MOLECULAR GENETICS OF AN IMPRINTED GENE CLUSTER ON
MOUSE DISTAL CHROMOSOME 7
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Karl Pfeifer, PhD, Head, Section on Genomic Imprinting Sangkyun Jeong, PhD, Visiting Fellow Kye Yoon Park, PhD, Research Fellow Elizabeth Sellars, BS, Postbaccalaureate Fellow |
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| Genomic imprinting is an unusual form of gene regulation in which expression of an allele is restricted based on its parental origin. Imprinted genes are not randomly scattered throughout the chromosome but rather are localized in discrete clusters. One cluster of imprinted genes is located on the distal end of mouse chromosome 7. The syntenic region in humans (11p15.5) is highly conserved in gene organization and in expression patterns. Mutations disrupting the normal patterns of imprinting at the human locus are associated with Beckwith Wiedemann syndrome, a developmental disorder exhibiting many types of tumor. In addition, inherited cardiac arrhythmia is associated with mutations in the maternal-specific Kcnq1 gene. Our unit uses mouse models to address the molecular basis for allele-specic expression in the cluster. In our studies, we hope to use imprinting as a tool with which to understand fundamental features of epigenetic regulation of gene expression. We are also using the mouse system to generate animal models for the several inherited disorders associated with the cluster. We have generated models to study defects in cardiac repolarization associated with loss-of-function mutations at Kcnq1 and to understand specifically the effect of beta-adrenergic-mediated stress on the cardiac phenotype. Molecular basis for allele-specic expression of the mouse H19 and Igf2 genes Park, Jeong, Sellars, Pfeifer Our studies on the mechanisms of genomic imprinting focus on the H19 and Igf2 genes, which lie at one end of the distal 7 imprinted cluster. Paternally expressed Igf2 lies about 70 kb upstream of the maternal-specific H19 gene. Using cell culture systems as well as transgene and knockout experiments in vivo, we have identified the enhancer elements responsible for activation of these two genes. They are largely shared and located downstream of the H19 gene. Parent-of-origin-specific expression of both genes depends on a shared element (called the H19DMR) located just upstream of the H19 promoter and thus positioned between the Igf2 gene and the shared enhancers. The CpG sequences within this element are methylated specifically on the paternally inherited chromosome. Our conditional ablation of the element in vivo demonstrates that the nonmethylated H19DMR (i.e., the copy on the maternal chromosome) is continually required for silencing of the maternal Igf2 allele. Knock-in experiments demonstrate that the H19DMR contains a methylation-sensitive transcriptional insulator. Thus, on the nonmethylated maternal chromosome, the active insulator within the H19DMR prevents activation of Igf2 by the downsteam enhancers. Methylation of the paternal chromosome inactivates the insulator and permits Igf2 expression. Unexplained by this model is the effect of several small DMRs proximal to the Igf2 transcription unit. Current studies are directed at the mechanistic significance of these elements. Imprinting of H19 occurs via a distinct genetic mechanism. The conditional ablation of the H19DMR indicates that it is not continuously required to silence the paternal allele. Rather, the H19DMR is required early in development to establish an epigenetic state at the H19 promoter that itself prevents transcription. Current studies indicate that the epigenetic program includes, but is not restricted to, the hypermethylation of the H19 promoter. To determine the elements that are necessary and sufficient for imprinting at the locus, we have moved the H19DMR and its mutated derivatives to heterologous loci. Our results demonstrate that the DMR alone is sufficient to imprint a normally nonimprinted chromosome. Moreover, this activity does not depend on germline differences in DMR methylation. Thus, the DMR likely marks its parental origin by a mechanism independent of DNA methylation. With genetic and molecular analyses of embryonic stem cells derived from mutant mice, we are now determining the epigenetic signals that constitute the genomic imprint. Kaffer CR, Grinberg A, Pfeifer K. Regulatory mechanisms at the mouse Igf2/H19 locus. Mol Cell Biol 2001;21:8189-8196. Kaffer CR, Srivastava M, Park KY, Ives E, Hsieh S, Batlle J, Grinberg A, Huang SP, Pfeifer K. A transcriptional insulator at the H19/Igf2 locus. Genes Dev 2000;14:1908-1919. Park KY, Pfeifer K. Epigenetic interplay. Nat Genet 2003;34:126-128. Srivastava M, Frolova E, Rottinghaus B, Boe SP, Grinberg A, Lee E, Love PE, Pfeifer K. Imprint control element mediated secondary methylation imprints at the Igf2/H19 locus. J Biol Chem 2003;278:5977-5983. Srivastava M, Hsieh S, Grinberg A, Williams-Simon L, Huang SP, Pfeifer K. H19 and Igf2 monoallelic gene expression is regulated in two distinct ways by a shared cis acting regulatory region upstream of H19. Genes Dev 2000;14:1186-1195. Mouse models for inherited long QT syndrome Casimiro, Pfeifer; in collaboration with Ebert, Knollman Inherited long QT syndrome (LQTS), which can result in syncope or sudden death, is characterized by an abnormal electrocardiogram indicative of repolarization defects. Romano-Ward syndrome (RWS) patients inherit the LQTS disorder generally as a dominant phenotype and show no other traits. Jervell and Lange-Nielsen syndrome (JLNS) patients display profound congenital deafness in addition to LQTS, with both phenotypes recessive. We have generated several mutations in the mouse Kcnq1 gene to model the human diseases. Ablation of the gene results in vestibular and auditory defects. Histological analyses suggest that the defects are due to deficiency in the K+-recycling pathway that is crucial for generating endolymph, the specialized fluid bathing the inner hair cells. ECG tracings of mutant mice indicate profound defects in cardiac repolarization when measured in vivo. However, the defects are not noted in isolated hearts ex vivo, indicating that the Kcnq1 protein plays a pivotal role in mediating critical extracardiac signals. Further analyses demonstrate that Kcnq1 function is specifically required to modulate cardiac function in the presence of beta-adrenergic stimulation. We have also generated three point mutations to model RWS. We have analyzed mutations in the central pore region and in the sixth membrane-spanning domain of Kcnq1. The phenotypes of these mutations are each a distinct subset of those seen in the null mutation and thus demonstrate that the Kcnq1 protein plays distinct roles in the heart versus the inner ear and in various aspects of cardiac function. While inherited LQTS is relatively rare, the genetic models represent excellent paradigms for addressing mechanisms for acquired LQTS, the single largest cause of death in Western societies. Biochemical and pharmacological studies both predicted that the key biological role of the Kcnq1 protein was its association with the helper protein, Kcne1, to form the IKS potassium channel. One of the most novel results of our studies is the discovery that that ablation of the Kcnq1 gene leads to cardiac defects in addition to those noted in Kcne1-decient mice. The results suggest a novel role for Kcnq1 in heart development and/or function. We have used our mutant mice as tools to detect a previously unappreciated potassium channel that was dependent on Kcnq1 but not on Kcne1. The role of this channel in mouse and human hearts is now under investigation. Casimiro M, Knollman B, Ebert S, Vary JC, Huang SP, Grinberg A, Pfeifer K. Targeted disruption of the Kcnq1 gene produces a mouse model of Jervell and Lange-Nielsen syndrome. Proc Natl Acad Sci USA 2001;98:2526-2531. Tosaka, T, Casimiro MC, Rong Q, Tella S, Oh M, Katchman AN, Pezzullo JC, Pfeifer K, Ebert SN. Nicotine induces a long QT phenotype in Kcnq1-decient mouse hearts. J Pharmacol Exp Ther 2003,in press. Beta-adrenergic hormone-synthesizing cells and development of the cardiac conduction system Boe, Pfeifer During early development, the heart is the primary (and probably the only) site of synthesis of the beta-adrenergic hormones norepinephrine and epinephrine. This cardiac-specific synthesis is transient and disappears by late gestation. Intriguingly, the cells synthesizing the beta-adrenergic hormones are located in positions that predict the location of the developing cardiac conduction network. To understand the fate of cells that synthesize these hormones, we generated a mouse with a mutated Pnmt locus such that that cre recombinase enzyme is synthesized in any cell normally making epinephrine. (Pnmt encodes phenylethanolamine N-methyltransferase, the enzyme that converts norepinephrine to epinephrine.) When crossed with appropriate tester strains, Pnmt-expressing cells and their descendants become beta-galactosidase-positive and thus can be readily identified and isolated. Early analyses indicate that epinephrine is synthesized by cells that give rise specifically to cardiac conduction cells, albeit a subset of these cells. Ongoing experiments will characterize the cell types. Parenthetically, our studies have generated a mouse that produces no epinephrine but shows normal levels of norepinephrine, thus allowing the specific roles of the two hormones to be dissected. Pfeifer K, Boe SP, Rong Q, Ebert SN. Generating a mouse model for studying the function and fate of intrinsic cardiac adrenergic cells. Annals NY Acad Sci 2003,in press. COLLABORATORS Steven Ebert, PhD, Georgetown University Medical Center, Washington DC |
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