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Originally published In Press as doi:10.1074/jbc.C500415200 on November 15, 2005

J. Biol. Chem., Vol. 281, Issue 1, 16-19, January 6, 2006
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Adult Tissue-specific Expression of a Dppa3-derived Retrogene Represents a Postnatal Transcript of Pluripotent Cell Origin*

Stephen J. Elliman{ddagger}1, Isaac Wu§1, and Daniel M. Kemp{ddagger}2

From the {ddagger}Diabetes and Metabolism Disease Area, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139 and the §Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received for publication, October 14, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Processed pseudogenes emerge by reverse transcription of spliced mRNAs followed by incorporation of the resultant cDNA into the genome. Their genesis requires that retrotransposition occurs within the germ line, a provision that significantly limits random distribution of source genes. We previously identified embryonic stem cell-specific genes as an enriched source of retropseudogene origin. Nanog, Oct4, and Dppa3 (Stella/PGC7) presented as source genes for >30 processed pseudogenes within the human genome. In the current study, we extended our previous analysis and focused on the pluripotent cell-specific Dppa gene family. Of the five Dppa genes characterized, four were associated with putative retropseudogenes as determined by nucleotide BLAST (basic local alignment sequence tool) searches of the respective mRNA transcripts against the human genome. A subset of the 11 Dppa3-derived hits were then screened against a human adult tissue cDNA panel for evidence of transcriptional activity. One of the putative Dppa3-derived retropseudogenes, Dppa3(d), located on human chromosome 16p13, tested positive for mRNA transcript in bone marrow, peripheral blood, pancreas, adrenal gland, and thyroid gland. Specificity against the source Dppa3 gene expression was sequence verified, and independent human tissue samples were obtained to confirm Dppa3(d) expression. These data substantiate the existence of human adult tissue-specific transcripts that originate via retrotransposition of the pluripotent cell-specific gene, Dppa3. Further studies may reveal an evolutionary role for this example of genetic diversity, but in the short term our observations serve a cautionary purpose regarding the use of Dppa3 transcripts in adult tissue-derived cells as a potential marker of pluripotency.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Processed pseudogenes (retropseudogenes) emerge by reverse transcription of processed mRNA and consequent genomic integration (1–3). Given the absolute requirement for germ line transmission to realize a processed pseudogene, we hypothesized that pluripotent tissue-specific gene transcripts may possess a competitive advantage over non-germ cell-specific genes with respect to inherited retrotansposition. Previously, we reported the excessive frequency and apparent selectivity of retrotransposition of pluripotent cell-specific genes. Six highly homologous pseudogenes for Oct4, 10 for Nanog, and 16 for Dppa3 (Stella/Pgc7) were identified by nucleotide BLAST3 (basic local alignment sequence tool) searches against the respective gene mRNA transcripts. Of 15 non-ES cell-specific transcription factor genes, only one had a single pseudogene hit in our screen, emphasizing the apparent selectivity (4). Oct4 and Nanog are both required to maintain pluripotency of embryonic cell-types (5–7), whereas the functional role of Dppa3 remains unclear. Studies suggest its function may be related to the cleavage stages of embryogenesis, rather than in germ cell specification (8–12). The nuclear localization of Dppa3 within the inner cell mass of blastocysts suggests a critical function, maybe as a transcriptional regulator (9).

Although the majority of retrotranspositions appear to manifest as nonfunctional genetic artifact, exceptions exist whereby retrogenes exhibit transcriptional activity (13, 14) and encode functional proteins (15, 16). The evolutionary implications of functional retrogenes are well defined (17), but the role of the embryonic cell transcriptome in this context remains to be established. Here, we extended our previous investigation to analyze additional pluripotent cell-specific genes for their predisposition to retrotransposition. In addition, we screened a panel of 24 adult human tissues to reveal transcriptional activity of these novel processed retrogenes. We report that one of the Dppa3-derived genomic sequences is transcriptionally active and displays a tissue-specific expression profile.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
BLAST was used to compare the cDNA of Nanog, Oct4, Dppa3 (Stella/PGC7), GDF3, and Dppa1, -2, -4, and -5 with the National Center for Biotechnology Information human genome data base (18). An E value limit was set to 1 x 10–4 for these searches, and the Ensembl genome browser was used to confirm all blast hits. Processed pseudogenes were selected based on the search criteria, which calculated similarity to the source genes in question.

Pseudogene sequences were compared with their corresponding source gene sequence and variations were exploited to design specific primers to each retropseudogene using Vector NTI Suite 9. All of the primers associated with one source gene were cross-checked with its source gene and all of its sister pseudogenes to confirm specificity. Primers were purchased from Sigma Genosys and resuspended to 1 µl/µg in ddH20 and then diluted 200-fold in ddH20. PCR against the human genome was performed using PCR SuperMix (Invitrogen).

Human rapid scan panels were purchased from OriGene Technologies. The panels contained 96 wells consisting of 24 tissues arrayed in quadruplicate. Each quadruplicate consisted of increasing concentrations of cDNA (1x, 10x, 100x, and 1000x). PCR using source- and retropseudogene-specific primers along with PCR SuperMix was carried out against these panels. In all panels screened in this study, {beta}-actin was amplified to validate the integrity of the cDNA samples.

RNA was prepared from donor-derived human blood and bone marrow (Cambrex) using RNeasy kits (Qiagen). First-strand cDNA synthesis was performed using the SuperScript III RT kit (Invitrogen). PCR was carried out using source and retropseudogene primers as well as PCR SuperMix. {beta}-Actin was employed as a control to validate the integrity of each sample.

Pancreas cDNA was obtained from BioChain, and RACE (rapid amplification of cDNA ends) cDNA was purchased from Ambion. cDNAs isolated from thyroid gland, heart and adrenal gland were obtained from BioChain.

Each reaction well contained 5 µl of forward primer, 5 µl of reverse primer, 1 µl of genomic DNA, and 39 µl of PCR SuperMix (Invitrogen). These samples were run at 94 °C (5 min), then through 35 cycles of the following three steps (1) 94 °C (1 min), (2) 55 °C (1 min), (3) 72 °C (1 min), and finally at 72 °C (5 min) followed by refrigeration. Annealing temperatures of 50 and 60 °C were also attempted for each primer pair. PCR products were then run on agarose gels, treated with ethidium bromide, and imaged. For the rapid scan screens, 5 µlof forward primer, 5 µl of reverse primer, and 39 µl of PCR SuperMix (Invitrogen) was added to each well on the cDNA panels. PCR and electrophoresis were carried out as detailed above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Previously, we BLASTed four known pluripotent cell-specific mRNA transcripts against the human genome and revealed their predisposition for retrotransposition and subsequent generation of processed pseudogenes (4). The frequency of retrotransposition within the "pluripotent gene" set was striking, compared with that of non-pluripotent cell-specific genes. In summary, three of the four stem cell-specific genes contributed to a total of 32 processed pseudogenes, whereas 15 non-stem cell-specific genes contributed to a single processed pseudogene.

In the current study, we shifted focus to a different set of recently identified pluripotent tissue-specific genes. The Dppa family of genes (Dppa1–5) are coordinately expressed in pluripotent tissues, including blastocyst, epiblast, and primordial germ cells (11). We observed by BLAST analysis that four of the five Dppa genes possess multiple, highly conserved retropseudogenes, dispersed throughout the human genome (Table 1). Briefly, mRNA transcript sequences of these genes were individually BLASTed against the National Center for Biotechnology Information human genome data base. The hits that represented the source genes contained the expected intron/exon gene organization, but all other sequences were contiguous, such that the conserved alignments reflected the mRNA transcript sequence rather than the genomic sequence. This suggested that these conserved sequences likely resulted from retrotransposition events (19). In the instances of Dppa1 and Dppa5, the mouse ortholog was used as source gene in the absence of an annotated human variant. However, these murine transcripts were BLASTed against the human genome. MEGABLAST was employed with an expected value significance cut-off equal to 0.01, and this relatively high stringency was used to predict the occurrence of gene retrotransposition. Due to this relative increase in stringency compared with our previous study, Dppa3 (Stella/Pgc7) associated with five less hits, totaling 11 (4). In total, four of the five Dppa genes were associated with 22 processed pseudogenes by this bioinformatic analysis. In summary, these data further consolidate our previous observation of apparent predisposition of pluripotent cell-specific genes to the generation of processed pseudogenes (38 from 8 ES cell-specific source genes, see Table 1).


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  		TABLE 1
MEGABLAST analysis of pluripotent cell-specific gene mRNA sequences against the human genome

 
Dppa3 (Stella/Pgc7) was associated with 11 processed pseudogenes, annotated Dppa3 (a–k) (Table 2). A non-extensive bioinformatic analysis was performed on each putative pseudogene to predict the likelihood of transcriptional activity. Start and stop codons conserved from the source gene represented a potential identifier, as did conservation of the full coding region (Table 2). Following this analysis, four processed pseudogenes of Dppa3 were selected to further characterize their functional potential (i.e. Dppa3 (a–d)).


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  		TABLE 2
Characterization of Dppa3 retropseudogenes

 
To selectively identify processed pseudogenes of Dppa3, specific PCR primers were designed to mismatch with other pseudogenes as well as the source gene (Table 3). All primer pairs generated a single product of the predicted size, and sequencing confirmed specificity (data not shown).


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  		TABLE 3
PCR primer design for Dppa3 and its retropseudogenes

 
To determine whether any processed pseudogenes of Dppa3 were transcriptionally active, we screened each pseudogene-specific PCR primer pair against a panel of 24 adult tissue-specific cDNA pools. Notably, the Dppa3 source gene primer pair failed to generate an amplification product in any of the adult tissues tested (data not shown), confirming the reported specificity of Dppa3 expression in pluripotent tissues (11). In contrast, RT-PCR against Dppa3(d) generated amplification products of the predicted size. Transcripts for Dppa3(d) were detected in bone marrow, peripheral blood, adrenal gland, pancreas, and thyroid (thyroid not clearly seen in Fig. 1). In each case, the amplified product was only detected in the 1000x cDNA reaction, due to the low expression level of the transcript in all positive tissue types.

To validate the expression of Dppa3(d) in these specific tissues, we obtained human RNA from alternative sources, from which to perform RT-PCR with the putative pseudogene-selective primers. Human bone marrow-derived RNA was extracted from mononuclear cells that were isolated from donated whole bone marrow aspirates. Peripheral blood was similarly donated by volunteers, from which mononuclear cells were isolated and prepared for RNA extraction. Other human tissues were acquired from various vendors (see Mateials and Methods) and RT-PCR was performed on all samples simultaneously (Fig. 2). Amplification of the Dppa3(d) product was successful in all of the re-tested adult tissue-types, confirming the data from the primary tissue panel screens. Importantly, the Dppa3 source gene primers were unable to amplify any product within these tissue samples, consistent with the reported absence of Dppa3 source gene transcript in human adult tissues (Fig. 2). The variation in amplification product intensity within this panel of tissues may be explained by the variety in sample source, cell-type homogeneity, as well as the non-quantitative nature of gel-based RT-PCR.

To confirm that the Dppa3(d) primers were not selective for the source Dppa3 gene-derived transcript, and that the primers for the source gene were legitimate, RT-PCR was performed on NT2 cell-derived RNA (a teratocarcinoma cell line). This cell line expresses Nanog and Oct4, and differentiates preferentially to neuronal lineages upon retinoic acid treatment (20). The Dppa3(d) primers failed to generate any product from NT2 cell RNA, whereas in bone marrow and pancreas (obtained from alternative sources to previous figures, see "Experimental Procedures"), robust transcripts were amplified under the same PCR conditions (Fig. 3A). In contrast, the Dppa3 source gene primer pair successfully amplified a product of the expected size from NT2 cell RNA, confirming expression of Dppa3 coordinate with the pluripotency-related genes Nanog and Oct4 (Fig. 3B). However, no amplification product was observed from bone marrow or pancreas cDNA using the source gene primers. Notably, in a single experiment, we did observe very weak positive amplification of Dppa3 source gene transcript in the bone marrow-derived mRNA (data not shown). To validate these data further, we sequenced the RT-PCR products and confirmed that the primers were exclusively amplifying the Dppa3 source gene or the Dppa3(d) gene as predicted in the various tissues and cell lines (Table 4).


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  		TABLE 4
Confirmation of RT-PCR specificity for Dppa3 source gene and Dppa3(d)

 



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  		FIGURE 1.
Tissue-specific expression of Dppa3(d) pseudogene. PCR primers pairs, specific to Dppa3(d), were used to amplify cDNA from 24 human adult tissues. 11 are represented here. {beta}-Actin serves as a positive control for sample integrity. Amplified Dppa3(d) product was 378 bp in size. Each tissue-specific cDNA was plated at four concentrations as depicted by the bar at the top of the panel (1x, 10x, 100x, and 1,000x concentrations). Each experiment was run in duplicate and the same hits were reproduced in each separate experiment.

 



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  		FIGURE 2.
Tissue-specific expression of Dppa3(d). RT-PCR was carried out using primers specific for {beta}-actin, Dppa3(d), or the source gene Dppa3. Adult tissue extracts included: adrenal gland (lane 1), bone marrow (lane 2), heart (lane 3), pancreas (lane 4), peripheral blood (lane 5), and thyroid gland (lane 6). Lane L represents the 100-bp ladder control.

 



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  		FIGURE 3.
Specificity of Dppa3 source gene primers. RT-PCR was performed on human bone marrow, pancreas, and NT2 teratocarcinoma cell-derived RNA using primers specific for Dppa3(d) (top panel) or the source gene Dppa3 (bottom panel). cDNA concentration of each sample is indicated in the figure. Lane L represents the 100-bp ladder control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Germ line transmission is a prerequisite for genes that give rise to processed pseudogenes, and elevated source gene transcription levels further promote such events (21). This primary feature of inherited retrotransposition inspired us to focus on pluripotent cell-specific genes as a potential source of processed pseudogenes in the human genome. Previously, we proposed a role of extensive retrotransposition as a potential signature event of ES cell-specific genes (4). We found that Nanog, Oct4, and Dppa3 (Stella/PGC7) each possess multiple processed pseudogenes, and our data suggested that retrotransposition frequency of ES cell-specific genes far exceeds that of non-ES cell-specific genes. Moreover, we proposed that this "pseudogene signature" may prove useful in identifying novel ES cell-specific genes in future. On this basis, we continued to develop the hypothesis to assess whether any of the ES cell-specific (or pluripotent cell-specific) genes were retrotransposed to form transcriptionally active genomic integrants. First, we identified further examples of processed pseudogenes originating from pluripotent cell-specific genes. The Dppa family of genes were identified as a cluster of genes whose developmental expression pattern was similar to that of Oct4 (11). Like Oct4, this cluster of genes also appeared predisposed to the generation of processed pseudogenes, consistent with the original tenet of the study. Next, we progressed to the question of whether any of the putative pluripotency gene-related retropseudogenes were transcriptionally active. We hypothesized that the creation of functionally active products of retrotransposition that originated from pluripotency-related genes may indicate far reaching implications in evolution.

Here, we reveal that retrotransposition of the pluripotent tissue-specific Dppa3 gene created a transcriptionally active daughter genomic sequence, which displays a discrete tissue-specific expression pattern. The functional role (if any) of this descendant transcript remains to be elucidated. As for precedent, the reported functional manifestations of other transcribed retrogenes include protein expression (22) as well as regulatory mechanisms mediated by duplex mRNA formation with parent transcripts that impart inhibitory (23) or stabilizing effects (24). However, as the tissue distribution of Dppa3(d) transcript is distinct from its source gene, a role in mRNA duplexing is unlikely. Furthermore, recent studies investigating the multipotency of adult tissue-derived cells have utilized RT-PCR detection of Dppa3, Oct4, and Nanog gene transcripts as markers of pluripotency (25). Given our present finding that a Dppa3-derived retrogene is expressed in bone marrow, it may be prudent to readdress the use of these pluripotency genes in this context. Moreover, further studies should reveal which specific cell types within bone marrow, peripheral blood, pancreas, thyroid, and adrenal gland express the Dppa3(d) retrogene, and these ongoing efforts will likely expedite its functional characterization.

Given the increased propensity for highly expressed genes within the germ line to propogate new, functional DNA, a clear advantage is provided for ES cell-specific genes or other pluripotency-related genes to promote genetic diversity and play a key evolutionary role. Whether this is the case for the Dppa3-derived retrogene, Dppa3(d), is tempting to speculate, but clarification will require further study.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Diabetes & Metabolism Disease Area, Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, MA 02139. Tel.: 617-817-0105; Fax: 617-871-7051; E-mail: daniel.kemp{at}novartis.com.

3 The abbreviations used are: BLAST, basic local alignment sequence tool; ES, embryonic stem; RT, reverse transcription. Back


   ACKNOWLEDGMENTS
 
We thank Jee-Hyung Lee for helpful discussions and advice.



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 INTRODUCTION
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 DISCUSSION
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