Human Molecular Genetics, 2000, Vol. 9, No. 5 835-841
© 2000 Oxford University Press
An imprinted antisense transcript at the human GNAS1 locus
Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St James’s University Hospital, Leeds LS9 7TF, UK
Received 17 December 1999; Revised and Accepted 14 January 2000.
DDBJ/EMBL/GenBank accession nos AJ251759–AJ251761.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Recent studies of the GNAS1 gene have shown a highly complex imprinted expression pattern, with paternally, maternally and biallelically derived pro- tein products, raising questions regarding how such transcriptional complexity is established and maintained. GNAS1 was originally identified as the gene encoding an important and widely expressed signal transduction protein, the
subunit of the stimulatory G protein Gs. Partial Gs deficiency results in the hormone resistance syndrome, pseudohypoparathyroidism type 1a. Gs is encoded by exons 1–13 of GNAS1 and, in most tissues at least, expression of this transcript is biallelic. Two large upstream exons, however, have monoallelic expression patterns, and in each case their transcripts splice onto GNAS1 exon 2. The most 5' of these is maternally expressed, and encodes neuroendocrine secretory protein 55 (NESP55), whose coding region does not overlap with that of Gs. The other exon, 14 kb further 3', is paternally expressed, and encodes XLs (extra large s-like protein), translated in-frame with Gs exons 2–13. This close proximity of two oppositely imprinted promoters suggested the likelihood of important regulatory interactions between them, and to investigate this possibility we have performed a search for other transcripts in the region. Here we show that the maternally methylated region up- stream of the XLs exon gives rise to a spliced polyadenylated antisense transcript, which spans the upstream NESP55 region. This antisense transcript is imprinted, and expressed only from the paternal allele, suggesting that it may have a specific role in suppressing in cis the activity of the paternal NESP55 allele. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Recent studies in both human and mouse have shown that GNAS1, located on human chromosome 20q13 and mouse distal chromosome 2, is a complex imprinted gene. As originally described (1), GNAS1 consisted of 13 exons, encoding the
subunit of the stimulatory guanine nucleotide-binding protein Gs. Gs is required for coupling of the ligand-activated forms of many seven-transmembrane-domain hormone receptors to the intra- cellular generation of cAMP. Consequently, null mutations of GNAS1 cause the hormone resistance syndrome pseudo- hypoparathyroidism type 1a (PHP1a) (2–4). This syndrome shows an anomalous autosomal dominant inheritance pattern, with maternal transmission being required for full phenotypic manifestation (5,6). However, despite these clinical pointers towards GNAS1 being an imprinted gene, human Gs-encoding transcripts were shown to be biallelically derived in a range of fetal tissues (7). Targeting of the murine Gnas locus provided fresh evidence that this gene was indeed imprinted, since paternally and maternally inherited heterozygous mutations result in different phenotypes (8). These bear little resemblance, however, to the PHP1a phenotype, so that the relevance of this animal model to human GNAS1 function remains unclear.
Recent studies of human GNAS1 have shown, however, that the locus is much more complex than previously realized. As well as Gs, this locus also encodes two other distinct polypeptides, XLs (9) and NESP55 (10). These proteins are encoded by alternatively spliced messages that initiate from either of two novel upstream exons, located ~35 kb (XLs) and 49 kb (NESP55) upstream of exon 1. All these alternative first exons splice onto the acceptor site of exon 2, within the Gs-encoding body of the gene (11,12).
Each of the novel upstream exons is embedded within a differentially methylated region, a common feature of imprinted genes. By analysis of a FokI polymorphism within the GNAS1 transcript, we also showed that both upstream exons are monoallelically expressed. Despite their proximity to each other, however, these two exons are oppositely imprinted, as the XLs transcript is expressed from its unmethylated paternal allele and the NESP55 transcript from its unmethylated maternal allele.
This arrangement, which is shown in Figure 1, is noteworthy for the very close proximity of two oppositely methylated and oppositely expressed promoter regions (the NESP55–XLs intron is ~12 kb). This in turn raises a number of questions concerning the mechanism by which such a mutually exclusive expression pattern is established and maintained. One specific possibility is that mutual suppression in cis between the NESP55 and XLs promoters might be exerted, as a consequence of transcription originating at one promoter and extending across the other promoter region. Precedent for such a mechanism comes from studies of the murine Igf2r locus, at which paternally derived antisense transcripts originating within a differentially methylated region in intron 2 are implicated in cis-suppression of the sense promoter, resulting in a maternal pattern of Igf2r expression (13). Such a mechanism operating at GNAS1 might therefore implicate antisense transcription from the XLs region in suppressing NESP55 promoter activity.
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In this report, we demonstrate that there is indeed a third imprinted transcript derived from the GNAS1 locus, which fulfils some of the expectations raised above. This is a spliced, paternally expressed antisense transcript, arising from a discrete site within the differentially methylated XL
s region, and traversing the oppositely imprinted upstream NESP55 exon. The observations suggest the differentially methylated antisense promoter region as a prime target for experimental manipulations aimed at understanding the regulation of GNAS1 imprinting. |
RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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To investigate possible regulatory interactions between the NESP55 (maternally expressed) and XL
s (paternally expressed) regions of GNAS1, we first characterized the genomic region containing these two exons. If important regulatory elements and/or transcripts exist in this region, it is possible that they would be conserved between human and mouse, both of which show a similar pattern of promoter-specific imprinting at this locus (11,12,14). We therefore sequenced the entire NESP55–XLs region in both human and mouse. These two sequences (GenBank accession nos AJ251760, 20 924 bp, and AJ251761, 21 927 bp) display conservation across the whole of the inter-exonic region. Outside the NESP55 and XLs exons, the highest degree of conservation (87% identity over 285 bp) occurs in a region ~3.5 kb upstream of the XLs exon. This highly conserved element lies at the upstream end of a 5.8 kb CpG-rich region, that also encompasses XLs itself and ~1 kb of sequence downstream (Fig. 2).
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The antisense transcript
Next, database searching using the 21 kb human genomic sequence was performed. This resulted in identification of an expressed sequence tag (EST T84962) matching the sequence of a region 1.0–1.3 kb 3' to the NESP55 exon. The corresponding cDNA clone (IMAGE 111764) was obtained and completely sequenced. Comparison with the genomic sequence showed that this cDNA (shown as clone 3 in Fig. 3b) represented a portion of a spliced polyadenylated message that was transcribed in the antisense direction relative to NESP55.
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To characterize this antisense transcript further, reverse transcription–polymerase chain reaction (RT–PCR) and 5'-RACE were performed using RNA from a normal female lymphoblastoid cell line. This allowed us to characterize a series of alternatively spliced transcripts, up to 1.1 kb in length (GenBank accession no. AJ251759). It should be noted that these RT–PCR experiments do not rely on strand-specific priming to demonstrate the antisense nature of the transcript. This is because the transcripts are all spliced, and comparison with the genomic sequence clearly demonstrates that they could have originated only from an antisense primary transcript.
The antisense RNA species identified in these experiments are shown in Figure 3. Five antisense exons were identified, four of which lie between the NESP55 and XLs exons, whereas the final exon, containing the polyadenylation signal for the transcript, lies ~19 kb upstream of NESP55 (Fig. 3a). These antisense transcripts are a heterogeneous population (Figs 3b and 4b), due to the occurrence of exon skipping and the use of alternative splice sites within exons (Fig. 3c). Despite this, two of the three 5'-RACE products characterized initiated at exactly the same nucleotide. The largest potential open reading frame in the largest transcript would encode a 97 amino acid polypeptide lacking homology to any described protein. This, in conjunction with the heterogeneity of the antisense transcripts, suggests that they lack coding potential.
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The highly conserved 285 bp genomic region previously identified in human and mouse genomic sequence comparisons was now seen to comprise the first 100 bp of antisense exon I and the region immediately upstream of it (Fig. 2a). This suggests that conservation of the antisense promoter and at least part of its transcript may be imposed by functional constraints.
Imprinting of the antisense transcript
Figure 2b summarizes the relationship between the exons of the antisense GNAS1 transcript and the CpG-rich regions of the human and mouse loci. As mentioned above, a large CpG-rich region (shown in Fig. 2b by the presence of multiple HhaI and HpaII restriction sites) that includes the XLs exon extends upstream as far as the location of the first antisense exon. We showed previously that the XLs exon and the region immediately downstream of it were methylated exclusively on the maternal allele (11). Therefore, we next assessed by Southern blotting whether the most upstream part of this CpG-rich region, adjacent to the first antisense exon, was also differentially methylated. Figure 5 shows that this region is indeed methylated on the maternal allele, in a fashion similar to that of XLs. The presence of a single non-imprinted NarI site 3 kb upstream of antisense exon I could indicate either that some CpGs scattered within the region are not imprinted or alternatively that the differentially methylated region has a bipartite structure. Distinguishing between these possibilities will require a more detailed analysis of the methylation pattern across the whole region.
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In order to investigate whether the antisense transcript also has an imprinted expression profile we searched for polymorphisms within its five exons. Sequencing of all five exons (1.1 kb total) in DNA from six unrelated individuals yielded only one polymorphism: a C
T transition in exon I. Screening a collection of fetal DNAs for informative heterozygotes showed that this polymorphism was very rare, occurring only once in 51 fetuses. RT–PCR of the antisense transcript was performed using RNA from this heterozygous fetus. The sequences of these RT–PCR products and of a genomic PCR product spanning the same region are shown in Figure 4a. Although the genomic sequence contains both alleles, RT–PCR products from muscle and spinal cord contain only the T allele, indicating that the antisense transcript is expressed from only one chromosome. Unfortunately, there was no parental DNA available for this heterozygous fetus, so we were unable to determine from this experiment alone the inheritance pattern of the polymorphism and hence which allele was expressed.
To determine the direction of the imprinting of the antisense transcript, we tested for the presence of the NESP55, XLs, Gs and antisense transcripts in RNA from a normal female lymphoblastoid cell line (LCL) and from the parthenogenetic lymphoblastoid cell line FD (11,12). Figure 4b shows that although the normal cell line expresses all four transcript species, the parthenogenetic cell line, as predicted by our previous data, expresses the maternally derived NESP55 transcript but the paternally derived XLs transcript is undetectable. The antisense transcript, like XLs, is easily detected in normal LCL (Fig. 4b, lanes 2 and 8) but undetectable by RT–PCR in the parthenogenetic cell RNA (lanes 3 and 9). This indicates that, like XLs, the antisense transcript is expressed from the paternal chromosome only.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Although differential methylation is a consistent marker for imprinted regions, other factors are becoming implicated in the regulation of imprinted genes. These include chromatin conformation (15–17), the presence of chromatin boundary elements (18,19), histone acetylation and deacetylation (20) and the presence of non-coding RNA transcripts (13,21–27). Non-coding RNA transcripts are also intimately involved in the hyperactivation of the single X chromosome in male Drosophila (28) and in the inactivation of one X chromosome in female mammals (29,30).
With the exception of the H19 transcript (27), non-coding imprinted transcripts seem to be located at least partly within protein-coding imprinted genes and are transcribed in the antisense direction relative to the coding transcript. These antisense transcripts can be spliced and relatively short (1–2 kb), or unspliced and extremely large (>60 kb) (22,25), but a common feature seems to be that they traverse one or more sense exons of the gene with which they are associated.
We postulated that the close proximity (12 kb) of oppositely imprinted promoters at the GNAS1 locus might require specific regulatory interactions between the two. In particular, we wondered whether the XLs region might have bidirectional promoter activity, with a paternally derived antisense transcript exerting a cis-suppressive effect on NESP55 transcription. The present report shows that such an imprinted antisense transcript does indeed exist, though the antisense promoter region appears likely to be separate from the XLs promoter. Careful site-directed manipulation of this region will be required to determine whether the functions of the antisense and XLs promoters can in fact be separated.
These results add another level to the complex expression profile of the GNAS1 locus. Antisense transcripts have been described in five other imprinted genes; UBE3A (21), KVLQT1 (called LIT1) (22,25), Igf2r (13), Igf2 (23) and Zfp127/ZFP127 (24,26). Interestingly, all of these antisense transcripts, like the GNAS1 antisense transcript, are expressed from the paternal chromosome, though whether this paternal exclusivity merely reflects the small number of genes that have been analysed remains to be seen. The transcriptional start sites of LIT1 (22,25) and Igf2r antisense (13) have been localized to differentially methylated regions (DMRs) within introns of their respective sense genes, and we have now shown that this is also the case for the GNAS1 antisense transcript. The removal of the Igf2r DMR2 from a YAC transgene (13) eliminated the production of Igf2r antisense transcripts and released expression of the sense Igf2r transcript from the paternal chromosome. Analysis of Beckwith–Wiedemann syndrome (BWS) patients showed that loss of imprinting of the LIT1 transcript was the most common genetic alteration in BWS (22). In both these examples, dysregulation of an antisense transcript appears to lead to a functional effect on the sense (protein-coding) gene. Our current efforts are therefore directed at assessing a comparable regulatory role for the GNAS1 antisense transcript. Its potential importance is suggested by the observation that it initiates in a 285 bp region that is highly conserved in the mouse. In addition, the paucity of polymorphic variation within the human antisense exons, and the discrete 5' ends of antisense transcripts, further support the idea that this non-coding RNA is under selective constraints.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Identification of genomic clones and sequence analysis
The isolation of the human P1-derived artificial chromosome (PAC 309F20) containing the GNAS1 locus has been described elsewhere (11). The mouse NESP55–XL
s region was isolated as follows: after comparison of the human NESP55 sequence and the partial mouse EST described by Ischia et al. (10) an RT–PCR product was generated from murine RNA using primers whose sequence was conserved in the NESP55 exon (NESP10) and in exon 2 (GNAS2R). This product was used to probe the RPCI21 mouse PAC library (HGMP Resource Centre, Hinxton, UK) and two positive clones were identified: 583L07 and 653B05. 583L07 was used to generate genomic subclones. For sequencing, subclones were generated using standard methods and sequenced using random transposon insertion (GPS-1 kit; New England Biolabs) to generate sequencing templates. Sequencing was performed using the ThermoSequenase radiolabelled terminator cycle sequencing kit and [33P]dideoxynucleotides from Amersham Pharmacia Biotech (Uppsala, Sweden) and by BigDye terminator sequencing on an ABI 377 sequencer (Perkin Elmer Applied Biosystems, Foster City, CA). Sequence assembly and comparisons were performed using the Genetics Computer Group (Madison, WI) package, hosted at the MRC HGMP site (http://www.hgmp.mrc.ac.uk ). Database searching was performed using BLAST (31) hosted at NCBI (http://www.ncbi.nlm.nih.gov ).
PCR and RT–PCR
RT was performed with Superscript RT-II (Gibco BRL, Gaithersburg, MD) using RNA isolated using the AGPC method (32). Standard RT used a primer containing a d(T)17 tract (RACE2) whereas 5'-RACE used an antisense specific primer (88F5), under conditions previously described (33). PCR used primers RACE3+88F6 to amplify the 5'-RACE transcripts (40 cycles of 94°C for 25 s, 62°C for 25 s and 72°C for 3 min) and 88R8+88F3 or 88R8+88F6 to amplify other antisense transcripts [‘hot start’ and then six cycles of 94°C for 30 s, 66°C for 30 s (–0.5°C per cycle) and 72°C for 90 s, and 34 cycles of 94°C for 30 s, 63°C for 30 s and 72°C for 90 s]. RT–PCR of specific GNAS1 transcripts (using primers NESP5/XL1F/GNAS1F + GNAS10R) used the same PCR conditions. PCR products were cloned into the Easy-T vector (Promega, Madison, WI). Primer sequences were:
RACE2, dGAGCTCGAGTCGACATCGA(T17);
RACE3, dGAGCTCGAGTCGACATCGATTT;
NESP5, dTCGGAATCTGACCACGAGCA;
NESP10, dGAGCTCGCCATAATTACAACG;
XL1F, dGGATGCCTCCGCTGGTTTCAG;
GNAS1F, dCCATGGGCTGCCTCGGGAACA;
GNAS2R, dGGATCCTCATCTGCTTCACAAT;
GNAS10R, dCACGAAGATGATGGCAGTCAC;
88F3, dGCCCCATCTCCAATTCTAAT;
88F5, dCAGGAACGTGGCACTAATGAG;
88F6, dGATCAGTCGTCGAAAAGGAGG;
88R8, dGAAGAGGTGCTGCTGGGGTG.
Methylation analysis
Southern blotting using standard methods was performed on parthenogenetic and matched normal 46,XX lymphoblastoid cell genomic DNA as described previously (11,12,34).
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ACKNOWLEDGEMENTS |
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We are very grateful to Dr L. Strain for the Southern blot shown in Figure 5. This work was supported by the Wellcome Trust (grant no. 053701).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 113 206 5681; Fax: +44 113 244 4475; Email: meddtb@gps.leeds.ac.uk
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 Kozasa, T., Itoh, H., Tsukamoto, T. and Kaziro, Y. (1988) Isolation and characterization of the human Gs
gene. Proc. Natl Acad. Sci. USA, 85, 2081–2085.
2 Levine, M.A., Ahn, T.G., Klupt, S.F., Kaufman, K.D., Smallwood, P.M., Bourne, H.R., Sullivan, K.A. and Van Dop, C. (1988) Genetic deficiency of the subunit of the guanine nucleotide-binding protein Gs as the molecular basis for Albright hereditary osteodystrophy. Proc. Natl Acad. Sci. USA, 85, 617–621.
3 Weinstein, L.S., Gejman, P.V., Friedman, E., Kadowaki, T., Collins, R.M., Gershon, E.S. and Spiegel, A.M. (1990) Mutations of the Gs-subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis. Proc. Natl Acad. Sci. USA, 87, 8287–8290.
4 Patten, J.L., Johns, D.R., Valle, D., Eil, C., Gruppuso, P.A., Steele, G., Smallwood, P.M. and Levine, M.A. (1990) Mutation in the gene encoding the stimulatory G protein of adenylate cyclase in Albright’s hereditary osteodystrophy. N. Engl. J. Med., 322, 1412–1419.[Abstract]
5 Davies, S.J. and Hughes, H.E. (1993) Imprinting in Albright’s hereditary osteodystrophy. J. Med. Genet., 30, 101–103.
6 Marguet, C., Mallet, E., Basuyau, J.P., Martin, D., Leroy, M. and Brunelle, P. (1997) Clinical and biological heterogeneity in pseudohypoparathyroidism syndrome—results of a multicenter study. Hormone Res., 48, 120–130.[Web of Science][Medline]
7 Campbell, R., Gosden, C.M. and Bonthron, D.T. (1994) Parental origin of transcription from the human GNAS1 gene. J. Med. Genet., 31, 607–614.
8 Yu, S., Yu, D., Lee, E., Eckhaus, M., Lee, R., Corria, Z., Accili, D., Westphal, H. and Weinstein, L.S. (1998) Variable and tissue-specific hormone resistance in heterotrimeric Gs protein -subunit (Gs) knockout mice is due to tissue-specific imprinting of the gs gene. Proc. Natl Acad. Sci. USA, 95, 8715–8720.
9 Kehlenbach, R.H., Matthey, J. and Huttner, W.B. (1994) XLs is a new type of G protein. Nature, 372, 804–809.[Medline]
10 Ischia, R., Lovisetti-Scamihorn, P., Hogue-Angeletti, R., Wolkersdorfer, M., Winkler, H. and Fischer-Colbrie, R. (1997) Molecular cloning and characterization of NESP55, a novel chromogranin-like precursor of a peptide with 5-HT1B receptor antagonist activity. J. Biol. Chem., 272, 11657–11662.
11 Hayward, B.E., Kamiya, M., Strain, L., Moran, V., Campbell, R., Hayashizaki, Y. and Bonthron, D.T. (1998) The human GNAS1 gene is imprinted and encodes distinct paternally and biallelically expressed G proteins. Proc. Natl Acad. Sci. USA, 95, 10038–10043.
12 Hayward, B.E., Moran, V., Strain, L. and Bonthron, D.T. (1998) Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins. Proc. Natl Acad. Sci. USA, 95, 15475–15480.
13 Wutz, A., Smrzka, O.W., Schweifer, N., Schellander, K., Wagner, E.F. and Barlow, D.P. (1997) Imprinted expression of the Igf2r gene depends on an intronic CpG island. Nature, 389, 745–749.[Medline]
14 Peters, J., Wroe, S.F., Wells, C.A., Miller, H.J., Bodle, D., Beechey, C.V., Williamson, C.M. and Kelsey, G. (1999) A cluster of oppositely imprinted transcripts at the Gnas locus in the distal imprinting region of mouse chromosome 2. Proc. Natl Acad. Sci. USA, 96, 3830–3835.
15 Schweizer, J., Zynger, D. and Francke, U. (1999) In vivo nuclease hypersensitivity studies reveal multiple sites of parental origin-dependent differential chromatin conformation in the 150 kb SNRPN transcription unit. Hum. Mol. Genet., 8, 555–566.
16 Hark, A.T. and Tilghman, S.M. (1998) Chromatin conformation of the H19 epigenetic mark. Hum. Mol. Genet., 7, 1979–1985.
17 Khosla, S., Aitchison, A., Gregory, R., Allen, N.D. and Feil, R. (1999) Parental allele-specific chromatin configuration in a boundary-imprinting-control element upstream of the mouse H19 gene. Mol. Cell. Biol., 19, 2556–2566.
18 Greally, J.M., Starr, D.J., Hwang, S., Song, L., Jaarola, M. and Zemel, S. (1998) The mouse H19 locus mediates a transition between imprinted and non-imprinted DNA replication patterns. Hum. Mol. Genet., 7, 91–95.
19 Lyko, F., Brenton, J.D., Surani, M.A. and Paro, R. (1997) An imprinting element from the mouse H19 locus functions as a silencer in Drosophila. Nature Genet., 16, 171–173.[Web of Science][Medline]
20 Hu, J.F., Oruganti, H., Vu, T.H. and Hoffman, A.R. (1998) The role of histone acetylation in the allelic expression of the imprinted human insulin-like growth factor II gene. Biochem. Biophys. Res. Commun., 251, 403–408.[Web of Science][Medline]
21 Rougelle, C., Cardoso, C., Fontes, M., Colleaux, L. and Lalande, M. (1998) An imprinted antisense RNA overlaps UBE3A and a second maternally expressed transcript. Nature Genet., 19, 15–16.[Web of Science][Medline]
22 Lee, M.P., DeBaun, M.R., Mitsuya, K., Galonek, H.L., Brandenburg, S., Oshimura, M. and Feinberg, A.P. (1999) Loss of imprinting of a paternally expressed transcript, with antisense orientation to KvLQT1 occurs frequently in Beckwith–Wiedemann syndrome and is independent of insulin-like growth factor II imprinting. Proc. Natl Acad. Sci. USA, 96, 5203–5208.
23 Moore, T., Costancia, M., Zubair, M., Bailleul, B., Feil, R., Sasaki, H. and Reik, W. (1997) Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2. Proc. Natl Acad. Sci. USA, 94, 12509–12514.
24 Jong, M.T.C., Carey, A.H., Caldwell, K.A., Lau, M.H., Handel, M.A., Driscoll, D.J., Stewart, C.L., Rinchik, E.M. and Nicholls, R.D. (1999) Imprinting of a RING zinc-finger encoding gene in the mouse chromosome region homologous to the Prader–Willi syndrome genetic region. Hum. Mol. Genet., 8, 795–803.
25 Mitsuya, K., Meguro, M., Lee, M.P., Katoh, M., Schulz, T.C., Kugoh, H., Yoshida, M.A., Niikawa, N., Feinberg, A.P. and Oshimura, M. (1999) LIT1, an imprinted antisense RNA in the human KvLQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids. Hum. Mol. Genet., 8, 1209–1217.
26 Jong, M.T., Gray, T.A., Ji, Y., Glenn, C.C., Saitoh, S., Driscoll, D.J. and Nicholls, R.D. (1999) A novel imprinted gene, encoding a RING zinc-finger protein, and overlapping antisense transcript in the Prader–Willi syndrome critical region. Hum. Mol. Genet., 8, 783–793.
27 Pfeifer, K. and Tilghman, S.M. (1994) Allele-specific gene expression in mammals: the curious case of the imprinted RNAs. Genes Dev., 8, 1867–1874.
28 Kelley, R.L., Meller, V.H., Gordadze, P.R., Roman, G., Davis, R.L. and Kuroda, M.I. (1999) Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell, 98, 513–522.[Web of Science][Medline]
29 Lee, J.T. and Lu, N. (1999) Targeted mutagenesis of Tsix leads to nonrandom X inactivation. Cell, 99, 47–57.[Web of Science][Medline]
30 Clemson, C.M., McNeil, J.A., Willard, H.F. and Lawrence, J.B. (1996) XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell Biol., 132, 259–275.
31 Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol., 215, 403–410.[Web of Science][Medline]
32 Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem., 162, 156–159.[Web of Science][Medline]
33 Hayward, B.E. and Bonthron, D.T. (1998) Structure and alternative splicing of the ketohexokinase gene. Eur. J. Biochem., 257, 85–91.[Web of Science][Medline]
34 Strain, L., Warner, J.P., Johnston, T. and Bonthron, D.T. (1995) A human parthenogenetic chimaera. Nature Genet., 11, 164–169.[Web of Science][Medline]
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 J. Liu, M. Chen, C. Deng, D. Bourc'his, J. G. Nealon, B. Erlichman, T. H. Bestor, and L. S. Weinstein From the Cover: Identification of the control region for tissue-specific imprinting of the stimulatory G protein {alpha}-subunit PNAS, April 12, 2005; 102(15): 5513 - 5518. [Abstract] [Full Text] [PDF]
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 J. Liu, J. G. Nealon, and L. S. Weinstein Distinct patterns of abnormal GNAS imprinting in familial and sporadic pseudohypoparathyroidism type IB Hum. Mol. Genet., January 1, 2005; 14(1): 95 - 102. [Abstract] [Full Text] [PDF]
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L. S. Weinstein, J. Liu, A. Sakamoto, T. Xie, and M. Chen Minireview: GNAS: Normal and Abnormal Functions Endocrinology, December 1, 2004; 145(12): 5459 - 5464. [Abstract] [Full Text] [PDF]
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S. Shirasawa, H. Harada, K. Furugaki, T. Akamizu, N. Ishikawa, K. Ito, K. Ito, H. Tamai, K. Kuma, S. Kubota, et al. SNPs in the promoter of a B cell-specific antisense transcript, SAS-ZFAT, determine susceptibility to autoimmune thyroid disease Hum. Mol. Genet., October 1, 2004; 13(19): 2221 - 2231. [Abstract] [Full Text] [PDF]
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 A. Lania, G. Mantovani, and A. Spada Genetics of Pituitary Tumors: Focus on G-Protein Mutations Experimental Biology and Medicine, October 1, 2003; 228(9): 1004 - 1017. [Abstract] [Full Text] [PDF]
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D. R. FitzPatrick, I. M. Carr, L. McLaren, J. P. Leek, P. Wightman, K. Williamson, P. Gautier, N. McGill, C. Hayward, H. Firth, et al. Identification of SATB2 as the cleft palate gene on 2q32-q33 Hum. Mol. Genet., October 1, 2003; 12(19): 2491 - 2501. [Abstract] [Full Text] [PDF]
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M. Bastepe and H. Juppner Pseudohypoparathyroidism and Mechanisms of Resistance toward Multiple Hormones: Molecular Evidence to Clinical Presentation J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4055 - 4058. [Full Text] [PDF]
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E. L. Germain-Lee, J. Groman, J. L. Crane, S. M. Jan de Beur, and M. A. Levine Growth Hormone Deficiency in Pseudohypoparathyroidism Type 1a: Another Manifestation of Multihormone Resistance J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4059 - 4069. [Abstract] [Full Text] [PDF]
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 C. Coombes, P. Arnaud, E. Gordon, W. Dean, E. A. Coar, C. M. Williamson, R. Feil, J. Peters, and G. Kelsey Epigenetic Properties and Identification of an Imprint Mark in the Nesp-Gnasxl Domain of the Mouse Gnas Imprinted Locus Mol. Cell. Biol., August 15, 2003; 23(16): 5475 - 5488. [Abstract] [Full Text] [PDF]
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O. El-Maarri, M. Seoud, P. Coullin, U. Herbiniaux, J. Oldenburg, G. Rouleau, and R. Slim Maternal alleles acquiring paternal methylation patterns in biparental complete hydatidiform moles Hum. Mol. Genet., June 15, 2003; 12(12): 1405 - 1413. [Abstract] [Full Text] [PDF]
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 H. Kiyosawa, I. Yamanaka, N. Osato, S. Kondo, and Y. Hayashizaki Antisense Transcripts With FANTOM2 Clone Set and Their Implications for Gene Regulation Genome Res., June 1, 2003; 13(6): 1324 - 1334. [Abstract] [Full Text] [PDF]
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R. Holmes, C. Williamson, J. Peters, P. Denny, RIKEN GER Group, GSL Members, and C. Wells A Comprehensive Transcript Map of the Mouse Gnas Imprinted Complex Genome Res., June 1, 2003; 13(6): 1410 - 1415. [Abstract] [Full Text] [PDF]
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 T. H. Vu, N. V. Chuyen, T. Li, and A. R. Hoffman Loss of Imprinting of IGF2 Sense and Antisense Transcripts in Wilms' Tumor Cancer Res., April 15, 2003; 63(8): 1900 - 1905. [Abstract] [Full Text] [PDF]
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G. Mantovani, E. Ballare, E. Giammona, P. Beck-Peccoz, and A. Spada The Gs{alpha} Gene: Predominant Maternal Origin of Transcription in Human Thyroid Gland and Gonads J. Clin. Endocrinol. Metab., October 1, 2002; 87(10): 4736 - 4740. [Abstract] [Full Text] [PDF]
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 M. Bastepe, Y. Gunes, B. Perez-Villamil, J. Hunzelman, L. S. Weinstein, and H. Juppner Receptor-Mediated Adenylyl Cyclase Activation Through XL{alpha}s, the Extra-Large Variant of the Stimulatory G Protein {alpha}-Subunit Mol. Endocrinol., August 1, 2002; 16(8): 1912 - 1919. [Abstract] [Full Text] [PDF]
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K. Nakabayashi, L. Bentley, M. P. Hitchins, K. Mitsuya, M. Meguro, S. Minagawa, J. S. Bamforth, P. Stanier, M. Preece, R. Weksberg, et al. Identification and characterization of an imprinted antisense RNA (MESTIT1) in the human MEST locus on chromosome 7q32 Hum. Mol. Genet., July 15, 2002; 11(15): 1743 - 1756. [Abstract] [Full Text] [PDF]
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I. S. Pedersen, P. Dervan, A. McGoldrick, M. Harrison, F. Ponchel, V. Speirs, J. D. Isaacs, T. Gorey, and A. McCann Promoter switch: a novel mechanism causing biallelic PEG1/MEST expression in invasive breast cancer Hum. Mol. Genet., June 1, 2002; 11(12): 1449 - 1453. [Abstract] [Full Text] [PDF]
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 T. Li, T. H. Vu, K.-O. Lee, Y. Yang, C. V. Nguyen, H. Q. Bui, Z.-L. Zeng, B. T. Nguyen, J.-F. Hu, S. K. Murphy, et al. An Imprinted PEG1/MEST Antisense Expressed Predominantly in Human Testis and in Mature Spermatozoa J. Biol. Chem., April 12, 2002; 277(16): 13518 - 13527. [Abstract] [Full Text] [PDF]
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 E. M. Shore, J. Ahn, S. J. de Beur, M. Li, M. Xu, R.J. M. Gardner, M. A. Zasloff, M. P. Whyte, M. A. Levine, and F. S. Kaplan Paternally Inherited Inactivating Mutations of the GNAS1 Gene in Progressive Osseous Heteroplasia N. Engl. J. Med., January 10, 2002; 346(2): 99 - 106. [Abstract] [Full Text] [PDF]
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 L. S. Weinstein, S. Yu, D. R. Warner, and J. Liu Endocrine Manifestations of Stimulatory G Protein {alpha}-Subunit Mutations and the Role of Genomic Imprinting Endocr. Rev., October 1, 2001; 22(5): 675 - 705. [Abstract] [Full Text] [PDF]
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H. Zheng, G. Radeva, J. A. McCann, G. N. Hendy, and C. G. Goodyer G{alpha}s Transcripts Are Biallelically Expressed in the Human Kidney Cortex: Implications for Pseudohypoparathyroidism Type 1b J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4627 - 4629. [Abstract] [Full Text] [PDF]
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M. Bastepe, J. E. Pincus, T. Sugimoto, K. Tojo, M. Kanatani, Y. Azuma, K. Kruse, A. L. Rosenbloom, H. Koshiyama, and H. Juppner Positional dissociation between the genetic mutation responsible for pseudohypoparathyroidism type Ib and the associated methylation defect at exon A/B: evidence for a long-range regulatory element within the imprinted GNAS1 locus Hum. Mol. Genet., June 1, 2001; 10(12): 1231 - 1241. [Abstract] [Full Text] [PDF]
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 A Wutz, H. Theussl, J Dausman, R Jaenisch, D. Barlow, and E. Wagner Non-imprinted Igf2r expression decreases growth and rescues the Tme mutation in mice Development, January 5, 2001; 128(10): 1881 - 1887. [Abstract] [PDF]
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S.-i. Horike, K. Mitsuya, M. Meguro, N. Kotobuki, A. Kashiwagi, T. Notsu, T. C. Schulz, Y. Shirayoshi, and M. Oshimura Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in Beckwith-Wiedemann syndrome Hum. Mol. Genet., September 1, 2000; 9(14): 2075 - 2083. [Abstract] [Full Text] [PDF]
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J. Liu, S. Yu, D. Litman, W. Chen, and L. S. Weinstein Identification of a Methylation Imprint Mark within the Mouse Gnas Locus Mol. Cell. Biol., August 15, 2000; 20(16): 5808 - 5817. [Abstract] [Full Text]
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