MOLECULAR GENETICS OF AN IMPRINTED GENE CLUSTER ON MOUSE DISTAL CHROMOSOME 7
, Head, Section on Genomic Imprinting
Claudia Gebert, PhD, Visiting Fellow
Qi Rong, MD, MS, Microbiologist
Shauna Bennett, BS, Student
John Van Druff, BS, Student
Genomic imprinting is an unusual form of gene regulation in which expression of an allele is restricted in accordance with the allele’s 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 expression patterns. Mutations disrupting 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 the syntenic region and 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 and have generated models to study defects in cardiac repolarization associated with Kcnq1 loss of function. More recently, we characterized the phenotype associated with loss of Calsequestrin2 gene function. These cardiac studies focus on the role of beta-adrenergic stimulation in cardiac arrhythmias.
Molecular basis for allele-specific expression of the mouse H19 and Igf2 genes
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. The 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 juxtaposed between the Igf2 gene and the shared enhancers. The CpG sequences within the shared element are methylated specifically on the paternally inherited chromosome. Our conditional ablation of the element in vivo demonstrates that the non-methylated H19DMR (i.e., the copy on the maternal chromosome) is continuously required for silencing the maternal Igf2 allele. Knockin experiments demonstrate 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 downstream 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. We are currently investigating the mechanistic significance of these elements and have so far shown that expression of Igf2 does not correlate with methylation of these sequences. Imprinting of H19 occurs via a distinct genetic mechanism. The conditional ablation of H19DMR indicates that the element 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 that itself prevents transcription. Current studies indicate that the epigenetic program includes but is not solely the hypermethylation of the H19 promoter.
To determine which elements are necessary and sufficient for imprinting at the locus, we moved H19DMR and its mutated derivatives to heterologous loci. Our results demonstrate that ICR alone is sufficient to imprint a normally non-imprinted chromosome. Moreover, such 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 performing 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 H19DMR acts as a transcriptional insulator. Several research 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 DNA 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 the insulator function, we have started 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 demonstrated that transcriptional activation is invariably associated with physical interactions of the promoter and enhancer elements. Insertion of the ICR/insulator abrogates the interactions and induces alternative long-range chromosomal interactions of itself with the enhancer and promoter elements. Curiously, induction of the alternative interactions with the insulator is enhancer-dependent.
Jeong S, Hahn Y-S, Rong Q, Pfeifer K. Accurate quantitation of allele-specific expression patterns by analysis of DNA melting. Genome Res 2007;17:1093-100.
Yoon YS, Jeong S, Rong Q, Park K-Y, Chung JH, Pfeifer K. Analysis of the H19ICR insulator. Mol Cell Biol 2007;27:3499-510.
Mouse models for inherited long QT syndrome
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 generally inherit the LQTS disorder as a dominant phenotype but show no other traits. Jervell syndrome 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 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 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. However, the defects are not noted 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 have also generated three point mutations to model RWS. We analyzed the mutations in the central pore region and in the sixth membrane-spanning domain. Each of the phenotypes of the mutations is a distinct subset of those seen in the null mutation and thus demonstrates that the Kcnq1 protein plays distinct roles in the heart versus inner ear and in various aspects of cardiac function. While inherited LQTS is relatively rare, our genetic models provide excellent paradigms for addressing mechanisms for acquired LQTS, the single largest cause of death in Western societies.
Biochemical and pharmacological studies 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 ablation of the Kcnq1 gene 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 on Kcne1. We are now investigating the role of the channel in mouse and human hearts.
Knollmann BC, Sirenko S, Rong Q, Katchman AN, Casimiro M, Pfeifer K, Ebert SN. Kcnq1 contributes to an adrenergic-sensitive steady-state K+ current in mouse heart. Biochem Biophys Res Commun 2007;360:212-8.
The role of calsequestrin2 in regulating cardiac function
Mutations in the CASQ2 gene, which encodes cardiac calsequestrin (Casq2), are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) and sudden death. That patients homozygous for loss-of-function mutations in CASQ2 are alive is extremely surprising, given the central role of Ca2+ ions in excitation-contraction coupling and the presumed critical roles of Casq2 in regulating Ca2+ release from the sarcoplasmic reticulum (SR) into the cytoplasm. To address this paradox, we generated a mouse model for loss of Casq2 gene activity. Comprehensive analysis of cardiac function and structure has generated several important insights into Casq2 function. First, Casq2 is not essential for providing sufficient Ca2+ storage in the SR of the cardiomyocyte. Rather, a compensatory increase in SR volume and surface area in mutant mice appears to maintain normal Ca2+ storage capacity. Second, Casq2 is not required for the rapid, triggered release of Ca2+ from the SR during cardiomyocyte contraction. Rather, the RyR receptor opens appropriately, resulting in the normal, rapid flow of Ca2+ into the cytoplasm, thus allowing normal contraction of the cardiomyocyte. Third, Casq2 is required for normal function of the RyR during cardiomyocyte relaxation. In the absence of Casq2, significant Ca2+ leaks occur through the RyR, 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, the compensatory changes in these two SR proteins plays in modulating the loss of Casq2 phenotype. To address these issues and to model cardiac disorders associated with late-onset (and not congenital) loss of Casq2 activity, we are beginning to analyze mouse models in which hormone treatment induces loss of Casq2 function in adult animals. We are also developing a mouse model in which Casq2 function can be restored to adult mice that develop without any Casq2 gene function.
Chopra N, Kannankeril PJ, Yang T, Hlaing T, Holinstat IA, Ettensohn K, Pfeifer K, Akin B, Jones LR, Franzini-Armstrong C, Knollmann BC. Modest reductions of cardiac calsequestrin increase sarcoplasmic reticulum Ca2+ leak independent of luminal Ca2+ and trigger ventricular arrhythmias in mice. Circ Res 2007;10:617-26.
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, contactkpfeifer@helix.nih.gov.
