Reverse Serial Analysis of Gene Expression (SAGE) Characterization of Orphan SAG - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:07 |只看该作者 |倒序浏览 |打印

作者：Mark Richardsa, Siew-Peng Tana, Woon-Khiong Chanb, Ariff Bongsoa作者单位：a Department of Obstetrics and Gynaecology, National University of Singapore, National University Hospital, Singapore;b Department of Biological Sciences, National University of Singapore, Singapore
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      【摘要】2 d1 ]: J) ?8 H4 s7 G, ~  m' J
   Serial analysis of gene expression (SAGE) is a powerful technique for the analysis of gene expression. A significant portion of SAGE tags, designated as orphan tags, however, cannot be reliably assigned to known transcripts. We used an improved reverse SAGE (rSAGE) strategy to convert human embryonic stem cell (hESC)-specific orphan SAGE tags into longer 3' cDNAs. We show that the systematic analysis of these 3' cDNAs permitted the discovery of hESC-specific novel transcripts and cis-natural antisense transcripts (cis-NATs) and improved the assignment of SAGE tags that resulted from splice variants, insertion/deletion, and single-nucleotide polymorphisms. More importantly, this is the first description of cis-NATs for several key pluripotency markers in hESCs and mouse embryonic stem cells, suggesting that the formation of short interfering RNA could be an important regulatory mechanism. A systematic large-scale analysis of the remaining orphan SAGE tags in the hESC SAGE libraries by rSAGE or other 3' cDNA extension strategies should unravel additional novel transcripts and cis-NATs that are specifically expressed in hESCs. Besides contributing to the complete catalog of human transcripts, many of them should prove to be a valuable resource for the elucidation of the molecular pathways involved in the self-renewal and lineage commitment of hESCs. 4 d0 z$ j4 }( S. ^5 q
      【关键词】 Reverse serial analysis of gene expression Human embryonic stem cells Transcriptome Antisense transcription POUF SOX NANOG' m) P6 R9 V; M# c# E% F. d: F
   INTRODUCTION
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Pluripotent human embryonic stem cells (hESC) cell lines are derived from fibroblast feeder layers via the isolation and extended serial propagation of the inner cell mass from supernumerary 5-day-old blastocysts .

SAGE is a sequence-based transcriptome profiling approach that provides qualitative and quantitative assessment of gene expression .

Another major source of uncertainty in SAGE tag-to-transcript assignment lies in the widespread presence of single-nucleotide polymorphisms (SNPs) within the human genome; SNPs occur as frequently as once every 100¨C300 bases .

Naturally occurring antisense transcripts (NATs) have been recently reported in a variety of metazoan species .

Without additional sequence information, it is difficult to characterize orphan SAGE tags from hESCs and identify the transcripts they represent. Several polymerase chain reaction (PCR)-based strategies have been developed, including reverse SAGE (rSAGE) , and used it to obtain additional 3' cDNA sequence information for a select group of orphan SAGE tags that are expressed specifically in hESCs. Our results identified novel transcripts unique in their expression to hESCs, transcripts that displayed alternative polyadenylation, and novel splice variants of known genes. More importantly, we found NATs for several pluripotency genes, including POU5F1 and NANOG. Collectively, the unique 3' ESTs derived from orphan hESC SAGE tags (HESTs) will be an important resource in downstream functional analyses and the concerted dissection of molecular pathways critical to the pluripotent phenotype of hESCs.! P5 B( L  c$ Z/ Z3 a" e

MATERIALS AND METHODS
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Culture of hESCs- g" l2 A. k! T' o3 o) l

hESCs (HES3 line, passages 19¨C25; ES Cell International, Singapore, http://www.escellinternational.com) were cultured on a feeder layer of mitomycin-C inactivated mouse embryonic fibroblasts (MEFs) as described previously .

Total RNA Isolation
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Total RNA was extracted from hESCs using TRIZOL (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), whereas total RNA from the various somatic and fetal tissues were obtained commercially (Clontech, Palo Alto, CA, http://www.clontech.com). Prior to rSAGE library construction or reverse transcription (RT)-PCR, total RNA was treated with DNase I (Ambion, Austin, TX, http://www.ambion.com) to remove any residual genomic DNA contamination, and PCR using ß-actin primers (forward, 5'-GATGCAGAAGGAGATCACTGC-3'; reverse, 5'-CACCTTCACCGTTCCAGTTT-3'), designed to span the last intron-exon boundary of the gene, was carried out to confirm the absence of genomic DNA.! X% x4 c; p1 x2 m
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cDNA Synthesis, NlaIII Digestion, and Linker Ligation
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A schematic for the rSAGE library construction with all primer and linker sequences is depicted in Figure 1. cDNA synthesis was carried out using the Superscript II double-stranded cDNA synthesis kit (Invitrogen) with 10 µg of total RNA from HES3 cells and a biotinylated primer was used (5'-biotin-ATTGGCGCGCCGCGAGCACTGAGTCAATACGAT30VN- 3'; Integrated DNA Technologies, Coralville, IA, http://www.idtdna.com). Double-stranded cDNA was digested with NlaIII (New England Biolabs, Ipswich, MA, http://www.neb.com) to generate 3' overhangs. The biotinylated cDNAs were immobilized on streptavidin-magnetic beads (Invitrogen). Annealed linkers, A1 (5'-AAGCAGTGGTATCAACGCAGAGTCATG-3') and A2 (5'-phosphate-ACTCTGCGTT-GATAC-CACGCTT-aminoC7-3') were ligated to the 5' end of NlaIII-digested cDNA before AscI (New England Biolabs) digestion was performed to release the 3' cDNA fragments from the streptavidin-magnetic beads.

Figure 1. Schematic diagram of the modified rSAGE protocol. Briefly, mRNA was isolated, and cDNA synthesis was performed with an anchored biotin-labeled RT primer. cDNAs were digested with NlaIII to reduce complexity of the library. An rSAGE linker was next ligated to cleaved 3' cDNAs bound to streptavidin beads, following which AscI digestion was performed to release the cDNAs. rSAGE library scale-up amplification was performed with the rSAGEF1 and rSAGER1 primers. An aliquot of the amplified rSAGE library was used in rSAGE amplifications with a serial analysis of gene expression tag-specific primer and the common Rev1 reverse primer. Abbreviations: HEST, human embryonic stem cell serial analysis of gene expression tag; PCR, polymerase chain reaction; rSAGE, reverse serial analysis of gene expression; RT, reverse transcription; TSP, tag-specific primer.
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PCR Scale-Up of rSAGE Library
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Amplification of the primary rSAGE library was performed with 1 µl of the NlaIII-digested cDNAs, 5 U of Platinum Taq Polymerase (Invitrogen), rSAGEF1 (5'-AAGCAGT-GGTAT-CAACGCAGAGT-3') and rSAGER1 (5'-GCGAGCACT-GAGTCAATACGC-3') primers (350 ng each). After an initial denaturation at 94¡ãC for 2 minutes, PCR was carried out for 25 cycles at 94¡ãC for 45 seconds, 57¡ãC for 1 minute, and 72¡ãC for 1 minute, with a final extension at 72¡ãC for 5 minutes.
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Selection of Orphan SAGE Tags and Design of Tag-Specific rSAGE Primers+ f" c+ i& p) I, q. E

The 200 orphan SAGE tags selected for rSAGE were identified through a pairwise comparison of HES3 SAGE data against pooled data from 21 human SAGE libraries . Typically, they included the entire 21 bases of the Long-SAGE tag or they included additional four to eight bases of the common linker (CGCAGAGT) and up to 19 bp of the Long-SAGE tag. If no appropriate LongSAGE tag was available (Tag IDs 1¨C77), the TSRPs were designed with seven bases of the common linker sequence (GCAGAGT) and the entire 14 bases of the SAGE tag, with the exception of Tag IDs 30 and 72.

rSAGE Amplification Reaction and Characterization of 3' rSAGE Fragments+ z% D# }4 ^9 ]
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Touchdown PCRs were performed using an initial denaturation cycle at 94¡ãC for 2 minutes, followed by four cycles at 94¡ãC for 45 seconds, 63¡ãC for 1 minute, and 72¡ãC for 1 minute; four cycles at 94¡ãC for 45 seconds, 60¡ãC for 1 minute, and 72¡ãC for 1 minute; 25 cycles at 94¡ãC for 45 seconds, 58¡ãC for 1 minute, and 72¡ãC for 1 minute; and a final extension step at 72¡ãC for 5 minutes. The reaction setup for rSAGE PCR was as follows: 1 µl of amplified rSAGE library, 1 U of Platinum Taq Polymerase, 350 ng of TSRP and rSAGER1 primer. The PCR products were run on 1.2% TAE agarose gel, and the bands were excised and purified using QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, http://www.qiagen.com). Purified PCR products (2¨C4 µl) were ligated into the pGEM-T Easy Vector (0.5 µl) (Promega, Madison, WI, http://www.promega.com) using T4 DNA ligase. The ligation reaction was incubated overnight at 16¡ãC and resuspended in 8 µl of sterile water. Electroporation was performed using 1 µl of the ligated products and 25 ml of pTOP10 cells (Invitrogen). The transformants were plated on selective media, and two to four clones were picked for each rSAGE PCR product. Plasmid DNA was extracted using QIA-prep Spin Miniprep Kit (Qiagen). Sequencing reactions were carried with Big Dye v3.1 (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com) and M13 Forward primer. The sequenced products were analyzed on an ABI 3100 DNA Sequencer (Applied BioSystems).

Sequence Analysis and Identification of Genuine rSAGE PCR Products

A bona fide 3' rSAGE product was defined as possessing the entire SAGE tag sequence, the rSAGER1 primer sequence and a poly(A) tract of >10 adenine residues. Sequences that lacked any one of the three were considered nonspecific amplification artifacts and omitted from further analysis. The rSAGE 3' EST sequences were searched against the GenBank Database (NR, dbEST, and human genome) using BLASTN (http://www.ncbi.nlm.nih.gov/BLAST/), the University of California Santa Cruz human genome browser database (May 2004 build) using the BLAT program (http://genome.ucsc.edu/cgi-bin/hgBlat) and the EMBL database using a web interface-based batch BLAST program (http://biomedicum.csc.fi:8010/cgi-bin/batchblast.cgi) .+ o  s# c$ G  _) o
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An rSAGE sequence was classified as novel if no matches to a transcript sequence (known gene, mRNA, or EST) were found. A sequence was considered to represent a known gene if it matched a full-length transcript sequence with >95% similarity in the same orientation. A sequence was classified as known EST if it matched an EST or open reading frame (ORF) with >95% similarity in the same orientation. A sequence was classified as an SNP alternative tag if it contained a single-bp mismatch within the SAGE tag sequence or NlaIII site. A sequence was classified as an insertion/deletion if it contained an insertion or deletion of fewer than three nucleotides within the SAGE tag sequence. A sequence was classified as an anti-sense transcript if it matched with high similarity to known transcripts in the opposite orientation. A sequence was classified as poly(A) if it was near the end of the poly(A) tract. Finally, a sequence was considered an alternative isoform if it matched the middle of known full-length transcripts in the same orientation and contained a poly(A) track immediately downstream of the matched region. Genomic coordinates of the 3' SAGE ESTs were annotated based on the University of California Santa Cruz genome browser annotation database (http://genome.ucsc.edu/).
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RT-PCR Confirmation of Novel 3' cDNAs

First-strand synthesis was performed using the SuperScript first-strand synthesis system (Invitrogen). One µl of first-strand reaction was used for each PCR together with 50 pmol of forward and reverse primers. Initial denaturation was carried out at 94¡ãC for 2 minutes, followed by 30 cycles of PCR (94¡ãC for 30 seconds, 55¡ãC for 30 seconds, 72¡ãC for 1 minute), and a final extension cycle at 72¡ãC for 5 minutes. PCRs were loaded on a 1.5% agarose gel and size fractionated. In instances where the 3' cDNA sequence obtained was short and no suitable primer pairs could be found, additional 5' genomic sequences were used to anchor the forward primers. In all cases, the reverse primer primed from the rSAGE 3' cDNA sequence. Primers used were as follows. ACTB: product 400 bp, 5'-TGGCACCACACCTTTCTACAAT-GAGC-3', 5'-GCACAGCTTCTCCTTAATGTCACGC-3'; POU5F1: product 247 bp, 5'-CGRGAAGCTG GAGAAG-GAGAAGCTG-3', 5'-CAAGGGCCGCAGCTTACACAT-GTTC-3'; HEST97: product 160 bp, 5'-CCTTTGTCATGAGC-CCTTGT-3', 5'-GGAATGAAAGAATGGTTG CTC-3'; HEST101: product 119 bp, 5'-AAGAGCCTGCTACG-GAACTG-3', 5'-TCACTAGAGGTTTCCAACACACTT-3'; HEST120: product 159 bp, 5'-AAATTTGGTGCTGTGAC TCG-3', 5'-GCGGGCTGAGTCGGATTT-3'; HEST123: product 200 bp, 5'-GGGTTATGT GTAGAAACCAAGTGA-3', 5'-TCTTAGAACTTATGATACACCCAGTTG-3'; HEST127: product 218 bp, 5'-GGGAAAAGATGGCAAGGTTA-3', 5'-AATATATTCGAGTCACATCA TGACA-3'; HEST146: product 171 bp, 5'GATGCCATCACTCAAACTAGACC-3', 5'-GACGTCCTATGCAGGCATTT-3'; HEST147: product 205 bp, 5'GGGGATTCGAGGTTC CTGTA-3', 5'-CATTTCAAG-GCACAATTTTAATAGC-3'; HEST149: product 196 bp, 5'-CCCAGGCTGAAGTGTAGTGA-3', 5'-CATTTACAATGGTA-CAAGGAGCA-3'. The universal reference RNA sample was obtained from Stratagene (La Jolla, CA, http://www.stratagene.com), and somatic tissue RNA samples were obtained from Clontech.

Orientation-Specific RT-PCR# d9 h) A! X4 @8 ^, _/ C
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To detect the NATs for POU5F1, NANOG, LIN28, TALE, TERF1, and TERA, orientation-specific first-strand cDNA synthesis was carried with the appropriate sense primers. Thereafter, Superscript II RT was heat-inactivated at 95¡ãC for 15 minutes. PCR was performed with 3 µl of the 20-µl first strand mix as described. Control experiments without reverse transcription (¨CRT controls) for each of the three antisense primers were performed to detect genomic DNA contamination. The primers used were as follows. POU5F1 NAT: product 184 bp, 5'-AGTTTGTGCCAGGGTTTTTG-3', 5'-TGTGTCCCAG-GCTTCTTTATTT-3'; NANOG NAT: product 278 bp, 5'-TCGGTATTGTTTGGGATTGG-3', 5'-TCATCGAAAC-ACTCGGTGAA-3'; LIN28 NAT: product 178 bp, 5'-GGAGGCCAAGAAAGGGAATA-3', 5'-CCGCCCCATA-AATT CAAGAT-3'; TALE NAT: product 80 bp, 5'-TTTTCA-GACTGTGCAATA CTTAGAGAA-3', 5'-TTAGACAG-TATGTGGGCATCC-3'; TERF1 NAT: product 169 bp, 5'-TGCGGAGT AGATGAGATGGA-3', 5'-AAGGCAATG-GAAAACAGGTAAA-3'; TERA NAT: product 131 bp, 5-TTT-TGGCTGCAGTATTGGTG-3', 5'-CATCCTACAGGC-AAAGAGAGG-3'.

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rSAGE Amplification, Specificity, Efficiency, and Size Distribution of 3' cDNAs

The original rSAGE (Kinzler/Vogelstein laboratories) , and our modified rSAGE strategy share several key features (Fig. 1). However, we have made several modifications to increase the efficiency of 3' cDNA conversion. For instance, changes in the design of the universal primers allowed the rSAGE library scale-up and the subsequent TSRP PCR amplification to be carried out at an increased melting temperature (Tm). The introduction of a longer poly(T) tract (T30) and the inclusion of VN dinucleotides in first strand RT-PCR primer allowed a better trapping and synthesis of full length mRNAs at their 3' ends, compared with a shorter poly(T) tract (T10) as used in the GLGI strategy that might result in the primer binding to internal poly(A) residues within mRNA transcripts. Finally, increasing the Mg2  concentration when no distinct rSAGE band was observed in the first round of PCR could occasionally enhance the specificity of the rSAGE amplification reaction.
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Of the 200 HES3 orphan SAGE tags that were selected for rSAGE conversion (supplemental online Table 1), 168 (84.0%) yielded PCR amplification products (Fig. 2A). The conversion rate of orphan LongSAGE tags into longer 3' cDNA fragments was much higher (93.4%) than that of the SAGE tags (69.2%). We attributed these improvements to the availability of additional sequences from the LongSAGE tags for the design of TSRPs, as well as better-designed universal primers (rSAGEF1 and rSAGER1) in our strategy (Fig. 1). In particular, we found the universal M13 primer used as the antisense primer in the original rSAGE strategy  was unsatisfactory for rSAGE because of its low Tm.

Figure 2. Results of reverse serial analysis of gene expression (rSAGE) amplification for 200 orphan serial analysis of gene expression (SAGE) tags. (A): Pie chart shows the distribution of rSAGE products. (B): rSAGE reactions were carried out using the tag-specific rSAGE and rSAGER1 primers, the products were analyzed on an agarose gel, and the bands were visualized with ethidium bromide. Most lanes show a single distinct amplified rSAGE band. A 100-bp ladder (M) was used as a molecular weight marker. The numbers at the top of the gel represent the SAGE Tag ID. Abbreviations: EST, expressed sequence tag; M, molecular weight marker; PCR, polymerase chain reaction.
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A representative agarose gel showing the rSAGE products is shown in Figure 2B. It is noteworthy that the majority (~90%) of the TSRPs yielded only a single distinct rSAGE band. Our results also support the notion that there is no strict correlation between the efficiency of target template amplification and the abundance of the SAGE tag .5 M1 S" v0 A' X% w" c
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From the 168 SAGE tags that yielded PCR amplification products, a total of 196 rSAGE products were cloned and sequenced. Of these, 148 (75.5%) were confirmed as specific rSAGE products following DNA sequencing, BLAST and BLAT confirmation (supplemental online Table 2). These 148 rSAGE 3' cDNA fragments have been submitted to GenBank (accession numbers DN604327  and for the present rSAGE library construction were grown on MEF feeders, we did not find contaminating murine RNA transcripts a significant problem in our 3' rSAGE conversion attempts.; f- o; |3 _! n- G! R

Overall, 16.0% of rSAGE reactions failed to give distinct amplification products. Taken together with the nonspecific rSAGE results, our main conclusion is that a SAGE tag does not always provide an ideal sequence for the design of thermodynamically favorable TSRPs for the efficient amplification of 3' cDNA by rSAGE. Thus, orphan SAGE tags that were AT-rich or contained sequences that were self-complementary often failed to generate specific rSAGE 3' cDNA fragments. Although it is possible that when the expression level of targeted templates is very low, partial annealing of the TSRPs with other highly expressed templates may result in nonspecific amplification  would allow most of the remaining orphan SAGE tags to be converted into longer 3' cDNA fragments for gene identification.+ e8 |& F( `6 z  w6 w9 k- O
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Analysis of 3' HESTs Generated from HES3 Orphan SAGE Tags

The size distribution of the 148 HESTs ranged from 36 to 538 bp, with 56.7% of them longer than 100 bp, which matched well to the reported data from GLGI-SAGE studies . We used both BLAT and BLAST searches to establish the identity of rSAGE cDNA sequences (Fig. 3A). Indeed, the BLAT transcript viewer made it easier to visualize and quickly identify NATs, novel introns, and new splice variants of known transcripts and to confirm SNPs within the SAGE tags. For several SAGE tags, rSAGE extension resulted only in poly(A) sequences, as a result of the NlaIII site occurring just adjacent to the poly(A) tract, and would require the use of a different tagging enzyme to reveal their true identity. More importantly, our rSAGE results have clearly identified 59 of these rSAGE 3' cDNA fragments as novel rSAGE 3'ESTs and 30 NATs, all of which are identified for the first time (Fig. 3A).4 L% G( o7 t. @3 Z2 ^8 L2 D0 z. n$ b/ R

Figure 3. Identity of the 148 rSAGE 3' cDNA fragments. (A): The distribution of the various categories of rSAGE products is summarized as a pie chart. (B): Human embryonic stem cell (hESC)-specific expression of eight HESTs were verified with semiquantitative reverse transcription-polymerase chain reaction (PCR) using total RNAs prepared from several peripheral adult tissues and fetal brain, universal reference RNA (Stratagene), undifferentiated HES3 and HES4 hESC lines, and D-HES3 cells. (C): Quantitative real-time PCR results for GJA1 SNP analysis. Abbreviations: bp, base pairs; CT, threshold cycle; EST, expressed sequence tag; FAM, 6-carboxyfluorescein; INDEL, insertion/deletion; rSAGE, reverse serial analysis of gene expression; SNP, single-nucleotide polymorphism.1 U8 c8 o: s% A8 V' O5 d- u0 g0 ?4 w
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The majority of the novel rSAGE 3'ESTs that mapped to specific chromosomal locations also contained the canonical polyadenylation signal, AATAAA or its functional variant , prove to be a reliable marker for monitoring the early differentiation of hESCs. Indeed, HEST149 expression was undetectable in the universal reference RNA sample, which is an RNA pool from several cancer tissues (Fig. 3B) and absent in several embryonal carcinoma lines such as GCT-27C4, GCT-27X1, and GCT-44 (unpublished results).. ]6 y3 S! P) ]8 A1 W) s
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Table 1. Chromosomal location and SAGE library representation of 18 novel 3' reverse SAGE expressed sequence tags with authentic polyadenylation signal  ~6 z4 c8 e' |+ r. L; z2 y
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In addition, a number of the novel 3' rSAGE cDNA fragments (e.g., HESTs 73, 92, 102, and 126) could not be matched reliably to the human genome and were also not the products of contaminating MEF cDNAs. Perhaps these HESTs represent transcripts from novel hybrid RNAs with a regulatory function or as yet undiscovered genes. The presence of consensus polyadenylation sites on several of these HESTs (e.g., 92, 102, and 126) is a good indication that these are authentic transcripts.& G8 m, {, a+ V+ U" r: n7 ?

Interestingly, four HESTs (112, 120, 128, and 170) showed high sequence similarity to the WiCell hESC ESTs , their exact functional role is unknown.
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The impact of SNPs on the correct assignment of SAGE tags to specific transcripts  is also illustrated by our rSAGE results. For instance, HEST49 matched the CHD8 with almost 100% sequence similarity and is the result of an SNP that created a new NlaIII restriction site upstream of the AATAAA polyadenylation site. The full-length cDNA sequence of CHD8 is 8,160 bp long, and this SNP would generate the C-most SAGE tag. The original C-most SAGE tag for CHD8 is GGC-CCCATTG (nts 7311¨C7320), which is also represented in the HES3 SAGE library (5 tpm). We also detected an SNP within the C-most SAGE tag of GJA1, which encodes the gap junction protein connexin 43. The putative C-most SAGE tag is TGT-TCTGGAG (nts 2916¨C2925). The rSAGE conversion of the orphan SAGE tag, TGTTTTGGAG, resulted in HEST113, which displayed a 97% sequence similarity to the 3' terminal region of the GJA1 coding region. Careful examination of corresponding EST and genomic DNA sequences indicated that this orphan tag most likely represented an SNP in the canonical GJA1 SAGE tag and not the hypothetical protein FLJ10407 as suggested by the predicted tag-to-gene mapping of SAGEGenie. The GJA1 SNP was verified using 6-carboxyfluorescein (FAM)- and VIC-labeled Taqman probes that were specific to the polymorphism (Fig. 3C).5 a8 D3 n9 ]8 \5 b) x9 V9 q
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The generation of longer 3' cDNA sequences by rSAGE has also helped to resolve some of the ambiguities in tag to gene assignments, at least in HES3 cells. For example, HEST119 (AGTGAGGATA) matched the hypothetical protein FLJ35155 (C3orf21), which is restricted in expression to hESC lines and tissues of cancerous origin. In addition, the SAGE tag for HEST114 (CATCCAAAAA) was incorrectly assigned to NPY and CEP2 by SAGEGenie and SAGEMap, respectively. Instead, rSAGE conversion confirmed that HEST114 matched to the hypothetical protein FLJ10884, a hypothetical protein restricted in its expression to the testis, placenta, and hESC lines, instead of NPY.' Z0 F* j9 _5 c, R/ j  T$ T0 F
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Antisense Transcription in hESCs
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BLAT and BLAST searches revealed that many of the HESTs were the products of antisense transcription. Interestingly, cis-NATs for several important ES-specific genes, such as NANOG (HEST16), POU5F1 (HEST88), and LIN28 (HEST168), were identified by our rSAGE results (supplemental online Table 2). Analyzing the chromosomal location of these cis-NATs and the corresponding sense tags from the HES3 library revealed the presence of sense-antisense (SA) gene pairs . Table 2 is a list of 18 SA SAGE tag pairs and the corresponding antisense HESTs that were experimentally obtained with rSAGE. Although several SA SAGE tag pairs can be mapped in trans to remote genomic loci, other pairs mapped in cis on contiguous oppositely oriented DNA strands (Fig. 4A). Besides POU5F1, NANOG, and LIN28, a number of other highly expressed hESC-specific genes, like TGIF/TALE (HEST109), ERH (HEST151), TERA (HEST155), and TERF1 (HEST193.2), also expressed cis-NATs. Furthermore, the representation of many of these co-expressed SA SAGE tag pairs decreased upon differentiation of the hESCs (Table 2). The SAGE tags for NANOG (TCATTACGAT) and POU5F1 (ATGTGGGATT) cis-NATs were found only in hESC SAGE libraries, indicating that the expression pattern of cis-NATs for NANOG and POU5F1 are even more restricted than their sense transcript counterparts., k$ t4 f- l6 m% N

Table 2. Sense-antisense SAGE tags pairs of antisense HESTs* h7 M  w0 g8 Y2 r* v
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Figure 4. Confirmation of natural antisense transcription in HES3 cells. (A): Illustration of the cis- and trans-serial analysis of gene expression AS tag pair concept. (B): Expression of POU5F1, NANOG, LIN28, TALE, TERA, and TERF1 cis-natural antisense transcripts (NATs). For amplification of cis-NATs, sense-specific primers were used for reverse transcription (RT) instead of oligo(dT) primer. During the subsequent polymerase chain reaction amplification, sense and antisense primers were used. Total RNA that had not been reverse-transcribed was used as a template control for genomic DNA contamination (¨CRT). Abbreviations: AS, antisense; bp, base pairs.5 p6 u" s& u3 v& R, R1 X+ s0 a- w4 h

To validate that the cis-NATs for POU5F1, NANOG, LIN28, TALE, TERF1, and TERA were specifically in hESCs, orientation specific RT-PCR  was carried out using total RNA isolated from HES3, a universal reference RNA sample (Stratagene), testis, and stomach (Fig. 4B). First strand cDNAs were prepared using primers specific to POU5F1, NANOG, LIN28, TALE, TERF1, and TERA, respectively. Specific RT-PCR products for all three cis-NATs were detected only when RT was included, thus confirming that these cis-NATs were specifically expressed in hESCs and not due to spurious PCR amplification.

HEST115 and 168 appeared to represent spliced SA transcripts from ILF2 and LIN28, respectively. Nucleotides (nts) 1¨C42 of HEST115 matched the ILF2 coding region in the antisense orientation (Chr1. The structure the cardiac troponin I "hybrid RNA," which the authors themselves have tentatively concluded to be formed from the transcription of the troponin mRNA in the cytoplasm, is very similar to what we have described for ILF2 and LIN28. The functional significance of these hybrid RNAs is currently unknown.
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DISCUSSION; C& u! g4 v# r& }' P8 U6 P2 X. u& N' {
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Unlike DNA microarray, SAGE does not require prior knowledge of the sequences to be analyzed. Hence, SAGE libraries provide discreet and unbiased directional gene expression data that are ideally suited for gene discovery and SA expression analysis .

Although the human transcriptome is necessarily less complex than the human genome, it is quite apparent that transcriptome complexity has been underestimated .
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A recent study reported that 41.5% of SA transcript overlaps occurred in the last exon or untranslated region (UTR) of the coding sequence . However, because there are several pseudogenes for POU5F1 and NANOG, the possibility of trans-NATs from these genomic loci remains to be determined.5 {$ D1 s' Z& `% g' D. ~* M

Several reports have hinted that the contribution of NATs in the human genome has been underestimated .
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Although certain human miRNAs (miR-1 and miR-124) have been recently demonstrated to influence and define tissue-specific gene expression profiles in HeLa cells . We failed to find SAGE tags representing UTF1, REX1, LEFTB, and GDF3 cis-NATs in human and mouse SAGE libraries. However, we detected cis-NATs for a number of key ES-specific genes (e.g., FGFR1, FGFR2, TDGF1, SOX2) in HES3 and SAGE libraries constructed from other hESC lines (Table 3). In addition, SAGE tags representing pou5f1, nanog, tera, and lin28 were also detected in mouse embryonic stem cells (mESCs). In summary, cis-NATs for a number of ES-specific genes, such as POU5F1 and NANOG, were shown to be expressed in both hESCs and mESCs, and it is possible that some of these cis-NATs might have a role in maintaining the "stemness" phenotype of ES cells.
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Table 3. Occurrence of SAGE tags of cis-natural antisense transcripts of selected embryonic stem-specific genes in human and mouse embryonic stem cell SAGE libraries% I2 i9 N# x( e6 O% e* w

Our study further underscores the importance of obtaining longer 3' cDNAs from orphan SAGE tags and the versatility of rSAGE as a powerful complementary tool to SAGE expression libraries for gene discovery. Lastly, the hESC-specific transcripts that we have described are clear targets for further study, and the conversion of the remaining orphan SAGE tags from HES3 and other hESCs would likely provide additional valuable resources, mainly in terms of novel transcripts, and uncover additional cis-NATs for the in-depth functional dissection of the molecular pathways involved in the self-renewal of pluripotent hESCs and their subsequent lineage commitment to their differentiated progenies.

ACKNOWLEDGMENTS
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This study was supported by Embryonic Stem Cell International Pte. Ltd. grant R-174-000-081-592 and National University of Singapore Academic Research Fund grant R-154-000-179-112.; \: C6 v8 B7 L" i' p

DISCLOSURES* `. k0 [2 }: e( K$ d9 T

The authors indicate no potential conflicts of interest.! e! d! v: v& u$ |
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