Multiple Retropseudogenes from Pluripotent Cell-specific Gene Expression Indicates a Potential Signature for Novel Gene Identification

Abstract

Oct4, Nanog, and Stella are transcription factors specifically expressed in embryonic stem (ES) cells and germ lineage cells that impart critical functions in the maintenance of pluripotency. Here, we report the excessive frequency and apparent selectivity of retrotransposition of ES cell-specific genes. Six highly homologous pseudogenes for Oct4, 10 for Nanog, and 16 for Stella were identified by nucleotide BLAST (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. We present a hypothesis whereby retrotransposition of ES or germ cellspecific genes may reflect an innate predisposition. This is based on the increased probability of germ-line transmission when retrotransposition occurs at a very early stage of development within cells known to contribute to the germ cell lineage. The parental genes for Nanog, Stella, and another embryonic gene, GDF3 are all located on chromosome 12p13 of the human genome, and on chromosome 6 in mouse. Here, we identified an Oct4 pseudogene at the same respective loci in both human and mouse genomes, suggesting functional relevance and indicative of epigenetic regulation. We tested whether the apparent susceptibility for ES cell-specific gene retrotransposition may be extrapolated to a more unified phenomenon, such that a bioinformatic approach may represent a potentially novel strategy for identification of genes with embryonic cell-specific functionality. A preliminary investigation indeed revealed a single gene, previously demonstrated to be responsible for multiple retropseudogenes via germ cell-specific expression in Xenopus.Embryonic stem cells are pluripotent and able to differentiate into all cell types of the body, including germ cells (1).Identifying the molecular determinants by which pluripotency is maintained is a critical persuit that will ultimately result in expediting therapeutic application. To date, four transcription factors have been identified that exhibit essential roles in maintenance of the pluripotent state. These are Oct4 (2), Nanog (3, 4), Stella (5), and GDF3 (6). The expression level of all four of these genes is down-regulated coincident with ES cell differentiation, and loss of expression leads reciprocally to differentiated cell states. Beyond maintenance of pluripotency, the functional roles of these genes have not been clearly defined, and the prospective identification of other novel ES cell genes will undoubtedly contribute to this endeavor. Notably, Oct4 is detected in adult stem cells such as bone marrowderived mesenchymal stem cells (7) and thought to play a role in maintaining the multipotency of these cells also. Recently, 10 pseudogenes of Nanog were reported within the human genome (8), likely a result of retrotransposition events. Characterized by extensive alignment and significant sequence identity with the Nanog mRNA transcript, these pseudogenes were s...

Retroposition of processed pseudogenes: the impact of RNA stability and translational control

“…Yet, only about 10% of cellular genes have one or more retroposed copies in the genome [21,23]. While the effects of germline expression [23,25,26] and genomic stability [23,27,28] have been studied previously, little is known about the influence of RNA stability and translation on retroposition of PPs. …”

The pluripotency regulator Oct4: A role in somatic stem cells?

“…These cells included the marrowisolated adult multi-lineage inducible (MIAMI) cells, multipotent adult progenitor cells (MAPC), bone marrow derived germ cells, very small embryonic-like (VSEL) cells, and other subpopulations of MSCs defined by cell surface markers and the ability to grown in specific media formulations. [22][23][24][25] The use of RT-PCR to define Oct4 positive cells has recently been called into question by several groups describing Oct4 retropseudogenes that are actively transcribed in both normal and transformed cells [26][27][28] (and Liedtke et al). A number of these pseudogenes encompass nearly the entire Oct4 coding sequence and are actively transcribed, confounding the interpretation of RT-PCR results since both minor contamination of isolated RNA with genomic DNA and pseudogene expression can contribute to false positive signals if primers are not carefully designed.…”

Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency

“…Furthermore, at least six pseudogenes are actively transcribed in hESC (17,25). Functional studies of OCT4 in hESC should therefore distinguish between the isoforms that are being evaluated (39).…”

“…An immunohistochemical study of OCT4 expression in early human embryos showed that OCT4A is expressed in the nuclei of the inner cell mass and trophectoderm, while OCT4B segregates to the trophectoderm cytoplasm (5), suggesting differential functions for OCT4A and OCT4B. To further complicate the regulation of OCT4 and interpretation of human OCT4 studies, there are six transcribed pseudogenes for OCT4, with up to 95% homology to the endogenous gene (17,25). Functional RNA interference studies in hESC demonstrated that OCT4 downregulation leads to trophectoderm and endoderm differentiation (13,20,32,45,46).…”