An imprinted transcript, antisense to Nesp, adds complexity to the cluster of imprinted genes at the mouse Gnas locus.

The Gnas locus in distal mouse chromosome (Chr) 2 is emerging as a complex genomic region. It contains three imprinted genes in the order Nesp-Gnasxl-Gnas. Gnas encodes a G protein alpha-subunit, and Nesp and Gnasxl encode proteins of unknown function expressed in neuroendocrine tissue. Together, these genes form a single transcription unit because transcripts of Nesp and Gnasxl are alternatively spliced onto exon 2 of Gnas. Nesp and Gnasxl are expressed from opposite parental alleles, with Nesp encoding a maternal-specific transcript and Gnasxl encoding a paternal-specific transcript. We now identify a further imprinted transcript in this cluster. Reverse transcription-PCR analysis of Nesp expression in 15. 5-days-postcoitum embryos carrying only maternal or paternal copies of distal Chr 2 revealed an isoform that is exclusively paternally, rather than maternally, expressed. Strand-specific reverse transcription-PCR showed that this form is an antisense transcript. The existence of a paternally expressed antisense transcript was confirmed by Northern blot analysis. The sequence is contiguous with genomic sequence downstream of Nesp and encompasses Nesp exons 1 and 2 and an intervening intron. We propose that Nespas is an additional control element in the imprinting region of mouse distal Chr 2; it adds further complexity to the Gnas-imprinted gene cluster.

Identification of imprinted loci by methylation-sensitive representational difference analysis: application to mouse distal chromosome 2.

Imprinted genes are distinguished by different patterns of methylation on their parental alleles, a property by which imprinted loci could be identified systematically. Here, representational difference analysis (RDA) is used to clone HpaII fragments with methylation differences on the maternal and paternal copies of distal chromosome (Chr) 2 in the mouse. Uniparental inheritance for this region causes imprinting phenotypes whose molecular basis is only partially understood. RDA led to the recovery of multiple differentially methylated HpaII fragments at two major sites of imprinted methylation: paternal-specific methylation at the Nesp locus and maternal-specific methylation at the Gnasxl locus. Nesp and Gnasxl represent oppositely imprinted promoters of the Gnas gene, which encodes the G-protein subunit, Gsalpha. The organization of the Nesp-Gnasxl-Gnas region was determined: Nesp and Gnasxl were found to be 15 kb apart, and Gnasxl was found to be 30 kb upstream of Gnas. Sites of imprinted methylation were also detected at the loci for neuronatin on Chr 2 and for M-cadherin on Chr 8. RDA was highly effective at identifying imprinted methylation, and its potential applications to imprinting studies are discussed.

A cluster of oppositely imprinted transcripts at the Gnas locus in the distal imprinting region of mouse chromosome 2.

Imprinted genes tend to occur in clusters. We have identified a cluster in distal mouse chromosome (Chr) 2, known from early genetic studies to contain both maternally and paternally imprinted, but unspecified, genes. Subsequently, one was identified as Gnas, which encodes a G protein alpha subunit, and there is clinical and biochemical evidence that the human homologue GNAS1, mutated in patients with Albright hereditary osteodystrophy, is also imprinted. We have used representational difference analysis, based on parent-of-origin methylation differences, to isolate candidate imprinted genes in distal Chr 2 and found two oppositely imprinted genes, Gnasxl and Nesp. Gnasxl determines a variant G protein alpha subunit associated with the trans-Golgi network and Nesp encodes a secreted protein of neuroendocrine tissues. Gnasxl is maternally methylated in genomic DNA and encodes a paternal-specific transcript, whereas Nesp is paternally methylated with maternal-specific expression. Their reciprocal imprinting may offer insight into the distal Chr 2 imprinting phenotypes. Remarkably, Gnasxl, Nesp, and Gnas are all part of the same transcription unit; transcripts for Gnasxl and Nesp are alternatively spliced onto exon 2 of Gnas. This demonstrates an imprinting mechanism in which two oppositely imprinted genes share the same downstream exons.