Regulated expression and developmental functions of the H19 long noncoding RNA

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发育障碍（包括贝克威斯·维德曼综合征（BWS）和拉塞尔·西尔弗综合征（RSS））、儿童癌症（包括肾母细胞瘤和横纹肌瘤）、心肌病以及许多成人癌症有关。 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以及位于 H19 启动子上游的 2.4 kb 印记控制区 (ICR)。使用条件删除和插入诱变，我们已经确定了与 ICR 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，该绝缘体可以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 会引起邻近序列染色质结构的变化，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 Beckwith Wiedemann 综合征表型的小鼠模型，该综合征与人类 Igf2/H19 基因座母体印记缺失相关。我们已经证明了 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 最近，我们对这些母性印记缺失小鼠的心脏功能障碍表型进行了表征。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。 H19 基因的主要产物是 2.2 kb 长的非编码 RNA (lncRNA)。我们研究的首要目标是了解这种 RNA 的分子和生化功能。 使用体外模型，我们发现 H19 RNA 通过与 p21 mRNA 分子的物理相互作用来介导细胞应激反应，从而调节 p21 稳定性和翻译效率。 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。 除了我们关于基因组印记的工作外，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。 在过去的几年中，我们已经证明，与 calsequestrin2 缺乏相关的小鼠心律失常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。

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.