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.

Project Start
Project End
Budget Start
Budget End
Support Year
17
Fiscal Year
2012
Total Cost
$924,196
Indirect Cost
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Gebert, Claudia; Correia, Lauren; Li, Zhenhu et al. (2017) Chromosome choice for initiation of V-(D)-J recombination is not governed by genomic imprinting. Immunol Cell Biol 95:473-477
Park, Ki-Sun; Pfeifer, Karl (2016) Making choices-how stochastic decisions determine disease progression. Genes Dev 30:485-6
Ray, Payal; De, Sandip; Mitra, Apratim et al. (2016) Combgap contributes to recruitment of Polycomb group proteins in Drosophila. Proc Natl Acad Sci U S A 113:3826-31
Gebert, Claudia; Rong, Qi; Jeong, Sangkyun et al. (2016) H19ICR mediated transcriptional silencing does not require target promoter methylation. Biochem Biophys Res Commun 476:121-6
De, Sandip; Mitra, Apratim; Cheng, Yuzhong et al. (2016) Formation of a Polycomb-Domain in the Absence of Strong Polycomb Response Elements. PLoS Genet 12:e1006200
Leikina, Evgenia; Defour, Aurelia; Melikov, Kamran et al. (2015) Annexin A1 Deficiency does not Affect Myofiber Repair but Delays Regeneration of Injured Muscles. Sci Rep 5:18246
Dey, Bijan K; Pfeifer, Karl; Dutta, Anindya (2014) The H19 long noncoding RNA gives rise to microRNAs miR-675-3p and miR-675-5p to promote skeletal muscle differentiation and regeneration. Genes Dev 28:491-501
Leikina, Evgenia; Melikov, Kamran; Sanyal, Sarmistha et al. (2013) Extracellular annexins and dynamin are important for sequential steps in myoblast fusion. J Cell Biol 200:109-23
Eun, Bokkee; Sampley, Megan L; Good, Austin L et al. (2013) Promoter cross-talk via a shared enhancer explains paternally biased expression of Nctc1 at the Igf2/H19/Nctc1 imprinted locus. Nucleic Acids Res 41:817-26
Xia, Jixiang; Varudkar, Namita; Baker, Candice N et al. (2013) Targeting of the enhanced green fluorescent protein reporter to adrenergic cells in mice. Mol Biotechnol 54:350-60

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