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Genomics 69, 295–304 (2000)doi:10.1006/geno.2000.6337, available online at http://www.idealibrary.com on

Tissue-Specific Expression of Antisense and Sense Transcriptsat the Imprinted Gnas Locus

Tao Li,* ,1 Thanh H. Vu,* ,1,2 Zhi-Lan Zeng,* Binh T. Nguyen,* Bruce E. Hayward,†David T. Bonthron,† Ji-Fan Hu,* and Andrew R. Hoffman* ,2

*Medical Service and GRECC, VA Palo Alto Health Care System, and Department of Medicine, Stanford University School of Medicine,Palo Alto, California 94304; and †Molecular Medicine Unit, University of Leeds, Clinical Sciences Building,

St. James’s University Hospital, Leeds LS9 7TF, United Kingdom

Received April 21, 2000; accepted July 13, 2000

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The mouse Gnas gene encodes an important signalransduction protein, the a subunit of the stimulatory

G protein, Gs. In humans, partial deficiency of Gsa, thea subunit of Gs, results in the hormone-resistance syn-drome pseudohypoparathyroidism type 1a. The mouseGnas (and the human GNAS1) locus is transcribedfrom three promoter regions. Transcripts from P1,which encode Nesp55, are derived from the maternalallele only. Transcripts from P2 encode Xlas and arederived only from the paternal allele, while tran-scripts from P3 encode the a subunit and are fromboth parental alleles. The close proximity of reciprocalimprinting suggests the presence of important puta-tive imprinting elements in this region. In this report,we demonstrate that the reciprocal imprinting occursin normal tissues of interspecific (Mus spretus 3C57BL/6) mice. Transcripts from P1 are most abun-dant in CNS (pons and medulla) in contrast to themore ubiquitous expression from P2 and P3. In theP1–P2 genomic region, we have identified an antisensetranscript that starts 2.2 kb upstream of the P2 exonand spans the P1 region. While the P1 transcript isderived from the maternal allele, the P1-antisense(Gnas-as) is derived only from the paternal allele inmost but not all tissues. Although both the Nesp55region and the Gnas-as transcripts are present in ce-rebral cortex, adrenal, and spleen, Gnas-as is abun-

ant in some tissues in which transcription from theesp55 region is negligible. Furthermore, the Nesp55

egion transcripts remain strictly imprinted in tissueshat lack Gnas-as. Our results suggest that multiplemprinting elements, including the unique Gnas-as,egulate the allelic expression of the Nesp55 regionense transcript. © 2000 Academic Press

Sequence data from this article have been deposited with theEMBL/GenBank Data Libraries under Accession No. AJ251761(mouse Nesp55–Xlas genomic region).

1 These authors made an equal contribution to this work.2 To whom correspondence should be addressed. Telephone: (650)

858-3930. Fax: (650) 856-8024. E-mail: arhoffman@stanford.edu or

thanhvu@stanford.edu.

295

INTRODUCTION

Human GNAS1 and mouse Gnas, which are locatedn human chromosome 20q13 and mouse distal chro-osome 2 (Gejman et al., 1991), respectively, encode

the a subunit of the stimulatory guanine nucleotide-binding protein Gs. This G protein couples hormonereceptors to stimulate adenylate cyclase, generatingintracellular cAMP. In humans, mutations of GNAS1ause the hormone-resistance syndrome pseudohypo-arathyroidism type 1a (PHP1a) (Levine et al., 1988;einstein et al., 1990), which is transmitted through

the maternal line (Davies et al., 1993), suggesting pre-ominant GNAS1 expression from the maternal allele.

In mice, targeted deletion of mouse Gnas suggestedhat the gene was imprinted. However, a phenotypeimilar to human PHP1a was not observed (Yu et al.,998).The mouse Gnas and human GNAS1 loci have sim-

lar genomic structures. The a subunit gene is tran-scribed from promoter P3 and encompasses 13 exons.Two other transcripts from upstream promoters P2and P1 encode two other distinct polypeptides: Xlas(Kehlenbach et al., 1994) and Nesp55 (Ischia et al.,1997) (Fig. 1). In humans, transcription from promoterP3 is derived from both alleles, while transcriptionfrom P2 (Xlas) and from P1 (Nesp55) is monoallelic.Transcription from P2 occurs only on the paternal al-lele, while in contrast, transcription from P1 (Nesp55)is exclusively from the maternal allele (Hayward et al.,1998). In the mouse, similar reciprocal imprinting ofP1 and P2 transcripts has been demonstrated in ma-ternal/paternal uniparental disomic mice (Peters et al.,1999).

The presence of the two reciprocally imprinted tran-scripts, P1 and P2, within a short chromosomal DNAsegment (;15 kb) makes the Gnas/GNAS1 locus anexcellent model for exploring the cis- and trans-ele-ments that govern the initiation and maintenance ofgenomic imprinting. We have recently demonstrated

the presence of a third imprinted transcript with an

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296 LI ET AL.

antisense orientation in the human GNAS1 P1–P2 re-gion (Hayward and Bonthron, 2000). In this report, wepresent a parallel study of the mouse Gnas, utilizingnterspecific (Mus spretus 3 C57BL/6) mice. We ana-yze the relative levels of expression from the multipleromoters in various normal tissues and the reciprocalmprinting of transcripts from P1 and P2, and we dem-nstrate the tissue-specific expression of the imprintedouse Gnas-as.

MATERIALS AND METHODS

Mouse resources. C57BL/6 and M. spretus mice were purchasedrom The Jackson Laboratory (Bar Harbor, ME). The F1 interspecificice (C57BL/6 females 3 M. spretus males) were bred in the Animalenter at the Palo Alto VA Medical Center. The F2 backcross mice

M. spretus and BALB/C) were described previously (Hu et al., 1997).Mice were sacrificed at age 1 and 12 months. Total nucleic acids,DNA, and RNA were prepared as described previously (Vu andHoffman, 1996).

Reverse transcription. Reverse transcription (RT) was performedwith MMLV reverse transcriptase (Gibco BRL) as described previ-ously (Vu and Hoffman, 1996) using RNA isolated by Tri reagent(Sigma). Both random hexamers and d(T)17 primers were used forcDNA synthesis. Total RNA was treated with DNase I (Gibco BRL)to eliminate any residual genomic DNA. Negative controls for thereverse transcription were performed without MMLV RT.

Promoter-specific allelic expression analysis. We sequenced theM. spretus liver cDNA by cloning RT-PCR fragments into a pCRIIvector according to the protocol suggested by the manufacturer (In-vitrogen, Carlsbad, CA). Transcripts specific for each promoter (P1,Nesp55; P2, Xlas; and P3, Gsa) were amplified by using promoter-pecific oligonucleotides (p#242 for P1, p#259 for P2, and p#240 for3) and the common primer p#290 in exon 12 (Fig. 1). PCR amplifi-ation was performed for 35 cycles at 95°C for 20 s and 65°C for 2in. The PCR products were diluted fivefold and aliquots (1 ml) were

radioisotope-labeled by 5 cycles of PCR (95°C for 20 s, 65°C for 2 min)using primer p#256 and a 32P-end-labeled primer, p#291. The 32P-end-labeled PCR products were digested with BstUI and then ana-lyzed on a 5% urea–polyacrylamide gel to reveal the M. spretus andthe C57BL/6 alleles. Sequences of the primers used for the promoter-specific allelic expression are as follows: p#242 (59-GTCAC TAATGGGTGA CTCCG T-39), p#259 (59-AGCTG CTTCG GTCTA TCCGAGTGTA-39), p#240 (59-CATGG GCTGC CTCGG CAACA-39), p#290(59-TGGAT GATGT CACGG CAGTC GTTGA-39), p#256 (59-ATGTGA CTGCC ATCAT CTTCG TGGTG-39), p#291 (59-ACACGGCGGA TGTTC TCAGT GTCCA-39).

Single-nucleotide allele-specific (SNAS) primer extension assay.We performed the SNAS assay according to the method describedpreviously (Vu and Hoffman, 1997). Briefly, transcripts were ampli-fied using primers p#296 (59-ATCAG TGGAG GCACC TCTCGGAGTC T-39) and p#324 (59-CCATT CTCTT AGGTG CTCAC CG-39)to amplify a 328-bp fragment encompassing the G-T polymorphicsite. The thermal cycling parameters were as follows: an initialdenaturation at 95°C for 1 min; 30 cycles of amplification at 95°C for15 s, 63°C for 15 s, and 72°C for 1 min; and a final extension at 72°Cfor 5 min. The PCR products were diluted fivefold with water andaliquots (1.0 ml) were labeled by primer extension using 32P-end-abeled primers p#322T* (59-AGCTT TAGAA AGTTC GCAGTGTTT T-39) and p#323G* (59-AGTTC GCAGT GGTTT G-39). The#322T* and p#323G* primers had specific T or G nucleotide at the9 end and therefore extended only on the specified T and G alleles by

5 cycles of the extension condition. Since the p#322T* primer was 10bases longer than the p#323G* primer, the T allele extended prod-ucts were 10 bases longer than the G allele extended products. Bothalleles for sense transcripts (103 and 93 bases) and antisense tran-

scripts (197 and 187 bases) were separated on a 5% urea–polyacryl-

amide gel. To eliminate the possibility of interference from minuteamounts of contaminating genomic DNA, we extensively digestedour RNA samples with DNase I (1 unit of DNase I for each 300 ngRNA, at 37°C for 1 h) and also carried out negative control experi-ments using mock reverse-transcribed (2RT) samples. The SNAS-PCR assays were repeated three times. b-Actin (135 bases) wasamplified by 20 cycles of PCR using primers p#5899 (59-CAGGTCATCA C(CT)AT(CT) GGCAA TGAGC-39) and p#5900 (59-CGGATGTC(AC)A CGTCA CACTT CATGA-39) as loading controls.

Multiplex PCR for P1, P2, and P3 transcripts. Amplification wasperformed for 30 cycles at 95°C for 15 s, 63°C for 15 s, and 72°C for1 min and a final extension at 72°C for 5 min. The common 39 primerp#289 was end labeled with [g-32P]ATP. Sequences of the primersused for the multiplex PCR are as follows: p#242 (59-GTCAC TAATGGGTGA CTCCG T-39), p#335 (59-TACRC ACCGC CTGCT GCTTCTAG-39), p#240 (59-CATGG GCTGC CTCGG CAACA-39), p#289 (59-CCTAG GAGTA GACGA AGTGT TACCA C-39) (R 5 mixed basesG 1 A).

Mapping the 59 end by primer extension. Primer extension wasused to map the 59 termini of the antisense transcript. The 32P-end-labeled primer p#375 (59-GTCCC TGACC TTCCC ACCTT GCCTTGGCGA TTTAG-39) (100,000 cpm) and 10 mg of spleen RNA (F1

ouse, 1 year of age) were precipitated and recovered by centrifu-ation. The precipitated nucleic acids were redissolved in 10 ml

hybridization buffer (40 mM Pipes, pH 6.4, 1 mM EDTA, 0.4 M NaCl,and 80% formamide). The hybridization mixture was denatured at85°C for 10 min and quickly transferred to 40°C. The mixture wasincubated overnight at 40°C. After ethanol precipitation, the primer–RNA hybrids were redissolved in reverse transcriptase buffer (50mM Tris–HCl, pH 7.6, 60 mM KCl, 10 mM MgCl2, 1 mM each dNTP,

unit/ml RNase inhibitor). The reaction was incubated with 30 unitsof murine leukemia reverse transcriptase (Life Technologies, Inc.)for 2 h at 42°C. The end-labeled cDNA primer extension productswere analyzed on a 5% urea–polyacrylamide gel.

Oligonucleotide primers for detection of antisense transcript. Se-uences of the primers used for PCR detection of antisense tran-cripts are as follows: p#260 (59-AGTCT GAGAC TTAGC GAGAGAGCC T-39), p#321 (59-TGTGC CTGCA CAAGG TGTCC ACATG-39), p#366 (59-TCATG TGGAC ACCTT GTGCA GGCAC A-39),

p#362 (59-GAATA CAGCT GGTCA ACTCT GGATG AA-39), p#36759-CTTTT CCAGC CAGGG GCAAC TCTGT ATC-39), p#363 (59-TGAC TGTCC TGTGT GAGGC AGGAT A-39), p#371 (59-CACTCCGGC GACCG TGGCC TTCTA-39), p#380 (59-TGGGT CTCAACACT AGCCC ACTCC CCT-39), p#369 (59-GAGAA CAGAAAGTT CCTAA AGTTG TTG-39), p#365 (59-GACCA TGGCT GAAGTAGTC ATCTC T-39), p#372 (59-AGCCA TGCCA TTCGA GACACACAG C-39), p#373 (59-GTATA GGCTC CCAGG CCTGG GCTC-39),#376 (59-TTGCT GGGTT GGTTG GAAGC GGTTG-39), and p#36459-TGAAC ACACT GCATG CCGGA CACTT-39).

DNA sequencing. DNA sequencing was performed on an ABI 377equencer using Big-Dye terminator chemistry (Perkin–Elmer).

RESULTS

Reciprocal Imprinting of P1 and P2in Various Tissues

To study the allelic expression of transcripts fromP1, P2, and P3 promoters in various tissues of the F1interspecific mice, we first attempted to identify poly-morphic sites within Gnas in the C57BL/6 and the M.spretus strains. In human and in mouse, transcriptsfrom P1, P2, and P3 encode three distinct polypeptides(Nesp55, P1; Xlas, P2; and Gsa, P3) by alternativesplicing of the promoter exons (Hayward et al., 1998;

Ischia et al., 1997; Kehlenbach et al., 1994; Peters et

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297IMPRINTED ANTISENSE OF MOUSE Gnas

al., 1999). The P1, P2, and P3 transcripts have exons2–13 in common (Fig. 1A). We sequenced the entireP1–P2 genomic region in both human and in Mus mus-culus (GenBank Accession Nos. AJ251760 and

J251761). To identify polymorphic sites, we furtherequenced the Gnas cDNA from M. spretus liver byT-PCR cloning. An informative site (position 1949 G

o C, GenBank Accession No. AF107848) that creates astUI restriction site in M. spretus DNA but not in57BL/6 DNA was identified in exon 12 (silent muta-

ion in Gnas peptides). This site was used to differen-iate the two parental alleles in all three sense tran-cripts. Transcripts specific for each promoter exonP1, Nesp55; P2, Xlas; and P3, Gsa) were amplified by

using primers that are specific for each promoter exonand a common primer p#290 in exon 13 (Fig. 1B). The

FIG. 1. Gene structure and promoter-specific allelic expression a1 (Nesp55), P2 (Xlas), and P3 (Gnas). Exons 6–13 are simplified.romoter-specific transcripts were amplified by RT-PCR using primroducts were labeled separately by nested PCR with a common primroducts (256 bp) with BstUI revealed M. spretus (99 bases) and C5n both M. spretus and C57BL/6 alleles. The polymorphic BstUI is malas (P2), and Gsa (P3), respectively, in various mouse tissues. In F57BL/6 (maternal) allele; transcripts from P2 were from the M. sp

ranscripts. The cDNA controls of C57BL/6 and M. spretus were fro

primers encompass a large genomic sequence (more P

than 10 kb). Only mature transcripts (cDNA) could beamplified while the genomic DNAs were not amplified(not shown). The PCR products were labeled by a sec-ond nested PCR (five cycles of amplification) withprimer p#256 (in exon 10) and 32P-end-labeled primerp#291 (in exon 13). Digestion of the PCR products (407bp) with BstUI revealed a C57BL/6 allele of 126 basesdue to the presence of an additional nonpolymorphicstUI site) and a M. spretus allele of 99 bases (Fig. 1B).he nonpolymorphic BstUI site served as an internalontrol, demonstrating complete digestion. As shownn Fig. 1C, in tissues of F1 mice that expressed thepecific transcripts, transcripts from P1 (Nesp55) wereerived from the maternal allele (C57BL/6, 126 bases)n cerebral cortex, medulla oblongata, pons, cerebel-um, kidney, and heart. In contrast, transcripts from

e Gnas locus. (A) Map of the Gnas locus showing transcription fromAlternative promoter exons (P1, P2, and P3) are spliced to exon 2.p#242, p#259, and p#240 and a common primer p#290. The PCR

p#256 and 32P-end-labeled p#291*. Digestion of the end-labeled PCR/6 alleles (126 bases). A BstUI site near primer p#256 was present

d (*). (C) Maternal, paternal, and biallelic expression of Nesp55 (P1),1 month of age) mouse, transcripts from P1 were derived from theus (paternal) allele, and both parental alleles were observed in P3erebral cortex.

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298 LI ET AL.

oblongata, cerebellum, and spleen) were derived fromthe paternal allele (M. spretus, 99 bases). Both paren-tal alleles were observed in transcripts from P3 (Gsa)n CNS (cortex, medulla, pons, and cerebellum) and ineripheral tissues (kidney, heart, lung, liver, muscle,ntestine, spleen, thymus, and ovary). In all tissuesxamined, the P3 transcripts from the maternal alleleundigested allele, 126 bases) appeared as strongerands than did those from the paternal allele (digestedllele, 99 bases). This might be due in part to theresence of heteroduplexes that would be resistant toestriction digestion, rather than incomplete digestion,ince the M. spretus allele in the same experimenthowed a complete digestion (Fig. 1C, P3)

issue-Specific Expression of P1, P2, and P3Transcripts

The levels of expression of P1 and P2 transcriptsere very low in various peripheral tissues. To esti-ate the relative abundance of P1, P2, and P3 tran-

cripts from various tissues we utilized a multiplexCR, as previously described (Vu and Hoffman, 1996).he transcripts were amplified simultaneously withhree promoter-specific primers (p#242, p#335, and#240, for P1, P2, and P3, respectively) and a common9 primer p#289 in exon 2 of Gnas (Fig. 2A). Under

identical PCR conditions in a single reaction tube, thethree promoter-specific primers competed for the end-labeled 39 primer. The relative abundance of the PCRproducts therefore reflects the relative abundance ofthe promoter-specific transcripts. In a control experi-

FIG. 2. Expression of Nesp55, Xlas, and Gnas in various mousproducts specific for P1, P2, and P3 differ in size (99, 71, and 190 basregion, Xlas region, and Gsa region in various tissues from a 1-monthp#335, and p#240) and a common 32P-end-labeled primer (p#289*), thLanes P1–P3: RT-PCR using single sets of primers and cDNA from

ment, single primer sets comprising a single promoter-

specific primer and the common end-labeled primerp#289 were used to amplify each promoter-specifictranscript in the CNS (pons) of the F1 mice (Fig. 2B,lanes P1, P2, and P3). A minor band of 165 bp in the P2lane (Fig. 2B) may represent alternative splicing in theXlas region, similar to the human XL-A20-A21 species.All three major transcripts appear in a multiplex PCRof the same CNS sample (Fig. 2B, pons). Transcriptsfrom P1 (Nesp55) are most abundant in CNS, espe-cially in the pons and medulla, and much less abun-dant in the cortex and in the cerebellum. This is inaccordance with a recent study showing that in ratbrain Nesp55 mRNA was detected only in specific neu-ronal cells and not in glial cells. The rat Nesp55 mRNAwas prominent in midbrain and brain stem, but wasabsent in the hippocampus and cerebellum (Bauer etal., 1999). In our PCR experiment the Nesp55 region isminimally transcribed in peripheral tissues. Liver,thymus, and ovary are nearly devoid of the Nesp55transcript (Fig. 2B). In contrast, transcripts from P2and P3 are ubiquitously expressed in all tissues exam-ined (Fig. 2B).

An Antisense Transcript Encompassingthe Nesp55 Region

We searched the GenBank database for mouse ESTsequences matching the 22-kb mouse genomic se-quence (Accession No. AJ251761) and found five ESTsequences within the P1–P2 region (Fig. 3A, ESTs1–5). Strikingly, all five sequences described had thetranscription in an orientation antisense to the P1–P2

issues. (A) Location of specific primers for a multiplex PCR. PCRrespectively). (B) Expression of transcripts derived from the Nesp55

F1 mouse. In a multiplex RT-PCR using 59-specific primers (p#242,CR products reflect the relative levels of P1, P2, and P3 transcripts.

e pons. Lane M, 10 and 100 base DNA ladder.

e tes,-olde P

transcripts. ESTs 4 and 5 (AV247408 and AI047184,

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299IMPRINTED ANTISENSE OF MOUSE Gnas

matching sequences 5077–5304 and 8873–9360, re-spectively, of the mouse genomic sequence) were in theP1–P2 intron sequence. The other three ESTs, 1, 2, and3 (AI561892, AA530580, and AI596956), were from asingle cDNA clone (IMAGE clone 932324, mouse heartlibrary). This putative antisense clone had a 1196-bpinsert and extended from nt 2132 through nt 3327 ofthe genomic sequence. The antisense cDNA sequencethus encompasses the Nesp55 region sequence andincludes a 95-bp intronic region within the Nesp55region sequence (nt 2203–2297) and 156-bp sequencedownstream of the second Nesp55 region exon. ANorthern blot probed with a single-strand RNA probethat was specific for the antisense sequence failed toshow a 1.2-kb band, but rather revealed a broad zonelarger than 5 kb (data not shown). Furthermore, thepresence of the two other antisense ESTs upstream ofthe putative antisense IMAGE clone suggests that theputative antisense transcript originates nearer to P2than nt 3327.

To locate more potential antisense transcripts in theP1–P2 region we scanned the region by amplifying two

FIG. 3. Identification of the antisense transcript associated witcanning antisense transcript in the 22-kb mouse genomic sequenceGenBank Accession No. AJ271761). Eleven primer sets (bars) wer

antisense transcript using seven primer sets (a–g). The cDNAs froantisense transcript. The PCR products from genomic DNA (lanes 1controls (lanes 15–21 and lanes 29–35) demonstrate the presence ofnot e (arrow). Both Nesp55 and Gnas-as were amplified by primer aNesp55. Xlas transcripts were amplified by primer sets f and g. (C) Sshows the PCR products of expected sizes. The RNA transcripts couldare shown in lanes 9–12 and in lanes 17–20. (D) Mapping of the 59 end(p#375) was extended by reverse transcriptase using 10 mg RNA fro

representative cDNAs (cerebral cortex and spleen) us-

ing a panel of seven primer sets (Fig. 3A). Three ofthese amplicon primers (c, d, and e) were upstream ofthe antisense cDNA clone (IMAGE clone 932324),while four other amplicons (a and b, f and g) in theNesp55 region and 59 Xlas region served as controls.Amplification of mouse genomic DNA showed specificbands of expected size for each amplicon (Fig. 3B, lanes1–7, amplicons a–g). The putative antisense transcriptwas detected by amplicons a, b, c, d, but not e (arrow),in both cerebral cortex and spleen cDNA samples (Fig.3B, lanes 8–11 and lanes 22–25). The P2 sense tran-script (Xlas) was detected by amplicons f and g (Fig.3B, lanes 13, 14 and 27, 28), which suggests the exten-sion of the 59 UTR of Xlas region to nt 15256 (GenBankAccession No. AJ 251761) or 282 bp upstream of theextended AUG site (nt 15538). Mock (2RT) PCRshowed no prominent bands (Fig. 3B, lanes 15–21 andlanes 29–35) except for a minor band from a contami-nating DNA.

The human and mouse P1–P2 genomic sequences(Accession No. AJ251760, 20,924 bp, and Accession No.AJ251761, 21,927 bp) display a high degree of sequence

esp55. (A) Locations of ESTs (1–5) and primer sets (a–k) used forive ESTs (arrows) were found by BLAST searching of the databasesed to scan the Nesp55–Xlas region. (B) RT-PCR scanning of the

cortex and spleen (1-month-old F1 mouse) were used to detect theare shown as positive controls. Mock (No-RT) samples as negativeA transcripts that could be amplified by primers a, b, c, and d, but

nes 8 and 22): the top band was P1-antisense and bottom band wasning in the putative 59-antisense region. Genomic DNA (lanes 1–4)

amplified by primer sets h and i, but not j and k. Mock (No-RT) PCRsthe antisense transcript by primer extension. 32P-end-labeled primermouse spleen. C, control using yeast tRNA. M, DNA marker.

h N. Fe um–7)RN(lacanbeof

conservation in the P1–P2 region. A 285-bp region lo-

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300 LI ET AL.

cated 2.2 kb upstream of the extended mouse Xlasregion exon shows 87% identity between the two se-quences. This region contains the 59 end of the humanantisense transcript (Hayward and Bonthron, 2000).To further pinpoint the 59 region of the putative mousentisense, we amplified the cortex and spleen cDNAsith four primer sets encompassing this region (Fig.A, amplicons h, i, j and k). Amplicons h and i areithin sequence homologous to the human antisense

ranscript, while amplicons j and k extend farther up-tream of the antisense. The mouse antisense tran-cript was detected mainly by amplicons h and i inpleen and in cerebral cortex (Fig. 3C, lanes 5, 6 and3, 14). This suggests that both the mouse and theuman antisense transcripts initiate within the regionf high sequence conservation. Finally, primer exten-ion experiments using primer p#375 and RNA frompleen indicated that transcription of the mouse anti-ense starts within ;15 bases of the human putativentisense transcription start site (Fig. 3D).

mprinting and Tissue-Specific Expressionof the Mouse Antisense

We have identified two polymorphic sites in theesp55 region: a nucleotide change that creates the

estriction site HaeIII (location nt 2289, GenBank Ac-ession No. AJ251761) in the C57BL/6 allele (but not inhe M. spretus allele) and an nt 2199 G–T (C57BL/–M. spretus) change. The polymorphic HaeIII site is

ocated within the 95-bp intron of the Nesp55 regionFig. 4). This 95-bp intron is spliced out of the Nesp55RNA (see Discussion below) but is retained in the

FIG. 4. Imprinting of Gnas-as determined by RFLP analysis. (A)95-base intron was used to differentiate the two parental alleles. PCis located in the Nesp55 intron. Therefore only the Gnas-as, contain

M. spretus allele (117 bp) and a C57BL/6 allele (91 bp). (B) Allelic e1 mouse were analyzed. The PCR products from genomic DNA areoth parental alleles were seen in F1 DNA. The cDNA controls ofetectable in lung, liver, muscle, and intestine. Paternal-specific (Mestis. M, 10 b and 100 base DNA ladder.

ntisense transcript because of the lack of consensus t

plicing sites in the antisense sequence. To study thellelic expression of the antisense in various tissues werst coamplified the Nesp55 region sense/antisenseranscripts by primers p#260 and p#324. The antisenseroducts were then selectively labeled by five cycles ofCR using the 32P-end-labeled primer p#339 that is

complementary to the intronic (antisense) sequence.Digestion with HaeIII reveals the M. spretus (117 bp)nd the C57BL/6 (91bp) allele, while the nonpolymor-hic HaeIII site serves as an internal digestion controlFig. 4A). As shown in Fig. 4, with the exception of twoissues (adrenal and testis), Gnas-as is transcribed ex-lusively from the paternal (M. spretus) allele. This isn contrast to the exclusively maternal expression ofhe sense Nesp55 mRNA (Fig. 1C), which excludes theheoretical potential interference of any unsplicedense transcript and/or contaminating genomic DNAn this antisense imprinting analysis. The biallelic ex-ression of antisense in adrenal and testis (this is alsohown in Fig. 5) has been observed in five independentCRs from a 1-year F1 mouse. The biallelic expression

ikely represents the tissue-specific (or polymorphic)oss of imprinting of the antisense in adult mice. Fur-her experiments on mice from different genetic back-rounds and/or different development stages would beeeded to address this tissue-specific loss of imprintingf the Gnas-as. As shown in Fig. 4, the antisense tran-cripts were expressed at different levels in variousissues and were undetectable in some tissues (lung,iver, muscle, and intestine).

To analyze simultaneously the levels of expressionnd imprinting of both the sense Nesp55 region and

ation of primers and PCR products. A polymorphic HaeIII site in theproducts (292 bp) were labeled by end-labeled primer p#339, whichthe Nesp55 intron, was visualized. Digestion with HaeIII revealedession of P1-antisense. Various tissues from 1-year-old interspecificwn as positive controls. The restriction digestion was complete and7BL/6 and M. spretus were from cerebral cortex. Gnas-as was notpretus) expression was evident in all tissues except in adrenal and

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301IMPRINTED ANTISENSE OF MOUSE Gnas

presence of the small (95 bp) intron and the availabilityof the second polymorphic site (nt 2199 G-T) in the firstexon of theNesp55 region. We performed a SNASprimer extension to differentiate the two parental al-leles (Fig. 5A). While the two allelic products differ insize by 10 bases (due to the different lengths of theallele-specific primers), the lengths of the sense andantisense products differ by 95 bases of the intronregion. Both sense and antisense were coamplified (andlabeled) by the same sets of primer. The relative levelsof sense and antisense transcripts were therefore re-flected in the amplified products. Because of the com-petitive nature of the assay, low-abundance Gnas-astranscripts were not seen in kidney and in stomach,while low-abundance P1 transcripts were competed outby Gnas-as transcripts in thymus and in testis(Fig. 5B).

Control genomic DNA demonstrates equal amountsof the C57BL/6 (maternal, 187 bp) allele and the M.spretus (paternal, 197 bp) allele in F1 mice (Fig. 5,DNA). The cerebral cortex cDNAs show the presence ofboth sense and antisense transcripts (C57BL/6, 93 and187 bp, and M. spretus, 103 and 197 bp). As expected,in F1 interspecific mice, the Nesp55 region sense tran-

FIG. 5. Simultaneous analysis of Nesp55 and Gnas-as imprintinsingle nucleotide polymorphism (2199T/G, Accession No. AJ251761) iby RT-PCR using common primers p#296 and p#324. The PCR prop#322T* and p#323G*, which were specific for the T and G alleles. BGnas-as (197-base M. spretus allele and 187-base C57BL/6 allele) we1-year-old F1 mouse. Nesp55 was always expressed from the maternaGnas-as was expressed predominantly from the paternal allele (197 btestis (biallelic expression in testis), but was not detectable in somintestine). Genomic DNAs from C57BL/6 (G/G homozygote), M. sppositive controls for the SNAS assay. The cDNA controls of C57BL/adder.

scripts are exclusively derived from the maternal allele c

(Fig. 5). In contrast, the Gnas-as transcripts are de-rived from the paternal allele except in adrenal andtestis, where they are derived from both parental al-leles. While the b-actin controls suggest that approxi-mately equal amounts of cDNA were analyzed, theabundance of Gnas-as transcripts was greater than thebundance of sense transcripts in thymus and testisFig. 5). Both sense and antisense transcripts are seenn cerebral cortex, adrenal, spleen, and thyroid, andecreased abundance of antisense transcripts was ob-erved in the tissues that express high levels of theense transcript (for example, pons, in which the faintands of 187 and 197 bases may represent genomicNA). Gnas-as is not detectable in a number of tissues,

ncluding lung, liver, muscle, and intestine (see alsoig. 4).

lteration of Imprinting of both Gnas-as and Nesp55by DNA Demethylation

DNA demethylating agents such as 5-aza-cytidine5-aza-C) can decrease the methylation of genomicNA in the nuclei and thereby alter the imprinting ofarious genes. We have shown that 5-aza-C treatment

A) Map of primers and PCR products by SNAS primer extension. Acated in exon 1 of Nesp55. Both Nesp55 and Gnas-as were amplifiedts were labeled by primer extension using 32P-end-labeled primersNesp55 (103-base M. spretus allele and 93-base C57BL/6 allele) andsimultaneously amplified. (B) Imprinting of Nesp55 and Gnas-as inllele (93 bases, C57/BL/6), but was not detected in thymus and testis.s, M. spretus) in most tissues. Gnas-as was abundant in thymus andtissues in which Nesp55 remained imprinted (lung, liver, muscle,s (T/T homozygote), and F1 (G/T heterozygote) were amplified asnd M. spretus were from cerebral cortex. M, 10 and 100 base DNA

g. (s loducothrel aasee

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an modulate the imprinting of the insulin-like growth

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302 LI ET AL.

factor 2 gene (Igf2),3 but not of H19, the reciprocallymprinted gene at the Igf2–H19 locus (Hu et al., 1997).t the Gnas locus, the reciprocal imprinting of Nesp55

egion and Xlas region transcripts is associated withallele-specific methylation of the CpG-rich regions inNesp55 (P1) and Xlas (P2) promoter exons (Peters et

l., 1999). The CpG-rich region upstream of Xlas, ex-tending to the putative antisense transcription site,has been shown to be methylated exclusively on thematernal allele in mouse and human (Peters, 1999;Hayward and Bonthron, 2000). In contrast, the pro-moter exon of the sense (Nesp55) region is methylatedexclusively on the paternal allele. We therefore as-sessed the allelic expression of the sense and antisensetranscripts in 5-aza-C-treated mice. As described pre-viously (Hu et al., 1997), 2-month-old interspecific mice(female F1 mice of M. spretus 3 M. musculus wererossed to M. musculus males) were injected intraperi-oneally with 5-aza-C (2.5 mg/g body weight) on two

occasions 3 days apart and were sacrificed 7 days later.Four of the treated F2 mice that were informative atthe Nesp55 locus were analyzed for the allelic expres-sion of the Gnas antisense and sense transcripts. While

ntreated mice demonstrated reciprocal imprinting ofhe Gnas-as and the Nesp55 transcripts, 5-aza-C-reated mice show biallelic expression of Nesp55 regionranscripts and allelic switching of Gnas-as transcriptsn some tissues (Fig. 6). We have not been able todentify a putative polymorphic site in the 59 region of

the P2 sense transcript that would allow us to assessthe potential alterations of P2 sense imprinting in thetreated mice. The alteration of imprinting of Nesp55transcripts and Gnas-as was observed in some but notll tissues of all treated mice. Although the mechanismy which 5-aza-C alters genomic imprinting in vivo hasot been delineated, we have previously observed sim-

lar modulation of imprinting of the Igf2 gene in 11-ay- and 2-month-old mice (Hu et al., 1997).

DISCUSSION

The Gnas locus represents an excellent model forxploring the mechanism of genomic imprinting. Thewo reciprocally imprinted P1 and P2 regions are lo-ated within a short chromosome segment of fewerhan 15 kb and are 35 kb upstream of the biallelicallyxpressed P3 region. The reciprocal imprinting and theenomic structure of the Gnas locus are conserved in

human and in mouse. Strikingly, the genomic sequenceof the P1–P2 region is also conserved between humanand mouse (Hayward and Bonthron, 2000). Presum-ably, all major cis- and trans-elements governing theimprinting of the Gnas locus are located within thehort segment of the P1–P2 DNA region. Reciprocalmprinting of two adjacent genes is also seen in thegf2–H19 loci (mouse and human), which are 90 kb

3 Abbreviations used: Igf2, mouse insulin-like growth factor II

ene; Igf2r, mouse Igf2 receptor gene.

part (Tilghman et al., 1993). However no apparentequence conservation in the Igf2–H19 intergenic re-

gion was observed between human and mouse (Jinno etal., 1996). The human IGF2 gene also demonstrated anunusual feature of promoter-specific imprinting,whereby imprinted promoters P2–P4 were preceded bya nonimprinted promoter P1 (Vu and Hoffman, 1994).An imprinting maintenance element has been locatedin the 59 region of the mouse Igf2 imprinted promotersHu et al., 1996), suggesting a critical role for theorder DNA between the imprinted and the nonim-rinted promoter regions.The Gnas/GNAS1 locus also shows a unique pattern

of protein expression. By alternative splicing to exon 2of the Gnas gene, transcripts from the three distinctpromoters have in common sequences from exons 2–13,yet encode three distinct proteins. While the Gsa pro-tein is functionally well characterized, the functions ofthe Xlas (the extra large Gsa-like) and Nesp55 pro-eins, both of which are expressed in neuroendocrineissues, remain to be elucidated.

Intriguingly, we now identify a third imprinted tran-cript in the P1–P2 region, both in mouse (this report)nd in human (Hayward and Bonthron, 2000). Themprinted transcript encompassing the P1 region hasn antisense orientation to the P1, P2, and P3 tran-cripts. The human and mouse P1-antisense tran-cripts (Gnas-as) initiate in a region conserved be-ween the two species, which is located ;3 kb

FIG. 6. Alteration of imprinting of both Gnas-as and Nesp55 byDNA demethylation. (A) Map of primers and SNAS products. Notethat in the informative F2 backcross mice reported previously (Hu et

l., 1997), the M. spretus allele is the maternal allele and theBALB/C allele is the paternal allele. (B) Alteration of imprinting ofGnas-as and Nesp55 in 5-aza-C-treated F2 mice. The 5-aza-C-treated F2 mice were reported previously (Hu et al., 1997). In sometissues of the treated mice, both the levels of expression and thepattern of allelic expression were altered. The maternal allele ofGnas-as (197 bp) was observed in lung, intestine, kidney, and spleenin three F2 mice. Both maternal (103 bp) and paternal (93 bp) allelesderived from the Nesp55 region were observed in various tissues ofthe three F2 mice.

upstream of the P2 transcription site. While human

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303IMPRINTED ANTISENSE OF MOUSE Gnas

Gnas-as transcripts are a heterogeneous populationith multiple alternative splicing events that spliceut the NESP55 region, the mouse Gnas-as encom-

passes the Nesp55 region. None of the splicing sites(except the first donor site) of the human Gnas-as areconserved in the mouse Gnas-as. However, there islikely to be a heterogeneous population of mouse anti-sense transcripts, which may account for the broad,fuzzy zone observed on Northern blots (data notshown). The mouse Gnas-as may be as large as 11.2 kb,covering 10 kb of the P1–P2 intronic region and theNesp55 region, although shorter transcripts may alsoexist. In an independent study, Wroe et al. have re-cently identified an imprinted antisense transcript inthe Nesp55 region, which they have named Nespas,hat is expressed in 15.5-day embryos carrying twoaternal copies of distal chromosome 2 (Wroe et al.,000). Nespas was identified by strand-specific PCRnd was confirmed by Northern blot as a 4.4-kb band inewborn heart. Consistent with our results, expressionf Nespas in adult tissues was very low; it was detecteds faintly fuzzy zones by Northern analysis. We haveurther characterized the transcription start site of thentisense, which we named Gnas-as, closer to pro-oter P2, in the conserved 285-base region located

.2-kb upstream of the extended Xlas region. We havealso analyzed the allelic expression of both Gnas-asand the Nesp55 transcript in various tissues in normalF1 interspecific mice.

The Gnas-as in human and in mouse is transcribedfrom the paternal allele in most tissues, in the samefashion as the P2 (Xlas) transcript. The ;3-kb DNAegion between Gnas-as and P2 is methylated on theaternal allele, which is consistent with the paternal

xpression of the Gnas-as. It is not yet known whetheroth Gnas-as and P2 transcripts are regulated by aingle or by two adjacent promoters within this 3-kbNA region. Bisulfite sequencing will reveal the meth-lation status of every CpG site in this region andherefore may reveal any discontinuity between theifferential methylation pattern of Gnas-as and the P2romoter region. In fact, while expression from P2 ismprinted in all tissues tested, biallelic expression ofnas-as was observed in some tissues (adrenal and

estis). Furthermore, expression of Gnas-as is tissue-pecific and is inversely correlated with the expressionf Nesp55 region (P1) sense transcripts in some tissuesthymus, testis). We postulate that the tissue-specificmprinting of Gnas-as might correlate with some spe-ific methylation pattern in the 3-kb region of someissues.

Antisense transcripts are transcribed in the direc-ion opposite to the protein-coding (sense) transcripts.hese antisense transcripts usually encompass exonsf the associated sense mRNA. Since the demonstra-ion in 1997 of the critical role of the imprinted Igf2rntisense in the imprinting of Igf2r (Wutz et al., 1997),our other imprinted genes have been found to be as-

ociated with antisense transcripts. These five anti-

sense transcripts are themselves imprinted, and allfive, surprisingly, are transcribed from the paternalallele. The antisense transcripts of Igf2 (Moore et al.,1997) and Zfp127/ZNF127 (Jong et al., 1999a,b) aretranscribed from the same parental allele as the sensetranscripts. In contrast, in the second group, compris-ing three imprinted genes, Igf2r (Wutz et al., 1997),

BE3A (Rougeulle et al., 1998), and KVLQT (Lee et al.,999; Mitsuya et al., 1999), antisense transcripts areranscribed from the allele opposite to the sense tran-cripts. In this latter group, the expression–competi-ion model (Barlow, 1997) can be applied to explain theutual allelic-exclusion expression. It is likely that the

ntisense must be transcribed at “competitive” levelso knock out the sense transcription on the same allele.

hile the expression levels of antisense in UBE3A andVLQT loci are not known, relatively high levels ofoth Igf2r antisense and Igf2r sense transcripts haveeen observed (Wutz et al., 1997; Hu et al., 1999).The Gnas-as in the Gnas locus in this report belongs

o the latter group. Both Gnas-as and Nesp55 region(sense) transcripts are abundant in various tissues(Fig. 5). The reciprocal allelic expression of Gnas-asand Nesp55 region transcripts in various tissues seemsto lend support for the expression–competition model.However, in some tissues [lung, liver, muscle, intestine(Fig. 5)], the imprinting of Nesp55 sense transcripts isstrictly maintained even in the absence of the Gnas-astranscripts. Furthermore, the imprinting of Gnas-as isalso maintained in the absence of the Nesp55 sensetranscripts (Fig. 5, thymus). We have also recentlyreported the lack of reciprocal imprinting of Igf2r an-tisense and Igf2r in mouse CNS tissues (Hu et al.,1999). Clearly, the tissue-specific imprinting ofGnas-as and the lack of coordination of reciprocal im-printing of the Nesp55 sense and Gnas-as in sometissues add another level of complexity to the imprint-ing at the Gnas locus. Our results suggest that multi-

le imprinting elements in the 15-kb P1–P2 region, inddition to the unique Gnas-as, may regulate the al-elic expression of the Gnas transcripts.

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