Regulated expression and developmental functions of the H19 long noncoding RNA

H19长非编码RNA的调控表达和发育功能

• 批准号: 10685191
• 负责人: Karl Eric Pfeifer
• 金额: $ 141.43万
• 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/10685191
• 关键词: 11p15.5; Ablation; Address; Adrenergic Agents; Adult; Affect; Age; Alleles; Animals; Arrhythmia; Beckwith-Wiedemann Syndrome; Behavior; Biochemical; Biological Models; Birth; CRISPR/Cas technology; Calcium ion; Calsequestrin; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell Cycle Regulation; Cell Differentiation process; Cell Nucleus; Cell physiology; Cells; Cellular Stress Response; Chromatin Structure; Chromosome 7; Chromosomes; DNA Modification Process; 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; Genomic Imprinting; Genomics; Germ Cells; Goals; Growth and Development function; H19 RNA; H19 gene; Health; Heart; Human; Hypertrophy; IGF2 gene; Inherited; Insertional Mutagenesis; MAP Kinase Gene; Malignant Childhood Neoplasm; Malignant Neoplasms; Mammals; Mediating; Mesenchymal; Messenger RNA; Methylation; MicroRNAs; Modeling; Molecular; Mothers; Mouse Strains; Mus; Muscle Cells; Mutation; Myocardial dysfunction; Nephroblastoma; Parents; Pathologic; Pathway interactions; Patients; Pattern; Phenocopy; Phenotype; Physiological; Pilot Projects; Proteins; RNA; Regulation; Research; Role; Silver; Skeletal Muscle; Stress; Syndrome; TP53 gene; Therapeutic Intervention; Tissues; Transcriptional Silencer Elements; Translations; Untranslated RNA; Work; 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; novel; postnatal; premature; prevent; programs; promoter; 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 developmental disorders (including Beckwith Wiedemann Syndrome (BWS) and Russell Silver Syndrome (RSS)), with pediatric cancers (including Wilms tumor and rhabdosarcoma), with cardiomyopathies, and with many adult cancers. Expression of both H19 and Igf2 is dependent upon a shared set of enhancer elements downstream of both genes and upon a 2.4 kb Imprinting Control Region (ICR) that lies just upstream of the H19 promoter. Using conditional deletion and insertional mutagenesis we have identified three functions associated with the ICR. 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 can 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 maternal loss of imprinting at the Igf2/H19 locus in humans. 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 More recently we have characterized cardiac dysfunction phenotypes in these maternal loss of imprinting mice. 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. The primary product of the H19 gene is a 2.2 kb long noncoding RNA (lncRNA). A top goal of our research tis to understand the molecular and biochemical functions of this RNA. Using in vitro models, we discovered a critical for H19 RNA in mediating cellular stress responses through physical interactions with p21 mRNA molecules that regulate p21s stability and translation efficiency. In brief summary, mice lacking H19 are more likely to respond to stress by activating senescence pathways. In addition to our work regarding genomic imprinting, a secondary 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 several 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.

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。在这两个基因中失去印迹突变的丧失与发育障碍有关（包括贝克维斯·威德曼综合征（BWS）和罗素银综合征（RSS）），儿科癌症（包括威尔姆斯肿瘤和色瘤），有心肌病，许多成人癌症。 H19和IGF2的表达都取决于基因下游的一组共享增强子元素以及位于H19启动子上游的2.4 Kb烙印控制区（ICR）。使用条件缺失和插入诱变，我们确定了与ICR相关的三个功能。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件的远距离相互作用，该绝缘子可以指导附近基因的父母特异性激活。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综合征表型的小鼠模型，与人类IGF2/H19基因座的孕产妇丧失印记相关。我们已经证明了肌肉细胞分化和肌肉再生的缺陷，其中IGF2/H19印迹中断了。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 最近，我们表征了这些印记小鼠的母体丧失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。 H19基因的主要产物是2.2 kb长的非编码RNA（LNCRNA）。我们研究的主要目标是了解该RNA的分子和生化功能。 使用体外模型，我们通过与调节P21稳定性和翻译效率的P21 mRNA分子的物理相互作用来介导细胞应激反应的H19 RNA至关重要。 简而言之，缺乏H19的小鼠更有可能通过激活衰老途径来应对压力。 除了我们在基因组印迹方面的工作外，二级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。 在过去的几年中，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。