H19--A MODEL FOR PARENTAL IMPRINTING IN THE MOUSE

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The purpose of this application is to investigate the function and mechanisms of parental imprinting in mammals, using as a model system the murine Igf2 and H19 genes. Igf2 encodes the fetal-specific growth factor insulin-like growth factor II, and is expressed throughout development primarily from the paternally inherited chromosome. The closely linked H19 gene is expressed in a very similar pattern to Igf2, but exclusively from the maternal chromosome. The imprinting and linkage of these genes is conserved in humans, where disruptions in their gene dosage have been implicated in Beckwith-Wiedeman syndrome, Wilm's tumor and susceptibility to childhood tumors such as rhabdomyosarcomas and hepatocellular carcinomas. A series of mutations will be generated in mice to test the role of these genes in these disorders. The first specific aim is to test a model to explain the reciprocal imprinting of Igf2 and H19. The model proposes that the genes compete in cis for common regulatory elements. The competition is biased differently on the two chromosomes by the presence of paternal-specific DNA methylation, which acts to silence the H19 promoter and facilitate Igf2 transcription. Experiments are proposed to test the role of specific enhancers which lie 3' to the H19 gene by mutating them via homologous recombination in embryonic stem cells. The effects of both maternal and paternal inheritance of the deletion of the enhancers on Igf2 and H19 will be examined. The basis for the preferential transcription of H19 on the apparently unmarked maternal chromosome will be investigated by transfecting both wild type and mutant yeast artificial chromosomes bearing the genes into tissue culture cells. The relaxation of imprinting of Igf2 in vivo will be studied to understand how imprinting can be erased in somatic cells. The second specific aim will focus on the function of the H19 gene, and its possible involvement in the mechanism of imprinting. A series of mutations in the gene and its transcriptional control regions will be generated through homologous recombination in embryonic stem cells. The phenotype of mice that have inherited either maternal and paternal null mutations will be determined. The basis for the late fetal lethality caused by extra copies of H19 will be studied by introducing both wild type and mutant copies of the gene into embryonic stem cells, in order to establish lines of mice which transmit extra copies of the gene. The nature of the proteins complexed in the H19 particle will be determined by purifying the particle and cloning the proteins contained within it. The third specific aim is to elucidate the function of imprinting in eutherian mammals by identifying how it evolved. The Igf2 and H19 genes will be identified and cloned from marsupials and monotremes, and tested for allele-specific expression. The likelihood that the H19 gene arose during mammalian speciation as a duplication of XIST, a gene which maps to the X chromosome inactivation center and is expressed exclusively from the inactive X chromosome, will be tested.