Regulation of Imprinting at the H19/Igf2 Locus

 Marisa S. Bartolomei, Joanne L. Thorvaldsen, Nora I. Engel, Andrew Fedoriw. Department of Cell & Developmental Biology, University of Pennsylvania School of Medicine, 415 Curie Blvd., Philadelphia PA  19104 USA.

The opposite imprinting of the linked mouse H19 and Igf2 genes is mediated through shared enhancers located 3’ to H19 and a 2 kb imprinting control region, designated the differentially methylated domain (DMD), that is located 2 kb upstream of the H19 start of transcription. The DMD is methylated on the repressed paternal H19 allele and hypersensitive to nucleases on the active maternal allele. We previously demonstrated that the DMD is required for H19 and Igf2 imprinted expression on both parental chromosomes. To explain these results, it has been postulated that the DMD acts as a methylation-sensitive boundary element that isolates H19 and the enhancers on the maternal chromosome, thereby preventing access of Igf2 to the enhancers. On the paternal chromosome, Igf2 is expressed because the DMD and H19 gene promoter are methylated, preventing boundary proteins from binding and H19 from being expressed. It was recently demonstrated that a putative boundary protein, CTCF, binds in a methylation sensitive manner to a conserved repetitive element in the DMD. Using conditional mutants that delete the DMD in liver, we have also shown that the DMD is required for the continued imprinting of Igf2 but is dispensable for H19 imprinting. To dissect the sequences required for the multiple functions of the DMD, we have generated new mutations at the endogenous locus. Mutation of 9 CpG dinucleotides within the CTCF binding sites disrupts repression and hypermethylation of the paternal H19 allele while maintaining the ability of these sites to bind CTCF. These experiments suggest that the sites governing repression of the paternal H19 allele and those that mediate insulator activity at this locus are distinct. Furthermore, these experiments show that mutation of only 9 CpG dinucleotides within a 2 kb region that contains over 50 CpG dinucleotides is sufficient to perturb the ability of the paternal allele to confer and maintain H19 repression. Experiments are also in progress to determine whether CTCF protects the maternal DMD from DNA methylation.

July 2003