- Molecular basis for allele specific expression of the mouseH19 and Igf2 genes.
- Mouse models for inherited long QT syndrome
- Understanding the role of cardiac calsequestrin in normal heart development and function.
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 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 the developmental disorder, Beckwith Wiedemann syndrome, and with many types of tumors. 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 specific expression in this region. In these studies we hope to use imprinting as a tool in 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 this region. We have generated models to study defects in cardiac repolarization associated with loss of function mutations at Kcnq1 and specifically to understand the effect of beta-adrenergic mediated stress on the cardiac phenotype.
Molecular basis for allele specific expression of the mouse H19 and Igf2 genes
Sydella Blatch, Bokkee Eun, Claudia Gebert, and Karl Pfeifer
Our studies on the mechanisms of genomic imprinting focus on the H19 and the 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. These are largely shared and located downstream of the H19 gene. Parent-of-origin specific expression of both genes is dependent upon a shared element (called the H19DMR) located just upstream of the H19 promoter and thus juxtaposed 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 the non-methyated 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 non-methylated maternal chromosome, activation of Igf2 by the downsteam enhancers is prevented by the active insulator within the H19DMR. 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 investigating the mechanistic significance of these elements. We have so far shown that expression of Igf2 does not correlate with methylation of these sequences. Imprinting of H19 is via a distinct genetic mechanism. The conditional ablation of the H19DMR indicates that it is not continuously required for silencing 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 solely the hypermethylation of the H19 promoter.
To determine just what elements 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 non-imprinted chromosome. Moreover, this activity is not dependent upon germline differences in DMR methylation. Thus the 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 do constitute the genomic imprint.
Finally, we are continuing a series of experiments to understand the molecular mechanisms by which the H19DMR can 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 Igf2 allele. To understand the molecular basis for insulator function, we have begun a series of experiments to characterize the 3D organization of the Igf2/H19 locus, comparing maternal and paternal and also wild type and mutant chromosomes. Specifically, we are examining the long-range interactions between the Igf2 and H19promoters and the shared enhancer elements and the effect that the presence of a working insulator has on these interactions. Our results do not support the idea that the primary role of transcriptional insulators is to organize the chromosomes into large structural domains. Rather our data support a model where insulators regulate transcription fairly directly by interacting with the regulated enhancers and promoters.
Mouse models for inherited long QT syndrome
Karl Pfeifer, in collaboration with Steve Ebert (University of Central Florida) and Bjorn Knollman (Vanderbilt University Medical Center).
Inherited long QT syndrome (LQTS) is characterized by an abnormal electrocardiogram indicative of repolarization defects and 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 the LQTS and 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 these 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, these defects are not noted in isolated hearts ex vivo indicating that the Kcnq1 protein plays a key role in mediating critical extracardiac signals. Further anlayses 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. 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 vs. the inner ear and in various aspects of cardiac function. While inherited LQTS is relatively rare, these 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 was dependent on Kcnq1 but not Kcne1. The role of this channel in mouse and human hearts is now under investigation.
Understanding the role of cardiac calsequestrin in normal heart development and function.
Cameron Johnson, Aditya Shirali and Karl Pfeifer, in collaboration with Bjorn Knollmann (Vanderbilt University Medical Center).
Cardiac calsequestrin (Casq2) is a low-affinity, high capacity Ca2+ binding protein located in the junctional sarcoplasmic reticulum (SR) of mammalian cardiomyocytes. Ca2+ ions play a critical role in cardiac function. The rapid release of calcium from the SR is the event that couples electrical excitation and muscle contraction. Release of Ca2+ from the SR into the cardiomyocyte cytoplasm occurs through the RyR receptor complex, a multiprotein complex consisting of the RyR ion channel and the associated triadin, junctin, and Casq2 proteins. To understand the specific role of Casq2 in this process, we have generated mutant mice carrying 1.1 kb deletion that removes 750 base pairs of upstream sequences (including the Casq2 promoter) and also the entire exon 1 of Casq2. Mice homozygous for this mutation lack any detectable Casq2 protein. Casq2null mice are susceptible to catecholaminergic ventricular arrhythmias and thus phenocopy the human disease associated with loss of CASQ2function. Our collaborators have characterized cardiomyocytes lacking Casq2. Exposure to catecholamines in Casq2-deficient myocytes causes increased diastolic SR Ca2+ leak, resulting in spontaneous SR Ca2+ releases and triggered beats. This cellular phenotype likely explains the in vivo arrhythmias. We noted several other curious adaptations of Casq2-null myocytes. First, mutant animals show striking increases in SR volume as well as an altered SR morphology. These changes likely explain the relatively normal Ca2+ storage capacity of the SR in mutant animals. Casq2-/- animals also show a very large reduction in triadin and junctin protein levels. We are currently investigating whether this secondary change is compensatory or if it contributes to the mutant phenotype.