GENOMIC IMPRINTING ON DISTAL MOUSE CHROMOSOME 7

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Research is directed at understanding the molecular mechanisms for regulated expression of a cluster of imprinted genes on the distal end of mouse chromosome 7. At least five imprinted genes (p57Kip2, Mash2, Igf2, Ins-2, and H19) have been mapped to this cluster in mouse and to the human syntenic region on chromosome 11p15.5. Disruption in imprinted expression of these genes has been associated with prenatal lethality in mice and with Beckwith-Wiedemann Syndrome and with a number of tumors in humans. In addition, the principle genetic defect increasing susceptibility to long QT syndrome maps here. Our goals are to understand the genetic and molecular mechanisms for allele restricted expression of these genes, to identify novel imprinted genes in the region, and to develop mouse models for the human diseases. Current studies are focused on three major research areas. First we wish to isolate genetic elements required for regulated expression of H19, a gene transcribed exclusively from the maternal chromosome. Using transgenic mice we have identified cis acting sequences required for normal developmental-specific expression patterns. We have also identified elements required for restriction of H19 expression to the maternal allele. This regulation is copy-number dependent, suggesting that additional elements involved in maternal-specific expression have not yet been identified. We have therefore generated new transgenic lines using Bacterial Artificial Chromosome clones to search for these elements. Our second aim is to understand the coordinate regulation of genes in the distal 7 cluster. Analysis of gene disruptions indicates that the H19 and Igf2 genes share enhancer elements. Curiously, the two genes are oppositely imprinted, i.e. Igf2 is expressed only from the paternal chromosome. Disruption of the H19 gene, including upstream imprinting control elements, results in the inappropriate expression of maternal Igf2. The activation of maternal Igf2 may be due to the deletion of the normally active maternal H19 promoter thus relieving a competition between Igf2 and H19 promoters which the H19 promoter normally wins. Alternately, the activation of maternal Igf2 may be mechanistically independent of inactivation of H19 but due to the deletion of the upstream imprinting regulatory element that we identified in our transgenic experiments. We are generating conditional deletion mutations specific to this H19 imprinting control element. These mutations, which leave the H19 promoter and coding sequences intact, will assay the relationship between H19 expression and Igf2 silencing on the maternal chromosome. Specifically, we expect our mutation to induce inappropriate expression of H19 from the paternal chromosome. Our key question, then, is what the effect of this paternal H19 expression will be at the Igf2 locus. Silencing of the normally active paternal Igf2 allele will support the notion that competition of the H19 and Igf2 promoters is responsible for the reciprocal imprinting patterns of the two genes. Biallelic expression of Igf2 will support a model that a common regulatory element controls expression of both H19 and Igf2. In this later case, our efforts will focus on understanding how this one element silences H19 specifically on the paternal chromosome but silences Igf2 specifically on the maternal chromosome. Finally, we seek to identify and characterize novel genes in the region. We have identified BAC and P1 clones that span the region from upstream of p57Kip2 to downstream of H19. Using these as probes, we have begun a search for novel transcripts in the region using direct sequencing, exon trap, and cDNA selection approaches. We have identified the mouse homolog of human KVLQT1. The products of KVLQT1 and of the minK gene together form a functional potassium channel. Mutations in this channel are responsible for about 60% of all long QT cases. The human KVLQT1 is expressed only from the maternal chromosome, at least in fetal tissue. We have physically and genetically mapped mouse vlqt1 to the distal 7 cluster. We have noted high levels of developmentally regulated expression in lung, kidney, gut, and placenta as well as in heart. The imprinting of Kvlqt1 is under strict developmental regulation. While the earliest embryonic expression is maternal specific, the paternal allele becomes increasingly active until the gene is essentially biallelic within one week after birth. If applicable to humans, these results demonstrating developmental loss of imprinting of Kvlqt1 can explain the lack of parental bias in inheritance of long QT.