Epigenetic mechanisms regulating the Igf2/H19 and Kcnq1 locus

印记代表了对正常孟德尔遗传学的一种奇怪的挑战。哺乳动物遗传了两组完整的染色体，一组来自母亲，另一组来自父亲，大多数常染色体基因将从母亲和父亲的等位基因中平等表达。然而，印记基因只在一条染色体上以亲本依赖的方式表达。因为沉默和活跃的启动子存在于单个细胞核中，所以活性的差异不能用转录因子的丰度来解释。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，印迹基因是理解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，由于亲本来源的限制表达，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，而且还会受到控制调控的表观遗传程序的破坏。因此，印记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。我们研究组正在研究小鼠7号染色体远端的一组基因。人类染色体11p15.5上的同线区域在基因组组织和单等位基因表达模式中是保守的。具体地说，我们正在剖析H19基因母系特异性表达和Igf2基因父系特异性表达的分子基础。这两个基因印记突变的丢失与Beckwith Wiedemann综合征(BWS)和Wilms肿瘤有关。H19和Igf2的表达依赖于这两个基因下游的一组共有的增强子元件。我们已经确定了H19启动子上游有一个2.4kb的ICR(印记控制区)。使用条件缺失和插入突变，我们已经确定了与该元件相关的三个功能。首先，这种元素的作用是区分它所插入的任何染色体的亲本来源。具体地说，该区域内的CPGS在父系遗传后变得高度甲基化。其次，该元件作为CTCF依赖的、甲基化敏感的转录绝缘体发挥作用。通过重组附近启动子和增强子元件的远程相互作用，这种绝缘体能够指导邻近基因的亲本特异性激活。最后，当父系遗传时，这种ICR也作为发育调节的消音器元件。具体地说，甲基化的ICR导致邻近序列染色质结构的变化，从而影响基因表达。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，并建立与母体和父体染色体相关的染色质结构。我们正在解决这些问题，既在生殖细胞中建立印记，也在体细胞组织中解决这些问题，在这些组织中，Igf2和H19的表达对正常、健康的细胞功能最为关键。 我们研究的第二个重点是建立心律失常的小鼠模型。我们首先专注于揭示印记的KCNQ1基因的生物学功能，该基因位于Igf2的上游。最近，我们建立了一个钙调素2缺乏症的小鼠模型。我们证明钙调蛋白2对心脏钙离子储存不是必需的，这可以通过扩大肌浆网(SR)的体积和表面积来维持。相反，钙调素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，钙调素2的缺失导致肌质网钙离子过早释放，导致电压变化，导致心肌细胞过早收缩，从而导致心律失常。这个小鼠模型的有效性最近得到了证实，我们用来成功改善小鼠心律失常的药物在对人类患者的初步研究中非常有效。在过去的一年里，我们已经证明，与钙调素2缺乏相关的心律失常随着年龄的增长而显著恶化。我们最近已经产生并正在分析Calequestrin 2的条件等位基因。使用这些模型，我们已经分析了Calequestrin 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情况。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，我们认为发育中的心脏具有应对钙代谢异常调节的机制，从而对心脏具有永久的保护作用。我们正在启动基因组方法，以确定这些机制，然后评估这些机制是否代表治疗靶点。我们现在还在确定在没有任何活性的calequestrin 2基因的情况下发育的动物恢复calequestrin 2基因功能的效果。综上所述，这些实验将帮助我们了解Calequestin 2基因活性如何调节肌浆网结构，并帮助我们开发针对先天性和获得性钙兴奋收缩偶联缺陷的人类患者的新疗法。

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 transcriptional 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. Specifically we are dissecting 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. A second focus of our research 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 a mouse model for Calsequestrin2 deficiency. We demonstrate 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 signficantly 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 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.