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

Abstract

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.

Figure 1

RDA applied to uniparental methylation. (a) Overview of RDA using MatDp.dist2 DNA as “tester” and PatDp.dist2 DNA as “driver,” so that HpaII fragments specifically unmethylated in MatDp.dist2 DNA are “targets.” MatDp.dist2 and PatDp.dist2 DNAs are digested with HpaII and ligated to adapters, and whole-genome PCR was performed to produce amplicons representing unmethylated HpaII fragments. Subtractive hybridization was performed by denaturing tester amplicon with an excess of driver amplicon and allowing reassociation to take place, thus driving fragments common to tester and driver into heteroduplexes. Postsubtraction PCR with tester-specific primer preferentially amplifies tester–tester duplexes as DPs, thereby enriching target fragments. A second subtraction with first DPs against driver amplicon produces greater enrichment of targets in second DPs (for a full explanation of RDA, see ref. 14). (b) Gel electrophoresis showing HpaII amplicons (Amps) from MatDp.dist2 (M) and PatDp.dist2 (P) embryo DNAs and first-round (MatDP1, PatDP1) and second-round (MatDP2, PatDP2) DPs. L indicates a 100-bp ladder. (c) Probes for MatDP clone 317 and PatDP clone 414 hybridized to Southern blots of MatDp.dist2 (M) and PatDp.dist2 (P) embryo DNAs digested with HindIII alone (−) or with HpaII or MspI. (d) Sequence alignment of MatDP clone 317 (mmNesp) with the bovine cDNA (btuNesp) and a consensus (est) of three mouse ESTs W15839, AA260302, and AA124617. The translational start site for Nesp is underlined; putative splice donor and acceptor sites are double underlined. The maternally unmethylated HpaII sites at either end of clone 317 are in italics. (e) Sequence alignment of PatDP clone 414 (mmGnαsxl) with genomic sequence lying between exons A20 and A21 in human XLαs [hsXLas; GenBank accession no. AJ224868 (17)]. Paternally unmethylated HpaII and AscI sites (in the mouse and human sequences, respectively) are italicized.

Figure 2

Differential expression of Nesp. (a) Northern blot analysis of mRNA extracted from MatDp.dist2 (M) and PatDp.dist2 (P) embryos and placenta derived from T26H. The Nesp-specific RNA probe detected a 3.9-kb transcript in MatDp.dist2 embryo and placenta at 15.5 and 12.5 dpc, respectively. The 3.9-kb transcript detected in placenta also hybridized with a Gnas RNA probe specific for exons 4–5. The 1.8-kb band is the primary Gnas transcript. (b) RT-PCR of oligo(dT)-primed cDNA derived from 15.5-dpc MatDp.dist2 (M), PatDp.dist2 (P), and phenotypically normal (wt) embryos from T26H intercross. Twenty-five cycles of amplification were performed by using the primers Nesp.f3 and ex2r. Hprt primers were included as an amplification control. Lane L is 1-kb ladder and lane G is the product amplified from C3H/HeH genomic DNA. The presence (+) and absence (−) of reverse transcriptase is indicated.

Figure 3

RT-PCR demonstrating differential expression of Gnasxl. Expression analysis was done on MatDp.dist2 (M), PatDp.dist2 (P), and phenotypically normal (wt) tissue derived from the reciprocal translocations T26H and T68H. Oligo(dT)-primed cDNA was amplified for 25 cycles with primers Xlf2 and ex2r; Hprt primers were included as an amplification control. Lane L is 1-kb ladder. The presence (+) and absence (−) of reverse transcriptase is indicated.

Figure 4

Schematic representation of the cluster of oppositely imprinted transcripts associated with Gnas in distal mouse Chr 2. Dark shaded blocks indicate Gnas exons known in mouse, exon 2 of Gnas being common to the overlapping genes; light shaded blocks show exons exclusive to Nesp (designated N1 and N2), and the hatched block represents the 3′ end of the exon called extra large (XL) that is specific to Gnasxl. M and P refer to maternally and paternally derived alleles, respectively. The approximate position of the methylation-sensitive restriction sites are represented by open circles if unmethylated or solid circles if methylated. Transcripts of Nesp and Gnasxl are represented as arrows and those of Gnas are not shown. Pulse field gel analysis of YACs containing the region indicate the following gene order: Nesp–Gnasxl–Gnas, with Nesp being 16 kb upstream of Gnasxl, which is between 30 and 41 kb upstream of Gnas exon 1 (data not shown). The figure is not to scale.