Tandem Repeat Hypothesis in Imprinting: Deletion of a Conserved Direct Repeat Element Upstream of H19 Has No Effect on Imprinting in the Igf2-H19 Region

Abstract

Igf2 and H19 are reciprocally imprinted genes on mouse distal chromosome 7. They share several regulatory elements, including a differentially methylated region (DMR) upstream of H19 that is paternally methylated throughout development. The cis-acting sequence requirements for targeting DNA methylation to the DMR remain unknown; however, it has been suggested that direct tandem repeats near DMRs could be involved. Previous studies of the imprinted Rasgrf1 locus demonstrate indeed that a direct repeat element adjacent to a DMR is responsible for establishing paternal allele-specific methylation at the DMR and therefore allelic expression of the Rasgrf1 transcript. We identified a prominent and conserved direct tandem repeat 1 kb upstream of the H19 DMR and proposed that it played a similar role in imprinted regulation of H19. To test our hypothesis, we generated mice harboring a 1.7-kb targeted deletion of the direct repeat element and analyzed fetal growth, allelic expression, and methylation within the Igf2-H19 region. Surprisingly the deletion had no effect on imprinting. These results together with deletions of other repeats close to imprinted genes suggest that direct repeats may not be important for the targeting of methylation at the majority of imprinted loci and that the Rasgrf1 locus may be an exception to this rule.

We thank The Babraham Institute Gene Targeting Facility for their help with generating the H19^ΔK mice, H. Sasaki for providing the cDH1 cosmid and the 189M11 Bac, and D. Adams for providing the DTA-containing plasmid. We thank G. Kelsey for comments on the manuscript and E. Walters for help with statistical analysis.

This work was supported by BBSRC and MRC.