Genomic imprinting is an unusual form of gene regulation in which expression of an allele is restricted in accordance with 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 localized on the distal end of mouse chromosome 7. The syntenic region in humans (11p15.5) is highly conserved in gene organization and expression patterns. Mutations disrupting the normal patterns of imprinting at the human locus are associated with the Beckwith Wiedemann syndrome, a developmental disorder, and with many types of tumors. In addition, inherited cardiac arrhythmia is associated with mutations in the maternal-specific Kcnq1gene. We use mouse models to address the molecular basis for allele-specific expression in the syntenic region and, in our studies, hope to use imprinting as a tool by 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 syntenic region. We have generated models to study defects in cardiac repolarization associated with loss-of-function mutations at Kcnq1and specifically to understand the effect of beta-adrenergic–mediated stress on the cardiac phenotype.
Molecular basis for allele-specific expression of the mouse H19and Igf2genes
Jeong, Rong, Yoon, Miller, Ettensohn, Pfeifer
Our studies on the mechanisms of genomic imprinting focus on the H19and Igf2genes, which lie at one end of the distal 7 imprinted cluster. Paternally expressed Igf2lies about 70 kb upstream of the maternal-specific H19gene. Using cell culture systems as well as transgene and knockout experiments in vivo, we identified the enhancer elements responsible for activation of the two genes and determined that the elements are largely shared and located downstream of the H19gene. Parent-of-origin–specific expression of both genes is dependent on a shared element (called the H19DMR) located just upstream of the H19promoter and thus juxtaposed between the Igf2gene and the shared enhancers. The CpG sequences within the enhancer element are methylated specifically on the paternally inherited chromosome. Our conditional ablation of the element in vivo demonstrates that the nonmethyated H19DMR(i.e., the copy on the maternal chromosome) is continually required for silencing of the maternal Igf2allele. Knockin experiments demonstrate that the H19DMRcontains a methylation-sensitive transcriptional insulator. Thus, on the nonmethylated maternal chromosome, the active insulator within the H19DMRprevents activation of Igf2by the downstream enhancers. Methylation of the paternal chromosome inactivates the insulator and permits Igf2expression. Still unexplained by our model is the effect of several small DMRsproximal to the Igf2transcription unit. We are currently investigating the mechanistic significance of these elements and have so far shown that expression of Igf2does not correlate with methylation of these sequences. Imprinting of H19occurs by a distinct genetic mechanism. The conditional ablation of the H19DMRindicates that it is not continuously required for silencing the paternal allele. Rather, the H19DMRis required early in development to establish an epigenetic state at the H19promoter that itself prevents transcription. Current studies suggest that the epigenetic program includes but is not solely the hypermethylation of the H19promoter.
To determine the elements necessary and sufficient for imprinting at the locus, we moved the H19DMRand its mutated derivatives to heterologous loci. Our results demonstrate that the DMRalone is sufficient to imprint a normally non-imprinted chromosome. Moreover, such imprinting is not dependent on germline differences in DMRmethylation. Thus, the DMRlikely marks its parental origin by a mechanism independent of DNA methylation. By using genetic and molecular analyses of embryonic stem cells derived from mutant mice, we are now determining the epigenetic signals that constitute the genomic imprint.
We are also continuing a series of experiments to understand the molecular mechanisms by which the H19DMRcan act as a transcriptional insulator. Several groups have demonstrated the presence of four CTCF binding sites within the H19DMR. CTCF is a DNA-binding protein previously demonstrated to interact with the chicken beta-globin insulator. The ability of CTCF to recognize DNA is methylation-sensitive, that is, CTCF cannot bind to the methylated paternally inherited DMR,thus explaining the activation of the paternal Igf2allele. To understand the molecular basis for insulator function, we have begun a series of experiments to characterize the three-dimensional organization of the Igf2/H19locus, comparing maternal and paternal and wild-type and mutant chromosomes. Specifically, we are examining the long-range interactions between the Igf2and H19promoters and the shared enhancer elements and the effect of the presence of a working insulator on these interactions. The experiments suggest that insulators act by competing with enhancer elements to interact with the cognate promoters. We are also identifying interactions between the H19DMR and these promoter and enhancer sequences.
Jeong SY, Pfeifer K. Shifting insulator boundaries. Nat Genet2004;36:1036-1037.
Park KY, Pfeifer K. Epigenetic interplay. Nat Genet2003;34:126-128.
Park KY, Sellars EA, Grinberg A, Huang SP, Pfeifer K. The H19DMRmarks the parental origin of a heterologous locus without gametic DNA methylation. Mol Cell Biol2004;24:3588-3595.
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/H19locus. J Biol Chem2003;278:5977-5983.
Mouse models for inherited long QT syndrome
Rong, Pfeifer; in collaboration with Ebert, Knollman
Inherited long QT syndrome (LQTS) is characterized by an abnormal electrocardiogram indicative of repolarization defects; it can result in syncope or sudden death. Romano-Ward syndrome (RWS) patients inherit the LQTS disorder generally as a dominant phenotype and show no other traits. Jervell and Lange-Nielsen syndrome patients display profound congenital deafness in addition to LQTS. Both phenotypes are recessive. We have generated several mutations in the mouse Kcnq1gene to model the human diseases. Ablation of the gene results in vestibular and auditory defects. Histological analyses suggest that the defects are attributable to deficiency in the K+ recycling pathway that is crucial for generating endolymph, the specialized fluid bathing the inner hair cells. When measured in vivo, ECG tracings of mutant mice indicate profound defects in cardiac repolarization. However, the defects are not noted in isolated hearts ex vivo, indicating that the Kcnq1 protein plays an important 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 and have analyzed mutations in the central pore region and in the sixth membrane-spanning domain. The phenotypes of the 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, our genetic models represent excellent paradigms for addressing mechanisms for acquired LQTS, the single greatest 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 in the formation of the IKS potassium channel. A novel result of our studies is the discovery that that ablation of the Kcnq1gene leads to cardiac defects in addition to those noted in Kcne1-deficient mice. The results demonstrate 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 is dependent on Kcnq1 but not Kcne1. The role of the channel in mouse and human hearts is now under investigation.
Casimiro MC, Knollman BC, Yamoah EN, Nie L, Vary JC, Sirenko SG, Greene AE, Grinberg A, Huang SP, Ebert SN, Pfeifer K. Targeted point mutagenesis of mouse Kcnq1: phenotypic analysis of mice with point mutations that cause Romano-Ward syndrome in humans. Genomics 2004;84:555.
Knollman BC, Casimiro MC, Katchman AN, Sirenko SG, Schober T, Rong Q, Pfeifer K, Ebert SN. Isoproterenol exacerbates a long QT phenotype in Kcnq1-deficient mice: possible roles for human-like isoform 1 and slow delayed rectifier K+ current. J Pharmacol Exp Ther 2004;310:311-318.
Beta-adrenergic synthesizing cells and development of the cardiac conduction system
Rong, Pfeifer; in collaboration with Ebert
During early development, the heart is the primary (and probably only) site of synthesis of the beta-adrenergic hormones norepinephrine and epinephrine. Such 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 the hormones, we generated a mouse with a mutated Pnmtlocus such that that Cre recombinase enzyme is synthesized in any cell normally making epinephrine. (Pnmtencodes phenylethanolamine N-methyltransferase, the enzyme that converts norepinephrine to epinephrine.) When crossed with appropriate tester strains, Pnmt-expressing cells and their descendants become positive for beta-galactosidase and thus readily identifiable and relatively easy to isolate. Our analyses indicate that epinephrine is synthesized by cells that indeed give rise to cardiac conduction cells. However, our analyses suggest that the intrinsic cardiac adrenergic cells represent a stem cell population that contributes extensively to fetal cardiac development. Thus, Pnmtexpression represents an excellent marker for potential cardiac stem cells. We recently generated a new mouse model in which the green fluorescent protein is expressed specifically in intrinsic cardiac adrenergic cells in the heart. We have purified cells from fetal mouse heart and are characterizing their ability to act as cardiac stem cells in vitro and in vivo.
Ebert SN, Rong Q, Boe S, Thompson RP, Grinberg A, Pfeifer K. Targeted insertion of the Cre-recombinase gene at the phenylethanolamine n-methyl transferase locus: a new model for studying the developmental distribution of adrenergic cells. Dev Dyn2004;231:849-858.
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 2004;1018:418-423.
Collaborators
Steven Ebert, PhD, University of Central Florida, Orlando, FL
Bjorn Knollman, MD, PhD, Georgetown University Medical Center, Washington, DC
For further information, contact kpfeifer@helix.nih.gov.