CHARACTERIZATION OF NOVEL PARENT-SPECIFIC EPIGENETIC MODIFICATIONS UPSTREAM OF THE IMPRINTED MOUSE H19 GENE

Genomic imprinting results in parent-specific monoallelic expression o f a small number of genes in mammals. The identity of imprints is unkn own, but much evidence points to a role for DNA methylation. The mater nal alleles of the imprinted H19 gene are active and hypomethylated; t he paternal alleles are inactive and hypermethylated. Roles for other epigenetic modifications are suggested by allele-specific differences in nuclease hypersensitivity at particular sites. To further analyze t he possible epigenetic mechanisms determining monoallelic expression o f H19, we have conducted in vivo dimethylsulfate and DNase I footprint ing of regions upstream of the coding sequence in parthenogenetic and androgenetic embryonic stem cells. These cells carry only maternally a nd paternally derived alleles, respectively, We observed the presence of maternal-allele-specific dimethylsulfate and DNase I footprints at the promoter indicative of protein-DNA interactions at a CCAAT box and at binding sites for transcription factors Sp1 and AP-2, Also, at the boundary of a region further upstream for which existent differential methylation has been suggested to constitute an imprint, we observed a number of strand-specific dimethylsulfate reactivity differences spe cific to the maternal allele, along with an unusual chromatin structur e in that both strands of maternally derived DNA were strongly hyperse nsitive to DNase I cutting over a distance of 100 nucleotides, We ther efore reveal the existence of novel parent-specific epigenetic modific ations, which in addition to DNA methylation, could constitute imprint s or maintain monoallelic expression of H19.