Epigenetic Properties and Identification of an Imprint Mark in the Nesp-Gnasxl Domain of the Mouse Gnas Imprinted Locus

Abstract

The Gnas locus in the mouse is imprinted with a complex arrangement of alternative transcripts defined by promoters with different patterns of monoallelic expression. The Gnas transcript is subject to tissue-specific imprinted expression, Nesp is expressed only from the maternal allele, and Gnasxl is expressed only from the paternal allele. The mechanisms controlling these expression patterns are not known. To identify potential imprinting regulatory regions, particularly for the reciprocally expressed Nesp and Gnasxl promoters, we examined epigenetic properties of the locus in gametes, embryonic stem cells, and fetal and adult tissues. The Nesp and Gnasxl promoter regions are contained in extensive CpG islands with methylation of the paternal allele at Nesp and the maternal allele at Gnasxl. Parental allele-specific DNase I-hypersensitive sites were found at these regions, which correlate with hypomethylation rather than actual expression status. A germ line methylation mark was identified covering the promoters for Gnasxl and the antisense transcript Nespas. Prominent DNase I-hypersensitive sites present on paternal alleles in embryonic stem cells are contained within this mark. This is the second gametic mark identified at Gnas and suggests that the Nesp and Gnasxl promoters are under separate control from the Gnas promoter. We propose models to account for the regulation of imprinting at the locus.

ACKNOWLEDGMENTS

C.C. and P.A. contributed equally to this work.

We are sincerely grateful to J. Richard Chaillet (Rangos Research Center, Pittsburgh) for advice in setting up bisulfite sequencing analysis from mouse germ cells. We thank Simon Ball (Harwell) for M. spretus stocks and Nick Allen (Babraham) for uniparental ES cell lines. We also thank Rachel Smith for helpful comments on the manuscript.

P.A. is supported by a Marie Curie Individual Fellowship from the European Community Programme in Human Potential (under contract HPMF-CT-2001-01122); C.C. and E.A.C. were supported by studentships from the MRC and BBSRC. G.K. is a senior fellow of the MRC.