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: 8941473
• 负责人: Karl Eric Pfeifer
• 金额: $ 103.44万
• 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/8941473
• 关键词: 11p15.5; 7-dehydrocholesterol reductase; Ablation; Address; Adrenergic Agents; Affect; Age; Alleles; Animals; Area; Arrhythmia; Beckwith-Wiedemann Syndrome; Behavior; Biological Models; Biological Process; Calcium ion; Calsequestrin; Cardiac; Cardiac Myocytes; Cell Differentiation process; Cell Nucleus; Cell physiology; Cells; Cholesterol; Chromatin Structure; Chromosomes; Chromosomes, Human, Pair 7; Collaborations; Coupling; DNA Modification Process; Defect; Deletion Mutagenesis; Development; Developmental Process; Disease; Disease Progression; Disease model; Distal; Drug usage; Elements; Epigenetic Process; Fathers; Fibroblasts; Functional RNA; 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; Heart; Human; In Vitro; Inherited; Insertional Mutagenesis; Late Effects; Mammals; Metabolism; Methylation; Modeling; Molecular; Mothers; Mus; Muscle Cells; Mutation; National Institute of Child Health and Human Development; Nephroblastoma; Parents; Pathway interactions; Patients; Pattern; Phenocopy; Phenotype; Pilot Projects; Proteins; Regulation; Research; Role; Sarcoplasmic Reticulum; Smith-Lemli-Opitz Syndrome; Structure; Surface; Therapeutic Intervention; Tissues; Transcriptional Silencer Elements; Work; adrenergic; base; cancer type; cell growth; cholesterol biosynthesis; clinically significant; coping mechanism; developmental disease; disease phenotype; enzyme activity; epigenome; flexibility; gene function; human disease; imprint; induced pluripotent stem cell; mature animal; mouse model; muscle regeneration; mutant; novel; premature; prevent; programs; promoter; relating to nervous system; research study; restoration; small molecule; stem cell technology; therapeutic target; transcription factor; transcriptome sequencing; 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. Finally, 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. Through RNA-seq experiments we are characterizing the molecular pathways downstream of the imprinting defect that are responsible for the disease phenotye. 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 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 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. Finally, to make full use of our expertise in stem cell technologies, we recently began a collaboration with FD Porter, also at the NICHD, to establish and characterize iPSC cells established using fibroblasts isolated from patients carrying mutations in the gene encoding 7-dehydrocholesterol reductase (DHCR7). Mutations in DHCR7 are associated with Smith-Lemli-Opitz syndrome (SLOS). Disruption in DHCR7 enzyme activity prevents the final steps in cholesterol biosynthesis and therefore leads to decreased cholesterol levels as well as the accumulation of cholesterol precursors. In the presence of exogenous cholesterol, wild type and DHCR7-mutant iPSCs are not distinguishable in terms of their in vitro differentiation capabilities. However, in the absence of added cholesterol, mutant cells show high levels of spontaneous differentiation to neural progeny. Rescue of the differentiation defect occurs not only with adding cholesterol but with several small molecules that alter the Wnt pathway.

印记代表了对正常孟德尔遗传学的一种奇怪的蔑视。哺乳动物继承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本和父本等位基因中同等表达。然而，印记基因仅从一条染色体以依赖于亲本的方式表达。由于沉默启动子和活性启动子存在于单个核中，因此活性差异不能用转录因子丰度来解释。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，印记基因是了解 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，由于亲本表达受到限制，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，而且还容易受到控制调节的表观遗传程序的破坏。因此，印记基因经常与人类疾病相关，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 11p15.5 号染色体上的同线性区域在基因组组织和单等位基因表达模式中是保守的。我们特别关注 H19 基因母本特异性表达和 Igf2 基因父本特异性表达的分子基础。这两个基因中印记突变的缺失与贝克威斯·维德曼综合征 (BWS) 和肾母细胞瘤有关。 H19 和 Igf2 的表达取决于两个基因下游的一组共享增强子元件。 我们在 H19 启动子上游鉴定了一个 2.4 kb ICR（印记控制区）。 使用条件删除和插入诱变，我们已经确定了与该元件相关的三个功能。 首先，该元件的作用是区分其所插入的任何染色体的亲本起源。 具体来说，该区域内的 CpG 在父系遗传后变得高度甲基化。 其次，该元件作为 CTCF 依赖性、甲基化敏感的转录绝缘子发挥作用。 通过重组附近启动子和增强子元件的长程相互作用，该绝缘体能够指导附近基因的亲本特异性激活。 最后，当父系遗传时，该 ICR 还充当发育调节的沉默元件。 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。 我们当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，并建立与母本和父本染色体相关的染色质结构。我们正在解决生殖细胞和体细胞中的这些问题，其中 Igf2 和 H19 的表达对于正常、健康的细胞功能至关重要。 最后，我们还致力于建立模拟与人类印记丧失相关的疾病表型的小鼠模型。 最近，我们证明了 Igf2/H19 印记被破坏的细胞中肌肉细胞分化和肌肉再生的缺陷。 通过 RNA-seq 实验，我们正在表征导致疾病表型的印记缺陷下游的分子途径。 第二个研究目标是生成心律失常的小鼠模型。 我们首先关注于揭示位于 Igf2 上游的印记 Kcnq1 基因的生物学功能。 最近，我们建立了 Calsequestrin2 缺乏症小鼠模型。 我们证明了 calsequestrin2 对于心脏钙离子储存不是必需的，可以通过扩大肌浆网 (SR) 体积和表面积来维持。 相反，calsequestrin 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，calsequestrin2 的损失会导致钙离子从 SR 中过早释放，从而导致电压变化，导致心肌细胞过早收缩，从而导致心律失常。 最近通过证明我们用来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，证实了该小鼠模型的有效性。 在过去的一年中，我们已经证明，与 calsequestrin2 缺乏相关的心律失常会随着年龄的增长而显着恶化。 我们最近生成并正在分析 calsequestrin 2 的条件等位基因。使用这些模型，我们分析了 calsequestrin 2 基因功能迟发性丧失的影响，从而模拟了一种常见的人类状况。 我们的结果表明，与发育后期基因功能丧失相关的表型要严重得多。因此，我们相信发育中的心脏具有应对 Ca++ 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来识别这些机制，然后评估这些机制是否代表治疗靶点。我们现在还正在确定恢复 calsequestrin 2 基因功能对在缺乏任何活性 calsequestrin 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-收缩耦合缺陷的人类患者开发新疗法。 最后，为了充分利用我们在干细胞技术方面的专业知识，我们最近开始与同样在 NICHD 的 FD Porter 合作，建立和表征 iPSC 细胞，这些细胞是使用从携带 7-脱氢胆固醇还原酶编码基因突变的患者中分离出的成纤维细胞建立的（DHCR7）。 DHCR7 突变与 Smith-Lemli-Opitz 综合征 (SLOS) 相关。 DHCR7 酶活性的破坏会阻止胆固醇生物合成的最后步骤，从而导致胆固醇水平降低以及胆固醇前体的积累。 在外源胆固醇存在的情况下，野生型和 DHCR7 突变型 iPSC 的体外分化能力没有区别。然而，在没有添加胆固醇的情况下，突变细胞表现出高水平的自发分化为神经后代。 挽救分化缺陷不仅需要添加胆固醇，还需要一些改变 Wnt 通路的小分子。