Karl Pfeifer, PhD, Head, Section on Genomic Imprinting
Claudia Gebert, PhD, Visiting Fellow
Sangkyun Jeong, PhD, Visiting Fellow
Qi Rong, MD, MS, Microbiologist
Kristen Ettensohn, BS, Student
John Van Druff, BS, Student
Young Soo Yoon, BS, Student

Genomic imprinting is an unusual form of gene regulation in which parental origin determines an allele's expression. 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 developmental disorders and many types of tumors. In addition, inherited cardiac arrhythmia is associated with mutations in the maternal-specific Kcnq1 gene. We use mouse models to address the molecular basis for allele-specific expression in this region of mouse chromosome 7. In these studies, we hope to use imprinting as a tool to understand fundamental features of epigenetic regulation of gene expression. We also generate mouse models for several inherited disorders. We generated models to study defects in cardiac repolarization associated with Kcnq1 loss of function and more recently characterized the phenotype associated with loss of Calsequestrin2 gene function. These cardiac studies all focus on the role of beta-adrenergic stimulation in cardiac arrhythmias.

Molecular basis for allele-specific expression of the mouse H19 and Igf2 genes

Gebert, Jeong, Rong, Yoon, Ettensohn, 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 identified the enhancer elements responsible for activation of the two genes. These elements are largely shared and located downstream of the H19 gene. Parent-of-origin-specific expression of both genes is dependent on a shared element (called H19ICR for H19 Imprinting Control Region) located just upstream of the H19 promoter and thus 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 this element in vivo demonstrates that non-methylated H19DMR (i.e., the copy on the maternal chromosome) is continually required for silencing the maternal Igf2 allele. Knockin experiments demonstrated that H19DMR contains a methylation-sensitive transcriptional insulator. Thus, on the non-methylated maternal chromosome, the active insulator within H19DMR prevents activation of Igf2 by the downsteam enhancers. Methylation of the paternal chromosome inactivates the insulator and permits Igf2 expression. Our model does not, however, explain the effect of several small DMRs (differentially methylated regions) proximal to the Igf2 transcription unit. Current studies are investigating the mechanistic significance of these elements. We have so far shown that expression of Igf2 does not correlate with methylation of these elements. Imprinting of H19 occurs via a distinct genetic mechanism. The conditional ablation of H19DMR indicates that it is not continuously required for silencing the paternal allele. Rather, H19DMR is required early in development to establish an epigenetic state at the H19 promoter, which itself prevents transcription. Current studies indicate that the epigenetic program includes but does not solely constitute the hypermethylation of the H19 promoter.

To determine what elements are necessary and sufficient for imprinting at the locus, we moved H19DMR and its mutated derivatives to heterologous loci. Our results demonstrated that ICR alone is sufficient to imprint a normally non-imprinted chromosome. Moreover, this activity is not dependent on germline differences in DMR methylation. Thus, DMR likely marks its parental origin by a mechanism independent of DNA methylation. By genetic and molecular analyses of embryonic stem cells derived from mutant mice, we are now determining the epigenetic signals that constitute the genomic imprint.

Finally, we are continuing a series of experiments to understand the molecular mechanisms by which H19DMR can act as a transcriptional insulator. Several groups have demonstrated the presence of four CTCF binding sites within 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 sensitive to methylation, that is, CTCF cannot bind to the methylated paternally inherited DMR, thus explaining the activation of the paternal Igf2 allele. To understand the molecular basis for insulator function, we have begun a series of experiments to characterize the three-dimensional organization of the Igf2/H19 locus, comparing maternal and paternal and wild-type and mutant chromosomes. We have demonstrated that transcriptional activation is invariably associated with physical interaction of the promoter and enhancer elements. Insertion of the ICR/insulator abrogates these interactions and induces alternative long-range chromosomal interactions of itself with the enhancer and promoter elements. Curiously, induction of these alternative interactions with the insulator is enhancer-dependent.

Mouse models for inherited long QT syndrome

Rong, Pfeifer; in collaboration with Ebert, Knollmann

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 (JLNS) patients display profound congenital deafness in addition to LQTS; both phenotypes are 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 attributable to a deficiency in the K+ recycling pathway, which 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 present in isolated hearts ex vivo, indicating that the Kcnq1 protein plays a key 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 also generated three point mutations to model RWS and analyzed mutations in the central pore region and 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 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-deficient mice. These 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 on Kcne1. We are now investigating the role of this channel in mouse and human hearts.

The role of calsequestrin2 in regulating cardiac function

Ettensohn, Van Druff, Pfeifer; in collaboration with Knollmann

Mutations in the CASQ2 gene, encoding cardiac calsequestrin (Casq2), are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) and sudden death. The existence of patients homozygous for loss-of-function mutations in CASQ2 is surprising given the central role of Ca2+ ions in excitation-contraction (EC) coupling and the presumed critical roles of Casq2 in EC coupling via its action as the major Ca2+ storage protein and its potential role in regulating ryanodine receptor (RyR) function. To address this paradox, we generated a mouse model for loss of Casq2 gene activity. Comprehensive analysis of cardiac function and structure generated several important insights regarding Casq2 function. First, Casq2 is not essential for providing sufficient Ca2+ storage in cardiomyocytes. Rather, a large increase in sarcoplasmic reticulum (SR) volume and surface area appears to maintain normal Ca2+ storage capacity. Second, Casq2 is not required for SR luminal sensing of Ca2+ levels. Third, Casq2 does regulate RyR Ca2+ release. In the absence of Casq2, significant Ca2+ leaks occur, leading to premature contractions and cardiac arrhythmias. Fourth, Casq2 function is required to maintain normal junctin and triadin levels. We do not yet understand what role, if any, these compensatory changes in these two SR proteins play in modulating the loss of Casq2 phenotype. To address these issues and to model cardiac disorders associated with induced (and not congenital) loss of Casq2 activity, we are beginning to analyze (1) mouse models in which loss of Casq2 function can be induced in adult animals and (2) mouse models in which Casq2 function can be restored to adult mice that developed without any Casq2 gene function.

Knollmann BC, Chopra N, Hlaing T, Akin B, Yang T, Ettensohn K, Knollmann BEC, Horton KD, Weissman NJ, Holinstat I, Zhang W, Roden DM, Jones LR, Franzini-Armstrong C, Pfeifer K. Casq2 deletion causes sarcoplasmic reticulum volume increase, premature Ca2+ release, and catecholaminergic polymorphic ventricular tachycardia. J Clin Invest 2006;116:2510-20.

COLLABORATORS

Steve Ebert, PhD, Burnett College of Biomedical Sciences, University of Central Florida, Orlando, FL
Björn Knollmann, MD, PhD, Vanderbilt University Medical Center, Nashville, TN

For further information, contact kpfeifer@helix.nih.gov.