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.

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。 我们还致力于建立模拟与人类 Igf2/H19 基因座印记丢失相关的 Beckwith Wiedemann 综合征表型的小鼠模型。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。我们已经证明，即使 Igf2 表达增加 <2 倍，也会因 MAPK 通路的过度激活而导致细胞周期调节的大规模破坏。此外，H19表达的减少会扰乱肌肉细胞中p53的正常调节，使它们无法再对Wnt刺激做出反应，因此不会发生正常的肥大。因此，H19 和 Igf2 基因印记的丧失与 BWS 的过度生长表型相关 我们现在正在表征这些突变动物的心脏功能障碍表型。在早期发育过程中，Igf2 的额外表达会导致生理性肥大。然而，肥大在出生后（当 Igf2 表达停止时）会减少，并且不会对健康产生长期影响。然而，H19 lncRNA 的缺失会导致出生后心脏出现病理性肥大和心脏功能下降。 遗传分析表明 H19 可以防止过早的内皮细胞向间质细胞的转变。在缺乏 H19 的情况下，内皮细胞错误表达间充质标记物，成年小鼠表现出明显的纤维化。 利用 CRISPR-Cas9 技术，我们培育出了在特定 H19 结构域中携带突变的新型小鼠品系。 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，利用体外模型，我们了解到通过 siRNA 或基因消融突然耗尽 H19 会导致细胞快速衰老。 H19 lncRNA 直接与 p21 mRNA 相互作用，使 mRNA 不稳定并抑制蛋白质翻译。 H19 缺失后，p21 mRNA 和肽水平迅速升高，从而诱导衰老。 第二个研究目标是生成心律失常的小鼠模型。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并不是必需的。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。 在过去的两年中，我们已经证明，与 calsequestrin2 缺乏相关的小鼠心律失常会随着年龄的增长而显着恶化。 人们已经知道这种与年龄相关的心脏表型增加发生在人类身上。我们现在正在完成基因组分析，以识别心律失常表型最强的老年小鼠中特别失调的基因和通路。 我们最近完成了对 calsequestrin 2 条件等位基因的分析。Casq2 缺陷小鼠与人类疾病的表型密切相关。也就是说，小鼠在基础条件下表现出正常的心功能（但心率降低），但在应激反应中出现多形性室性心动过速（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，心律失常的存在取决于分析时 Casq2 的状态，在 Casq2 基因功能方面受心脏发育史的影响最小。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 肽的同时丢失。这一发现的实际意义在于，仅在 CCS 中挽救 Casq2 的疗法可能足以预防 CPVT。与 CPVT 表型相反，心率表型仅依赖于 CCS 中 Casq2 的丢失。更有趣的是，心率取决于 CCS 的发育史。也就是说，心率由两个因素决定：1) 分析时 Casq2 的状态；2) 关于 Casq2 基因功能的 CCS 发育历史。总而言之，我们的数据表明心率和 CPVT 之间的关系很复杂，但支持以下观点：基础心率降低是缺乏 Calsequestrin 的心脏中应激性心律失常风险增加的主要原因。