Identification of a Methylation Imprint Mark within the Mouse Gnas Locus

Genomic imprinting is an epigenetic phenomenon affecting a small number of autosomal genes that results in differences in gene expression between the maternal and paternal allele and explains why both paternal and maternal genomes are required for normal development (3, 12, 38). Mutation, deletion, and dysregulation of imprinted genes have been implicated in several human diseases, such as the Beckwith-Wiedemann, Prader-Willi, and Angelman syndromes and Albright hereditary osteodystrophy, and in carcinogenesis. Virtually all imprinted genes have regions in which CpG dinucleotides are differentially methylated between the maternal and paternal alleles. Loss of imprinting in mice lacking a DNA methyltransferase gene (28, 29) strongly suggests that allele-specific methylation differences are critical for the maintenance of imprinting. The presence of differentially methylated regions (DMRs) in the male or female germline (methylation imprint marks) that are required to establish differential methylation at other loci within the same imprinted region in later development (12) strongly suggests that methylation is important for establishing the maternal and paternal epigenotypes (methylation and expression patterns). Heterozygous inactivating mutations within GNAS1, the human gene at 20q13 that codes for the heterotrimeric G protein α-subunit GSα, lead to multihormone resistance only when inherited through the maternal germline (13, 42). GSα is a ubiquitously expressed protein that is required for hormone-stimulated cyclic AMP generation. Maternal-specific expression of GSα in hormone target tissues likely explains the hormone resistance that results from maternal inheritance of GNAS1 mutations. Imprinting of GSα has not been confirmed in humans (9, 17, 18). However, we showed, using Gnas knockout mice (Gnas in distal chromosome 2 is the mouse orthologue of GNAS1) that GSα is imprinted in mice in a tissue-specific manner, being maternally expressed in some tissues (e.g., renal proximal tissues and adipose tissue) and biallelically expressed in most other tissues (43, 47, 48). Gnas has at least four alternative promoters and first exons that generate multiple imprinted mRNAs (Fig. ​(Fig.1).1). The gene was originally defined by 13 coding exons for GSα (exons 1 to 13) (27). Three additional Gnas mRNAs result from splicing of alternative first exons to exon 2. Alternative promoters located 30 and 45 kb upstream of GSα exon 1 are oppositely imprinted and produce mRNAs encoding XLαs, a Golgi-specific isoform of GSα, and the chromogranin-like protein NESP55, respectively (17, 18, 26, 34). XLαs is expressed from the paternal allele, and its promoter region is methylated on the maternal allele, whereas NESP55 is expressed from the maternal allele and its promoter is methylated on the paternal allele (17, 18, 34). Both proteins are expressed primarily in neuroendocrine tissues, and little is known about their function (21, 25). These two promoter regions have been named Gnasxl and Nesp (18). A fourth alternative promoter and first exon (which we call exon 1A) located 2.5 kb upstream of GSα exon 1 generates mRNAs that are probably untranslated and are of unknown function (22, 36). The imprinting status of the exon 1A promoter has not been determined. FIG. 1 Schematic diagram showing the maternal (Mat) and paternal (Pat) alleles of Gnas. Alternative first exons which splice into exon 2 to generate alternative mRNAs encoding NESP55, XLαs, an unknown gene product, and GSα are shown as boxes ... Given the close proximity of the oppositely imprinted Gnasxl and Nesp promoters and the similar tissue distribution of their mRNAs, it is possible that they coordinately regulate each other, as has been suggested for other closely linked imprinted genes (2, 3). However two observations make it unlikely that either promoter or their mRNAs are involved in the tissue-specific imprinting of GSα. First, the mRNAs of both promoters are expressed in several human tissues in which the GSα transcript is biallelically expressed (17, 18). Second, we were unable to amplify either transcript in mouse tissues in which the GSα transcript is imprinted (e.g., brown adipose tissue [BAT]) (S. Yu and L. S. Weinstein, unpublished data). To define the mechanisms that lead to imprinting of the Gnas locus in general and tissue-specific imprinting of GSα in particular, we determined the methylation status of the GSα promoter and of the region immediately upstream of the promoter. In this paper, we show first that imprinting of GSα is not associated with methylation of its promoter. Second, we identify a region upstream of the GSα promoter that is methylated on the maternal allele in all tissues examined. Within this region is exon 1A (22, 36), which generates mRNAs only from the paternal allele. This upstream region is a methylation imprint mark, as methylation in this region is established during gametogenesis and maintained throughout pre- and postimplantation development.