Genomic imprinting is an unusual form of gene regulation in which expression of an allele is restricted in accordance with its parental origin. Thus, some genes, such as H19, are expressed only from the maternally inherited chromosome while the paternal allele is silent. In contrast, the neighboring Igf2 gene is expressed almost exclusively from the paternal allele. 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 expression patterns. Mutations disrupting the normal patterns of imprinting at the human locus are associated with a 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 studies involve the use of imprinting as a tool for understanding fundamental features of epigenetic regulation of gene expression and rely on the mouse system for generating animal models for the several inherited disorders associated with this region, specifically the effect of b-adrenergic–mediated stress on the cardiac phenotype.

Molecular Basis for Allele-Specific Expression of the Mouse H19 and Igf2 Genes
Park, Boe, Pfeifer
Our studies of imprinting focus on expression of the H19 and Igf2 genes. Paternally expressed Igf2 lies about 70 kb upstream of the maternal-specific H19 gene. By using cell culture systems and conducting transgene and knock-out experiments in vivo, we have identified the enhancer elements responsible for activation of the two genes. The elements 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 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-methylated 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 transcriptional insulator that is inactivated, and thus permissive for enhancer-promoter interactions, by DNA methylation. Therefore, on the non-methylated 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 investigating 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 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 provide evidence that the epigenetic program includes, but is not solely, the hypermethylation of the H19 promoter.

To determine precisely the elements that are necessary and sufficient for imprinting at the locus, we have moved the H19DMR and mutated derivatives to a normally unimprinted locus. Mice carrying the mutation are currently under analysis, and results indicate that both forms of gene regulation, the developmentally dependent modification of neighboring sequences and transcriptional insulation, can be examined by the above approach.

Mouse Models for Inherited Long QT Syndrome
Casimiro, Pfeifer; in collaboration with Ebert, Knollman
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 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. When measured by ECG, in vivo tracings of mutant mice are indicative of profound defects in cardiac repolarization. However, the defects are not noted in isolated hearts ex vivo, demonstrating that the Kcnq1 protein plays a central role in mediating critical extracardiac signals. Further analyses demonstrate that Kcnq1 function is specifically required to modulate cardiac function in the presence of b-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 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 of acquired LQTS, the single largest cause of death in Western societies.

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 b-adrenergic hormones, norepinephrine and epinephrine. This cardiac-specific synthesis is transient and disappears by mid-gestation. Intriguingly, the cells synthesizing the badrenergic 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 bgalactosidase–positive and thus can be readily identified and isolated. Early analyses indicate that epinephrine is synthesized by cells that specifically give rise to cardiac conduction cells, albeit a subset of such cells. Ongoing experiments will characterize cell types. Parenthetically, our studies have generated a mouse that has no epinephrine but shows normal levels of norepinephrine, thus allowing dissection of the specific roles of these two hormones.