是必要的。 最后，使用体外模型，我们了解到，siRNA或通过遗传消融对H19的突然耗尽会导致细胞衰老的快速发作。 H19 lncRNA直接与p21 mRNA相互作用，并破坏mRNA稳定并抑制蛋白质翻译。 损失H19后，P21 mRNA和肽水平迅速升高以诱导衰老。 第二个研究目标是为心律不齐而产生小鼠模型。最近，我们生成了鼠标模型以用于CalSequestrin2缺乏症。我们证明了CalSequestrin2对于心脏钙离子储存并不是必需的。相反，在β-肾上腺素能刺激的条件下，CALSequeStrin的主要功能似乎是SR钙离子释放通道的调节。因此，Cal -sequestrin2的损失导致SR过早钙离子释放，导致电压变化导致心肌细胞过早收缩，从而导致心律不齐。最近，通过证明我们用来成功改善小鼠心律不齐的药物在对人类患者的试验研究中非常有效，这是该小鼠模型的有效性。 在过去的两年中，我们证明了与CalSequestrin2缺乏症相关的小鼠心律不齐随着年龄的增长而发生明显恶化。 人们已经知道，这种依赖年龄的心脏表型的增加发生在人类中。现在，我们正在完成基因组分析，以鉴定在心律不齐表型最强的老鼠中特异性失调的基因和途径。 我们最近完成了对CALSequestrin 2的条件等位基因2的分析。CASQ2缺陷型小鼠与人类疾病紧密相关。那就是小鼠在基础条件下表现出正常的心脏功能（但心率降低），但会响应压力而产生多态性心室心动过速（CPVT）。这些小鼠的表型分析表明，CPVT表型与发育史无关。也就是说，心律不齐的存在取决于在分析时Casq2的状态，而对Casq2基因功能，心脏发育史的影响很小。此外，我们的数据表明，CPVT表型取决于心脏传导系统（CCS）和工作心肌细胞中CASQ2肽的同时损失。这一发现的实际意义在于，仅在CCS中拯救CASQ2的疗法可能足以防止CPVT。与CPVT表型相反，心率表型仅取决于CCS中CASQ2的丢失。更有趣的是，心率取决于CCS的发育历史。也就是说，心率取决于两个因素：1）CASQ2在分析时的状态和2）CCS发育历史在CASQ2基因功能方面。总的来说，我们的数据表明心率与CPVT之间的关系很复杂，但支持这样的想法：减少基础心率是增加压力引起的心律失常的核心因素。