Oct4 Pseudogenes Research Articles

Efficient Ex Vivo Expansion of Human Umbilical Cord Blood-Derived Small CD133+lin-CD45- Stem Cells Expressing Pluripotency Markers in Presence of Histone Deacethylase Inhibitors—As a New Challenging Strategy to Obtain Multipotent Cells for Regenerative Medicine

Background. The field of regenerative medicine is still looking for a pluripotent or multipotent stem cell with trans-germ-layer differentiation potential that could be employed in repairing damaged organs and expanded into transplantable hematopoietic stem cells (HSCs). Our group as first isolated a population of small CD133+lin-CD45- early-development stem cells from hematopoietic tissues, including bone marrow (BM), mobilized peripheral blood (mPB), and umbilical cord blood (UCB). Based on expression of early-development markers, these cells were named “very small embryonic-like stem cells” (VSELs). Currently, several groups worldwide who have carefully followed a multicolor-staining cell-sorting strategy described by us (Current Protocols in Cytometry 2010, 9.29.1-9.29.15) have successfully isolated these cells and shown their contribution to all three germ layer lineages in appropriate tissue/organ injury models in vivo, including their specification into HSCs. However, efficient ex vivo expansion of these cells has been a problem due to erasure of certain parentally imprinted genes involved in insulin/insulin-like growth factor signaling (e.g., Igf-H19, Rasgrf1), which is a mechanism very well known for keeping for example the primordial germ cells in a quiescent state. In order to endow cells with proliferative potential, these erased loci must be again re-methylated by “de novo DNA methyltransferases - DNMTL3, DNMT3A and DNMT3B” (DNMTs). Hypothesis. Based on our own observations that i) DNMT3L are highly expressed in VSELs and ii) certain inhibitors of histone deacetylases (HDACs) such as valproic acid (VPA) or nicotinamide (NA) activate DNMTs and promote DNA re-methylation of erased loci in early development stem cells (Cell Reports 2017, 18, 1930-1945) we hypothesized that HDACs inhibitors could be employed for ex vivo expansion of these cells in highly defined media without third party supportive feeder layer cells . Materials and Methods. Human umbilical cord blood- or mobilized peripheral blood-derived VSELs highly purified by FACS were placed in DMEM supplemented with 10% serum in the presence of VPA or NA + factors that as we demonstrated in vivo may promote egress of VSELs fromquiescent state such as follicle stimulating hormone (FSH), luteinizing hormone (LH), bone morphogen protein 4 (BMP-4), kit ligand (KL), fibroblast growth factor-2 (FGF-2) and insulin-like growth factor 2 (Igf2) (Stem Cells & Development 2015, 24, 927-937 ). Cells were cultured for 2-3 months and every 7 days, medium was exchanged. Cells in the expansion cultures were counted and studied for i) remethylation of erased parentally imprinted loci, ii) expression of Oct-4 (using specific primers to avoid amplification of Oct-4 pseudogenes) as well as for expression of the germline markers Stella, Fragilis, and Blimp-1. After 1 month of expansion, aliquots of these cells were tested for their hematopoietic potential. Results. We found that over time these small cells (slightly smaller than red blood cells) became enlarged and after 2 weeks began to undergo symmetric and some of them asymmetric divisions (Figure 1). The number of cells in the cultures increased up to 2000 times, and while the majority of cells retained small-cell morphology and expressed markers of pluripotency or multipotency at the protein and mRNA levels, some became enlarged in size and expressed markers typical of cells involved in germ line development (Stella, Blimp-1, and Fragilis). Moreover, cells proliferating in our ex vivo-expansion system re-methylated paternally erased loci (e.g., at the Igf2-H19 locus). Importantly, they did not expand indefinitely and after 3-4 months started to lose pluripotency markers and to cease proliferation and did grow teratoma in immunodeficient mice. Conclusions. We provide an evidence that small CD133+lin-CD45- cells are a population of highly quiescent cells that are kept in a dormant state by erasure of certain paternally imprinted growth-promoting genes and that these loci can be remethylated de novo by the presence of HDAC inhibitors such as VPA or NA in a DNMT3L-dependent manner. This data also support a hypothetical link between primordial germ cells and hematopoiesis. We propose that the currently established expansion system for VSELs isolated from postnatal tissues provides a new and exciting opportunity to employ cells isolated from adult tissues in regenerative medicine. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An Overview on the Complexity of OCT4: at the Level of DNA, RNA and Protein

OCT4 plays critical roles in self-renewal and pluripotency maintenance of embryonic stem cells, and is considered as one of the main stemness markers. It also has pivotal roles in early stages of embryonic development. Most studies on OCT4 have focused on the expression and function of OCT4A, which is the biggest isoform of OCT4 known so far. Recently, many studies have shown that OCT4 has various transcript variants, protein isoforms, as well as pseudogenes. Distinguishing the expression and function of these variants and isoforms is a big challenge in expression profiling studies of OCT4. Understanding how OCT4 is functioning in different contexts, depends on knowing of where and when each of OCT4 transcripts, isoforms and pseudogenes are expressed. Here, we review OCT4 known transcripts, isoforms and pseudogenes, as well as its interactions with other proteins, and emphasize the importance of discriminating each of them in order to understand the exact function of OCT4 in stem cells, normal development and development of diseases.

Chromatin Regulation at Parental Gene Promoters by Pseudogene Sense lncRNAs.

There is accumulating evidence that pseudogenes can produce functionally relevant lncRNAs in a tightly controlled manner. This class of transcripts has been demonstrated to play an important role in development and disease, by controlling parental gene expression. Classically, pseudogene derived lncRNAs compete with parental transcripts for miRNAs or factors that control parental mRNA metabolisms. Recently, pseudogene lncRNAs were demonstrated to take over the control of classic chromatin modifying enzymes and alter parental gene promoter activity or genome wide gene expression. Here, we discuss a new mechanism of parental gene expression controlled by the mOct4P4 lncRNA, a sense transcript derived from the murine Oct4 pseudogene 4. mOct4P4 lncRNA specifically interacts with the RNA binding protein FUS and the Histone Methyltransferase SUV39H1 to target heterochromatin formation at the parental Oct4 promoter in trans. In addition, we will address key issues for the functional dissection of epigenetic control of parental gene promoters by pseudogene lncRNAs.

FUS-dependent loading of SUV39H1 to OCT4 pseudogene-lncRNA programs a silencing complex with OCT4 promoter specificity

The resurrection of pseudogenes during evolution produced lncRNAs with new biological function. Here we show that pseudogene-evolution created an Oct4 pseudogene lncRNA that is able to direct epigenetic silencing of the parental Oct4 gene via a 2-step, lncRNA dependent mechanism. The murine Oct4 pseudogene 4 (mOct4P4) lncRNA recruits the RNA binding protein FUS to allow the binding of the SUV39H1 HMTase to a defined mOct4P4 lncRNA sequence element. The mOct4P4-FUS-SUV39H1 silencing complex holds target site specificity for the parental Oct4 promoter and interference with individual components results in loss of Oct4 silencing. SUV39H1 and FUS do not bind parental Oct4 mRNA, confirming the acquisition of a new biological function by the mOct4P4 lncRNA. Importantly, all features of mOct4P4 function are recapitulated by the human hOCT4P3 pseudogene lncRNA, indicating evolutionary conservation. Our data highlight the biological relevance of rapidly evolving lncRNAs that infiltrate into central epigenetic regulatory circuits in vertebrate cells.

Systematic expression alteration analysis of master reprogramming factor OCT4 and its three pseudogenes in human cancer and their prognostic outcomes

OCT4 is a master transcription factor that regulates the pluripotency of pluripotent stem cells and cancer stem cells along with other factors, including SOX2, KLF4, and C-MYC. Three different transcripts, OCT4A, OCT4B, and OCT4B1, are known to be generated by alternative splicing and eight OCT4 pseudogenes have been found in the human genome. Among them, we examined OCT4 and three pseudogenes (POU5F1P1, POU5F1P3, and POU5F1P4) because of their high expression possibility in cancer. In addition, previous studies indicated that OCT4 expression is augmented in cervical cancer and associated with poor prognosis, whereas OCT4 is down-regulated and correlated with good clinical outcomes in breast cancer. Because of these conflicting reports, we systematically evaluated whether expression of OCT4 and its pseudogenes can serve as oncogenic markers in various human cancers using the Oncomine database. Moreover, copy number alterations and mutations in OCT4 gene and its pseudogenes were analyzed using cBioPortal and the relationship between expression of OCT4 and pseudogenes and survival probability of cancer patients were explored using Kaplan-Meier plotter, OncoLnc, PROGgeneV2, and PrognoScan databases. Multivariate survival analysis was further conducted to determine the risk of the expression of the occurrence of OCT4 and its pseudogenes on certain cancer types using data from the Kaplan-Meier plotter. Overall, an association between expression of OCT4 and pseudogenes and cancer prognosis were established, which may serve as a therapeutic target for various human cancers.

The Long Noncoding RNA Lncenc1 Maintains Naive States of Mouse ESCs by Promoting the Glycolysis Pathway.

SummaryThe naive embryonic stem cells (nESCs) display unique characteristics compared with the primed counterparts, but the underlying molecular mechanisms remain elusive. Here we investigate the functional roles of Lncenc1, a highly abundant long noncoding RNA in nESCs. Knockdown or knockout of Lncenc1 in mouse nESCs leads to a significantly decreased expression of core pluripotency genes and a significant reduction of colony formation capability. Furthermore, upon the depletion of Lncenc1, the expression of glycolysis-associated genes is significantly reduced, and the glycolytic activity is substantially impaired, as indicated by a more than 50% reduction in levels of glucose consumption, lactate production, and extracellular acidification rate. Mechanistically, Lncenc1 interacts with PTBP1 and HNRNPK, which regulate the transcription of glycolytic genes, thereby maintaining the self-renewal of nESCs. Our results demonstrate the functions of Lncenc1 in linking energy metabolism and naive state of ESCs, which may enhance our understanding of the molecular basis underlying naive pluripotency.

POU5F1B promotes hepatocellular carcinoma proliferation by activating AKT

Hepatocellular carcinoma (HCC) is one of the most common tumors, so far, there still aren’t good therapeutic methods. Looking for new targets makes a top priority. In this study, we found a OCT4 pseudogene, POU5F1B was significantly upregulated in HCC cells and tissues, patients with high POU5F1B had shorter survival time compared to patients with low POU5F1B expression. POU5F1B overexpression promoted HCC proliferation, while its knockdown inhibited HCC proliferation determined by MTT assay, soft agar growth assay, BrdU incorporation assay, and cell cycle assay. Mechanism analysis suggested POU5F1B could activate AKT, AKT inhibition in HCC cells with POU5F1B overexpression significantly inhibited cell proliferation compared to cells only with POU5F1B overexpression, suggesting POU5F1B promoted HCC proliferation by activating AKT. Finally, we analyzed the relationship between POU5F1B expression and AKT activity in HCC tissues, and found POU5F1B expression was positive correlated with activated AKT. Taken together, our findings suggested POU5F1B promoted HCC proliferation by activating AKT, and provided a new target for HCC therapy.

Differential Expression of OCT4 Pseudogenes in Pluripotent and Tumor Cell Lines.

The human OCT4 gene, the most important pluripotency marker, can generate at least three different transcripts (OCT4A, OCT4B, and OCT4B1) by alternative splicing. OCT4A is the main isoform responsible for the stemness property of embryonic stem (ES) cells. There also exist eight processed OCT4 pseudogenes in the human genome with high homology to the OCT4A, some of which are transcribed in various cancers. Recent conflicting reports on OCT4 expression in tumor cells and tissues emphasize the need to discriminate the expression of OCT4A from other variants as well as OCT4 pseudogenes. In this experimental study, DNA sequencing confirmed the authenticity of transcripts of OCT4 pseudogenes and their expression patterns were investigated in a panel of different human cell lines by reverse transcription-polymerase chain reaction (RT-PCR). Differential expression of OCT4 pseudogenes in various human cancer and pluripotent cell lines was observed. Moreover, the expression pattern of OCT4-pseudogene 3 (OCT4-pg3) followed that of OCT4A during neural differentiation of the pluripotent cell line of NTERA-2 (NT2). Although OCT4-pg3 was highly expressed in undifferentiated NT2 cells, its expression was rapidly down-regulated upon induction of neural differentiation. Analysis of protein expression of OCT4A, OCT4-pg1, OCT4-pg3, and OCT4-pg4 by Western blotting indicated that OCT4 pseudogenes cannot produce stable proteins. Consistent with a newly proposed competitive role of pseudogene microRNA docking sites, we detected miR-145 binding sites on all transcripts of OCT4 and OCT4 pseudogenes. Our study suggests a potential coding-independent function for OCT4 pseudogenes during differentiation or tumorigenesis.

Specific detection of OCT4 isoforms in inflammatory bowel disease.

BackgroundDevelopmentally early cells are mobilized into peripheral blood in Crohn’s disease (CD) patients. OCT4, is considered to be important in sustaining the pluripotency of stem cells. OCT4 splicing variants are differentially expressed in pluripotent and non-pluripotent cells. Our study aims to investigate the expression pattern of OCT4 variants and SOX-2, an essential factor implicated in self-renewal and pluripotency, in tissue and blood samples from patients with IBD.MethodsPeripheral blood and tissue samples were collected from patients with active CD and ulcerative colitis (UC), and from healthy individuals. OCT4 expression was documented by Western blot, immunohistochemistry and by reverse transcription-real-time PCR. OCT4 isoform determination was documented using specific primers. SOX-2 expression levels were also evaluated.ResultsOCT4 protein levels were significantly higher in CD tissue samples than in CD blood samples, and in UC tissue samples. OCT4 protein was localized mainly in the cytosol. In all samples, only the OCT4 pseudogenes and the OCT4B1 variant were detected. OCT4B1 expression levels were elevated in both tissue and blood samples from CD and UC cases compared to healthy controls. In CD patients only SOX-2 mRNA levels were found slightly increased compared to healthy controls.ConclusionOur results suggest that OCT4 is expressed in patients with IBD. Furthermore, we found the presence of the OCT4B1 isoform in IBD in both tissue and blood samples. Our results have shown, that developmentally early cells might be mobilized into peripheral blood as result of tissue damage, indicating a possible role of these cells in repair of injured intestinal tract.

Epigenetic silencing of Oct4 by a complex containing SUV39H1 and Oct4 pseudogene lncRNA.

Pseudogene-derived, long non-coding RNAs (lncRNAs) act as epigenetic regulators of gene expression. Here we present a panel of new mouse Oct4 pseudogenes and demonstrate that the X-linked Oct4 pseudogene Oct4P4 critically impacts mouse embryonic stem cells (mESCs) self-renewal. Sense Oct4P4 transcription produces a spliced, nuclear-restricted lncRNA that is efficiently upregulated during mESC differentiation. Oct4P4 lncRNA forms a complex with the SUV39H1 HMTase to direct the imposition of H3K9me3 and HP1α to the promoter of the ancestral Oct4 gene, located on chromosome 17, leading to gene silencing and reduced mESC self-renewal. Targeting Oct4P4 expression in primary mouse embryonic fibroblasts causes the re-acquisition of self-renewing features of mESC. We demonstrate that Oct4P4 lncRNA plays an important role in inducing and maintaining silencing of the ancestral Oct4 gene in differentiating mESCs. Our data introduces a sense pseudogene–lncRNA-based mechanism of epigenetic gene regulation that controls the cross-talk between pseudogenes and their ancestral genes.

All the Tested Human Somatic Cells Express Both Oct4A and Its Pseudogenes but Express Oct4A at Much Lower Levels Compared with Its Pseudogenes and Human Embryonic Stem Cells.

Oct4 pseudogenes and isoforms seriously confuse the detection of the pluripotency-associated Oct4A expression in somatic cells, which in many cases was not accurately determined. This confusion has recently been discussed, but the wrong conclusions have continuously been made. Most studies failed to detect the expression of Oct4 pseudogenes and isoforms in somatic cells but detected only Oct4A, for which the detection signals incorrectly came from its pseudogenes and isoforms. Some studies detected the expression of only Oct4 pseudogenes in somatic cells but failed to detect Oct4A. The other studies failed to detect the expression of any Oct4 genes. Oct4A is more homologous to its pseudogenes than its isoforms, and it is much more difficult to distinguish Oct4A from its pseudogenes, so this study focused on them. In this study, the strict experimental procedures were followed. Three pairs of Oct4A-specific polymerase chain reaction (PCR) primers were carefully designed and tested by sequencing reverse transcription-polymerase chain reaction (RT-PCR) clones, which showed that only one of them was truly specific to Oct4A. RT-PCR was also performed with the primers amplifying both Oct4A and its pseudogenes, and several hundreds of PCR clones from each cell type were sequenced to reliably distinguish the low-abundant Oct4A from its high-abundant pseudogenes. Western blot, immunocytochemistry, and flow cytometric analyses were performed with three Oct4 antibodies to confirm the results of Oct4 mRNA expression. This study undoubtedly made the correct conclusions about Oct4 expression in human somatic cells and showed that all the tested human somatic cells expressed both Oct4A and its pseudogenes but expressed Oct4A at much lower levels compared with its pseudogenes.

OCT4 pseudogene 5 upregulates OCT4 expression to promote proliferation by competing with miR-145 in endometrial carcinoma.

OCT4 plays a critical role in the maintenance of stem cell pluripotency and proliferation, and is overexpressed in multiple human tumors, including endometrial cancer. OCT4 expression can be modulated by miR-145 and the OCT4 pseudogene 5 (OCT4-pg5), which share similar binding sites in the OCT4 3'-untranslated region. The goal of the present study was to evaluate the interaction between miR-145 and OCT4‑pg5 on OCT4 expression in endometrial cancer. We assessed OCT4-pg5 expression in 14benign endometrium and 29endometrial carcinoma samples. Furthermore, miR-145 mimic transfection was performed to explore its effect on OCT4-pg5 and OCT4 expression, and small interfering RNA (siRNA)-mediated knockdown of OCT4 was conducted to determine whether the effect of OCT4-pg5 on cellular growth was OCT4-dependent. We observed that OCT4-pg5 was abnormally activated in the endometrial carcinomas, and that overexpression of OCT4-pg5 contributed to enhanced cell proliferation and OCT4-PI3K/AKT-cyclinD1 signaling. Moreover, the miR-145 mimic depleted OCT4 expression, whereas elevated OCT4-pg5 restored OCT4 expression and OCT4-PI3K/AKT-cyclinD1 signaling. In conclusion, these data indicate that OCT4-pg5 can act as an RNA sponge to protect OCT4 transcripts from being inhibited by miR-145, providing novel insight into the control of OCT4 expression.

Expression and Differentiation between OCT4A and Its Pseudogenes in Human ESCs and Differentiated Adult Somatic Cells

The POU5F1 gene codes for the OCT4 transcription factor, which is one of the key regulators of pluripotency. Its transcription, alternative splicing, and alternative translation leading to the synthesis of the active, nuclear localized OCT4A has been described in detail. Much less, however, is known about actively transcribed OCT4 pseudogenes, several of which display high homology to OCT4A and can be expressed and translated into proteins. Using RT-PCR followed by pseudogene-specific restriction digestion, cloning, and sequencing we discriminate between OCT4A and transcripts for pseudogenes 1, 3 and 4. We show that expression of OCT4 and its pseudogenes follows a developmentally-regulated pattern in differentiating hESCs, indicating a tight regulatory relationship between them. We further demonstrate that differentiated human cells from a variety of tissues express exclusively pseudogenes. Expression of OCT4A can, however be triggered in adult differentiated cells by oxygen and FGF2-dependent mechanisms.

The OCT4 pseudogene POU5F1B is amplified and promotes an aggressive phenotype in gastric cancer

POU5F1B (POU domain class 5 transcription factor 1B), a processed pseudogene that is highly homologous to OCT4, was recently shown to be transcribed in cancer cells, but its clinical relevance and biological function have remained unclear. We now show that POU5F1B, which is located adjacent to MYC on human chromosome 8q24, is frequently amplified in gastric cancer (GC) cell lines. POU5F1B, but not OCT4, was also found to be expressed at a high level in GC cell lines and clinical specimens. In addition, the DNA copy number and mRNA abundance for POU5F1B showed a positive correlation in both cancer cell lines and GC specimens. Overexpression of POU5F1B in GC cells promoted colony formation in vitro as well as both tumorigenicity and tumor growth in vivo, and these effects were enhanced in the additional presence of MYC overexpression. Furthermore, knockdown of POU5F1B expression with a short hairpin RNA confirmed a role for the endogenous pseudogene in the promotion of cancer cell growth in vitro and tumor growth in vivo. POU5F1B overexpression induced upregulation of various growth factors in GC cells as well as exhibited mitogenic, angiogenic and antiapoptotic effects in GC xenografts. Finally, amplification of POU5F1B was detected in 17 (12%) of 145 cases of GC and was a significant predictor of poor prognosis in patients with stage IV disease. In conclusion, we found that the POU5F1B pseudogene is amplified and expressed at a high level in, as well as confers an aggressive phenotype on, GC, and that POU5F1B amplification is associated with a poor prognosis in GC patients.

The stem cell markers Oct4A, Nanog and c-Myc are expressed in ascites cells and tumor tissue of ovarian cancer patients

The aim of this study was to examine the expression of established stem cell markers in ascites and tumor tissue obtained from ovarian cancer patients. Mononuclear cells present in ascites were collected by density gradient centrifugation. Intracellular flowcytometry was used to assess the putative presence of stem cell markers. RT-PCR was used to detect full length Oct4A, a splice variant Oct4B, implicated in glioma and breast cancer, Oct4 pseudogenes and c-Myc. Genes were cloned and sequenced to determine putative mutations. Confocal laser scanning microscopy was performed to localize the markers in ascites cells as well as in tumor tissue. Material from carcinomas other than epithelial ovarian carcinoma served as control. A small quantity of cells in ascites and in tumor tissue of ovarian cancer patients was detected that expresses c-Myc, Oct4A and Nanog. Besides Oct4A, present in the nucleus, also the cytoplasmic resident Oct4B splice variant was detected. Remarkably, c-Myc was found partially in the cytoplasm. Since no mutations in c-Myc were found that could explain the cytoplasmic localization, we hypothesize that this is due an IL-6 induced c-Myc shuttle factor. The expression of stem cell genes was detected in a small proportion of tumor cells present in ascites as well as in tumor tissue. IL-6 plays an important role in the induction of c-Myc.

Differential Expression of Oct4 Variants and Pseudogenes in Normal Urothelium and Urothelial Cancer

The transcription factor octamer-binding protein 4 (Oct4; encoded by POU5F1) has a key role in maintaining embryonic stem cell pluripotency during early embryonic development and it is required for generation of induced pluripotent stem cells. Controversy exists concerning Oct4 expression in somatic tissues, with reports that Oct4 is expressed in normal and in neoplastic urothelium carrying implications for a bladder cancer stem cell phenotype. Here, we show that the pluripotency-associated Oct4A transcript was absent from cultures of highly regenerative normal human urothelial cells and from low-grade to high-grade urothelial carcinoma cell lines, whereas alternatively spliced variants and transcribed pseudogenes were expressed in abundance. Immunolabeling and immunoblotting studies confirmed the absence of Oct4A in normal and neoplastic urothelial cells and tissues, but indicated the presence of alternative isoforms or potentially translated pseudogenes. The stable forced expression of Oct4A in normal human urothelial cells in vitro profoundly inhibited growth and affected morphology, but protein expression was rapidly down-regulated. Our findings demonstrate that pluripotency-associated isoform Oct4A is not expressed by normal or malignant human urothelium and therefore is unlikely to play a role in a cancer stem cell phenotype. However, our findings also indicate that urothelium expresses a variety of other Oct4 splice-variant isoforms and transcribed pseudogenes that warrant further study.

OCT4 pseudogenes present in human leukemia cells

The transcription factor OCT4 is expressed in embryonic stem cells (ESCs) and has been considered as a main regulator in maintaining pluripotency of ESCs. Several studies have showed OCT4 expression in human solid tumor and adult stem or progenitor cells. However, whether OCT4 is expressed in normal hematopoietic system including the peripheral blood and bone marrow remains controversial. Furthermore, the functional expression of OCT4 in leukemia cells and its potential significance in leukemia studies have been poorly defined. The aim of this study was to examine and analyze the genuine expression of OCT4 in human leukemia cells by means of RT-PCR, flow cytometry, PCR product sequencing and alignment with NCBI BLAST and DNAMAN software. The full lengths of the putative OCT4 genes were amplified in 2/9 leukemia cell lines and 7/49 leukemia patients' samples. However, many base mutations in putative OCT4 positive samples were found. Sequence alignment analysis showed a higher similarity between the putative OCT4 PCR products and the pseudogenes in chromosomes 1 and 8. The positive rates of OCT4 protein detected with flow cytometry were low, and almost all of them were less than 10% of positivity. A very small fraction of leukemia stem cells with OCT4 protein expression was found. We conclude that OCT4 pseudogenes in chromosomes 1 and 8 present in the panel of leukemia cells tested and the OCT4 protein is rarely detected with flow cytometry in leukemia cells.

Expression of OCT4 pseudogenes in human tumours: lessons from glioma and breast carcinoma

The POU family transcription factor OCT4 is required for maintaining the pluripotency of embryonic stem cells and for generating induced pluripotent stem cells. Although OCT4 is clearly shown to be expressed in some pluripotent germ cell tumours, its expression in human somatic tumours remains controversial. Some studies have shown that OCT4 is expressed in adult stem cells, somatic cancers and, further, cancer stem cells, while other studies failed to make such an observation. It is thus important to ascertain whether OCT4 is expressed in human somatic tumours. By using RT-PCR and sequencing analysis, three OCT4 pseudogenes, viz. OCT4-pg1, OCT4-pg3 and OCT4-pg4 but excluding the OCT4 gene, were found to be expressed in two types of human solid tumours, glioma and breast carcinoma, from which cancer stem cells had earlier been isolated. The protein expression of these pseudogenes was further demonstrated by immunochemistry and western blotting. Along with this, it was shown that OCT4 pseudogenes lacked OCT4-like activities. The expression of OCT4 splicing variant and various pseudogenes at both the mRNA and protein levels in human somatic tumours might call into question the reliability of the results regarding OCT4 expression and function in tumourigenesis. Hence, in investigations of OCT4 expression in cancers and stem cells, different approaches with appropriate controls would be desirable to exclude possibility of false-positive results.

Transcriptional regulation of Oct4 by a long non-coding RNA antisense to Oct4-pseudogene 5

Long non-coding RNAs (lncRNAs) have been shown to epigenetically regulate certain genes in human cells. Here we report evidence for the involvement of an antisense lncRNA in the transcriptional regulation of the pluripotency-associated factor Oct4. When an lncRNA antisense to Oct4-pseudogene 5 was suppressed, transcription of Oct4 and Oct4 pseudogenes 4 and 5 was observed to increase. This increase correlated with a loss of silent state epigenetic marks and the histone methyltransferase Ezh2 at the Oct4 promoter. We observed this lncRNA to interact with nucleolin and PURA, a 35-kD single-stranded DNA and RNA binding protein, and found that these proteins may act to negatively regulate this antisense transcript.

Human haematopoietic stem cells express Oct4 pseudogenes and lack the ability to initiate Oct4 promoter-driven gene expression

The transcription factor Oct4 is well defined as a key regulator of embryonic stem (ES) cell pluripotency. In recent years, the role of Oct4 has purportedly extended to the self renewal and maintenance of multipotency in adult stem cell (ASC) populations. This profile has arisen mainly from reports utilising reverse transcription-polymerase chain reaction (RT-PCR) based methodologies and has since come under scrutiny following the discovery that many developmental genes have multiple pseudogenes associated with them. Six known pseudogenes exist for Oct4, all of which exhibit very high sequence homology (three >97%), and for this reason the generation of artefacts may have contributed to false identification of Oct4 in somatic cell populations. While ASC lack a molecular blueprint of transcription factors proposed to be involved with 'stemness' as described for ES cells, it is not unreasonable to assume that similar gene patterns may exist. The focus of this work was to corroborate reports that Oct4 is involved in the regulation of ASC self-renewal and differentiation, using a combination of methodologies to rule out pseudogene interference. Haematopoietic stem cells (HSC) derived from human umbilical cord blood (UCB) and various differentiated cell lines underwent RT-PCR, product sequencing and transfection studies using an Oct4 promoter-driven reporter. In summary, only the positive control expressed Oct4, with all other cell types expressing a variety of Oct4 pseudogenes. Somatic cells were incapable of utilising an exogenous Oct4 promoter construct, leading to the conclusion that Oct4 does not appear involved in the multipotency of human HSC from UCB.