Epigenetic mechanisms regulating the Igf2/H19 and Kcnq1 locus

调节 Igf2/H19 和 Kcnq1 位点的表观遗传机制

基本信息

批准号：
9339250
负责人：
Karl Eric Pfeifer
金额：
$ 104.76万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/9339250
关键词：
11p15.5 Ablation Address Adrenergic Agents Affect Age Alleles Animals Area Arrhythmia Beckwith-Wiedemann Syndrome Behavior Binding Sites Biochemical Biochemistry Biological Models Biological Process Biotin Calcium ion Calsequestrin Cardiac Cardiac Myocytes Cell Cycle Regulation Cell Differentiation process Cell Nucleus Cell physiology Cells Chromatin Structure Chromosomes Chromosomes, Human, Pair 7 Code Coupling DNA Modification Process Defect Deletion Mutagenesis Development Developmental Process Disease Disease Progression Disease model Distal Drug usage Elements Epigenetic Process Fathers Gene Cluster Gene Expression Gene Expression Profile Gene Mutation Genes Genetic Genetic Enhancer Element Genetic Transcription Genomic approach Genomics Germ Cells Goals Growth and Development function H19 RNA H19 gene Heart Human Hypertrophy Inherited Insertional Mutagenesis Late Effects MAP Kinase Gene Mammals Metabolism Methylation MicroRNAs Mitogens Modeling Molecular Mothers Mus Muscle Cells Mutation Myocardial dysfunction Nephroblastoma Parents Pathway interactions Patients Pattern Peptides Phenocopy Phenotype Pilot Projects Play Proteins Regulation Research Role Sarcoplasmic Reticulum Skeletal Muscle Structure Surface Therapeutic Intervention Tissues Transcriptional Silencer Elements Untranslated RNA Work aptamer base cancer type cell growth clinically significant coping mechanism developmental disease disease phenotype epigenome flexibility gene function human disease imprint mature animal mouse model muscle regeneration mutant novel novel therapeutics premature prevent programs promoter research study restoration therapeutic target tool transcription factor transcriptome sequencing voltage

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 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 diseases phenotypes associated with loss of imprinting 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 prevents muscle cells from responding to Wnt stimulation and thus prevents normal hypertrophy. We are now characterizing cardiac dysfunction phenotypes in these mutant animals. Through RNA-seq experiments we are characterizing the molecular pathways downstream of the imprinting defect that are responsible for the disease phenotypes. Igf2 encodes a peptide mitogen whose biochemistry is well understood. However, H19 encodes a long non-coding RNA and its biochemical roles remain unclear. We are generating novel mouse models with small mutations in the H19 RNA coding regions that delete putative miRNA encoding sequences and also putative let7 miRNA binding sites and will determine if these sequences play any role in the H19 functions described above. We are also generating a novel mouse model where H19 RNA is tagged with an aptamer that mimics biotin. We will use this tool to identify proteins that interact with H19. A second research goal 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 mouse models for Calsequestrin2 deficiency. We demonstrated 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 significantly 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 believe that 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 mechanisms 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.

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。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CPG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。我们还在努力建立与人类失去印迹相关的模仿表型的小鼠模型。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。另外，H19的表达降低可防止肌肉细胞对Wnt刺激的反应，从而防止正常肥大。我们现在正在表征这些突变动物中心脏功能障碍表型。通过RNA-Seq实验，我们表征了负责疾病表型的印记缺陷下游的分子途径。 IGF2编码一种肽有丝分裂原，其生物化学已被充分理解。但是，H19编码长的非编码RNA，其生化作用尚不清楚。我们正在H19 RNA编码区域中生成具有小突变的新型小鼠模型，这些区域删除了推定的miRNA编码序列，并推定了LET7 miRNA结合位点，并将确定这些序列是否在上述H19函数中起任何作用。我们还生成了一种新型的小鼠模型，其中H19 RNA用模仿生物素的适体标记。我们将使用此工具来识别与H19相互作用的蛋白质。第二个研究目标是为心律不齐而产生小鼠模型。我们首先专注于发现位于IGF2上游的印迹KCNQ1基因的生物学功能。最近，我们生成了鼠标模型，以实现CalSequeStrin2缺乏症。我们证明了CalSequestrin2对于心脏钙离子储存不是必不可少的，可以通过肌浆网（SR）体积和表面积的扩展来维持。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。在过去的一年中，我们证明了与CalSequestrin2缺乏症相关的心律不齐随着年龄的增长而显着恶化。我们最近已经生成，现在正在分析CalSequestrin 2的条件等位基因。使用这些模型，我们分析了CalSequestin 2基因功能的后期丧失损失的效果，从而建模了共同的人类条件。我们的结果表明，与基因功能在发育后期的丧失相关的表型要严重得多。因此，我们认为，发展的心脏具有应对可以永久保护心脏的CA ++代谢异常调节的机制。我们正在启动将识别这些机制的基因组方法，然后评估这些机制是否代表治疗靶标。现在，我们还确定了在没有任何活跃的CALSequeStrin 2基因的情况下恢复CALSequeStrin 2基因功能对动物的恢复的影响。这些实验共同有助于我们了解CALSequesin 2基因活性如何调节肌质网的结构，还可以帮助我们为在CA ++激发促进偶联的先天性和获得性缺陷的人类患者开发新的疗法。