A small number of genes in the mammalian genome are regulated by a process called genomic imprinting, whereby the maternal and paternal alleles are differentially expressed. The process requires a gamete-specific mark, most likely DNA methylation of specific sequences in the vicinity of imprinted genes. The mark is inherited from parents, and maintained in their progeny throughout life. Based on mutations in both humans and mice in imprinted genes, it is likely that the process evolved in mammals to regulated prenatal and possibly early postnatal growth. In human disruptions in imprinting have been implicated in three genetic syndromes, Beckwith-Wiedemann, Prader Willi and Angelman syndromes. Furthermore, loss of imprinting of specific genes has been observed in a large number of human tumors. This application proposes experiments to understand the number of human tumors. This application proposes experiments to understand the mechanism of genomic imprinting, as well as the function and evolution of the process. The laboratory is studying a cluster of imprinted genes on the distal end of mouse Chromosome 7 that include four maternally expressed genes that encode the growth factors insulin and insulin-like growth factor II. A model was proposed to explain the reciprocal imprinting of H19 and Igf2 in which the promoters of the two genes, which lie 90 kb apart, compete for transcriptional enhancers. Although there is substantial evidence in support of this model, the basis for the competition on the maternal chromosome is not completely understood. The investigators propose to generate two conditional mutations of the H19 gene, one in the promoter and a second in the 5' flank, to assess the relative importance of transcription of H19 versus a requirement for lack of methylation of the gametic mark in its 5' flank, for the imprinted silencing of Igf2. The nature of the gametic mark, and its acquisition of stable DNA methylation will be explored using both transfections and transgenic mice experiments. The proteins that act to prevent its methylation in the female germline and in somatic cells will be identified and cloned. The generality of the competition model will be explored by studying the genes at the telomeric end of the cluster. Gametic imprints will be identified and tested by transgenesis. To explore the importance of linkage for the imprinting of the genes in the region, specific translocations will be engineered in mice to dissociate the linkage of the genes. Finally the evolution of imprinting will be studied in the marsupial, Monodelphis, and in two strains of the wild mouse Peromyscus, where dramatic perturbations in growth are observed in reciprocal F1 hybrids.
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