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

Scarola, Michele; Comisso, Elisa; Pascolo, Rhena; Chiaradia, Riccardo; Maria Marion, Rosa; Schneider, Claudio; Blasco, Maria A.; Schoeftner, Stefan; Benetti, Roberta

doi:10.1038/ncomms8631

Download PDF

Article
Open access
Published: 09 July 2015

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

Michele Scarola¹,
Elisa Comisso¹,
Rhena Pascolo¹,
Riccardo Chiaradia¹,
Rosa Maria Marion⁴,
Claudio Schneider^1,2,3,
Maria A. Blasco⁴,
Stefan Schoeftner^1,5 &
…
Roberta Benetti^1,2,3

Nature Communications volume 6, Article number: 7631 (2015) Cite this article

7709 Accesses
78 Citations
3 Altmetric
Metrics details

Subjects

Abstract

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.

Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF

Article 27 March 2024

Sandi Radko-Juettner, Hong Yue, … Charles W. M. Roberts

Transcription bodies regulate gene expression by sequestering CDK9

Article Open access 08 April 2024

Martino Ugolini, Maciej A. Kerlin, … Nadine L. Vastenhouw

Long non-coding RNAs: definitions, functions, challenges and recommendations

Article 03 January 2023

John S. Mattick, Paulo P. Amaral, … Mian Wu

Introduction

Oct4 is a member of the POU family of transcription factors that controls early embryonic differentiation and is essential to maintain mESC pluripotency and self-renewal^1,2,3,4,5,6. Oct4 is expressed in germ cells, the inner cell mass of pre-implantation embryos, embryonic stem cells (ESC) but also in different types of human cancer^7,8,9,10. Oct4 is essential to establish the core transcriptional network maintaining pluripotency and self-renewal in mESCs^1,2,3,4. Importantly, ectopic expression of the key mESC self-renewal transcription factors Oct4, Sox2, Klf4 and c-Myc induces pluripotency in differentiated cells, resulting in induced pluripotent stem (iPS) cells¹¹. Out of this basic set of transcription factors, Oct4 is the only indispensible factor for iPS cell generation^11,12. This highlights a central role of Oct4 for ESC pluripotency and self-renewal. Given the key role of Oct4 in mESC self-renewal, discovering pathways that control Oct4 expression has primary importance. Induction of ESC differentiation leads to rapid transcriptional and epigenetic silencing of Oct4 (refs 13, 14, 15, 16, 17, 18). Other pathways that regulate Oct4 expression involve post-translational modifications or miRNAs^{19,20,21,22,23,24,25}. A growing body of evidence indicates an important role of pseudogene-derived RNAs in regulating the expression of their ancestral gene, either by modulating chromatin structure via anti-sense transcription, short interfering RNA (siRNA) formation or sponging miRNAs from their ancestral genes by acting as competing endogenous RNAs (ceRNAs)^{26,27,28,29,30,31,32}. Recent studies demonstrate the existence of various human OCT4 pseudogenes and anticipate a relevance for pseudogene-derived ncRNAs in the regulation of the ancestral OCT4 gene in ESCs but also cancer^{33,34,35,36,37,38,39,40,41,42,43,44}. To this end, the human OCT4 pseudogene OCT4-pg4 was demonstrated to sponge OCT4-specific miRNAs in hepatocellular carcinoma cells⁴⁵; human OCT4-pg5 anti-sense transcripts reduce OCT4 promoter activity and mouse OCT4-pg1 impacts on mesenchymal stem cell differentiation via a still unknown mechanism^39,46.

However, the role of Oct4 pseuodogenes in controlling mESC pluripotency and self-renewal remains unknown.

Here we identify a panel of novel, processed murine Oct4 pseudogenes and report on a trans-epigenetic cross-talk between an X-linked Oct4 pseudogene and the ancestral Oct4 gene, located on chromosome 17. We found that a long, non-coding RNA transcribed in sense orientation from Oct4 pseudogene 4 (Oct4P4) recruits the H3K9-specific HMTase SUV39H1 to induce epigenetic repression of the promoter of the ancestral Oct4 gene in trans. This mechanism reduces the expression of key self-renewal transcription factors and promotes mESC differentiation. Importantly, functional disruption of the Oct4P4–SUV39H1 silencing complex leads to a re-acquisition of self-renewal features, such as Oct4 expression and the re-activation of telomerase-dependent telomere maintenance mechanisms. This indicates that Oct4P4 is essential to prevent the re-activation of self-renewal circuits in differentiated cells.

Results

Oct4 pseudogenes expression during mESC differentiation

Recent studies report the existence of eight OCT4 pseudogenes in the human and one Oct4 pseudogene (Oct4-pg1, here called ‘Oct4P1’) in the mouse genome^36,38,39,40. Performing genome blast analyses using the mouse Oct4 transcript 1 (Pou5f1) complementary DNA (cDNA) as reference sequence, we identified four additional candidate pseudogenes (Oct4P2, Oct4P3, Oct4P4 and Oct4P5) that show high homology to portions of the open reading frame and untranslated regions (UTRs) of the ancestral Oct4 transcript 1 (Pou5f1) (Fig. 1a; Supplementary Fig. 1a). Using primers specific for each Oct4 pseudogene, we were able to PCR amplify all candidate pseudogenes from random or oligo-dT primed cDNA prepared from mESC total RNA (Fig. 1b). No PCR product was obtained when cDNA synthesis was carried out in the absence of primers or reverse transcriptase, thus excluding RNA self-priming or contamination of PCR reactions with genomic DNA. The identity of all Oct4 pseudogene-derived RNAs was sequence validated (Supplementary Fig. 1b). We conclude that Oct4P2, Oct4P3, Oct4P4 and Oct4P5 represent novel, processed Oct4 pseudogenes that are expressed in mESCs. Interestingly, Oct4P4 shows homology to the entire mature Oct4 mRNA but has acquired a 334 bp insertion that shows high homology to the LTR element of the ERVL-MaLRs retrotransposon family (Fig. 1a). Remarkably, this insert is subjected to RNA splicing (Supplementary Fig. 1c). To test whether Oct4 pseudogenes are subjected to transcriptional regulation, we prepared total RNA from self-renewing mESC (EB T0) and mESCs that were subjected to embryoid body (EB) in vitro differentiation for 3 (EB D3), 5 (EB D5) or 7 (EB D7) days (see Supplementary information). As expected, the ancestral Oct4 gene is subjected to efficient transcriptional repression during 7 days of EB differentiation (Fig. 1c). We found that EB differentiation is paralleled by a significant reduction of Oct4P1 and activation of Oct4P2 and Oct4P4 expression. EB differentiation does not significantly impact on Oct4P3 and Oct4P5 expression (Fig. 1c). Remarkably, Oct4P4 long non-coding RNA (lncRNA) levels are 150–300-fold higher in primary mouse embryonic fibroblasts (pMEFs) or immortalized mouse embryonic fibroblast compared with undifferentiated mESCs (Fig. 1c). Considering the lower RNA content of mESC compared with pMEFs⁴⁷, we found that Oct4P4 lncRNA levels are more than 600-fold higher in pMEFs compared with mESCs (Supplementary Fig. 1e). Information on subcellular localization of lncRNAs can provide important information about a possible function as nuclear-restricted epigenetic regulator or cytoplasmatic ceRNA^27,48. Pseudogene-specific quantitative reverse transcription–PCR (RT–PCR) using total RNA from nuclear and cytoplasmatic fractions of undifferentiated mESCs revealed that Oct4P2 and Oct4P4 are predominantly located in the nucleus (Fig. 1d). Oct4P1, gapdh and the ancestral Oct4 mRNA show enrichment in the cytoplasm; Oct4P3 and Oct4P5 did not show specific enrichment in nucleus or cytoplasm of mESCs (Fig. 1d). Together, these data indicate that a subset of RNAs derived from Oct4 pseudogenes show controlled intracellular localization and tight transcriptional regulation, anticipating a potential role in the control of mESC self-renewal. Unique properties of the Oct4P4 lncRNA such as extended homology to the ancestral Oct4 gene, nuclear localization, splicing, efficient upregulation during mESC differentiation and the virtual absence of relevant ORFs (Supplementary Fig. 1d) prompted us to investigate a possible role of Oct4P4 in the control of mESC differentiation and self-renewal.

**Figure 1: Characterization of murine *Oct4* pseudogenes.**

Oct4P4 lncRNA represses the expression of ancestral Oct4

Next, we were interested in testing whether the existence of Oct4P4 sense and anti-sense transcripts could provide a possible inroad in understanding the role of Oct4P4 in mESC. Performing sense/anti-sense-specific RT–PCR, we were not able to detect anti-sense transcripts originating from the Oct4 and Oct4P4 genes. We therefore concluded that a putative function of Oct4P4 in mESCs must arise from Oct4P4 sense transcripts (Supplementary Fig. 2a). Investigating a possible role for Oct4P4 in mESC biology, we stably overexpressed sense Oct4P4 lncRNA in self-renewing mESCs. We found that stable overexpression of Oct4P4 resulted in a 23% reduction of alkaline phosphatase activity, a classic marker of mESC self-renewal potential (Fig. 2a,b). Remarkably, Oct4P4 overexpression reduced AP activity to a similar extent like RNA interference (RNAi)-mediated depletion of endogenous Oct4 (Supplementary Fig. 2b,c). Importantly, Oct4P4 overexpression caused a 50% reduction of OCT4 protein levels (Fig. 2c). Reduced alkaline phosphatase activity and OCT4 protein levels were found to be paralleled by significantly reduced mRNA expression of key pluripotency transcription factors such as Oct4, Sox2, Klf4 and Gdf3 and increased expression of early mESC differentiation markers such as Fgf5, Brachyury, Nestin and Otx2 (Fig. 2d). Together, this indicates that ectopic expression of Oct4P4 impairs mESC self-renewal by interfering with transcriptional regulatory circuits that control pluripotency.

Figure 2: ***Oct4P4*** **impairs mESC self-renewal potential.**

To identify functionally relevant lncRNA domains, we created a panel of Oct4P4 expression vectors lacking Oct4P4 sequence elements with lowest sequence homology to the ancestral Oct4 cDNA (Fig. 2e). We found that stable overexpression of Oct4P4Δ1 that lacks a 449 nucleotide region comprising the 334 bp ERVL-MaLRs retrotransposon homology sequence was still able to reduce Oct4 mRNA and protein levels, recapitulating the effect of full-length Oct4P4 (Fig. 2f). In contrast, overexpression of Oct4P4Δ2, lacking sequences with high homology to the Oct4 5′ and 3′ UTR regions were not able to reduce Oct4 expression in mESCs (Fig. 2f). Oct4P4Δ1 overexpression significantly reduces the expression of pluripotency transcription factors. In contrast, Oct4P4Δ2 overexpression does not impact on self-renewing transcriptional circuits in self-renewing mESCs (Fig. 2g). These data suggest that Oct4P4 5′ and 3′UTR portions are important to reduce Oct4 expression. We therefore created additional Oct4P4 deletion constructs centred on regions located at the 5′ and 3′ termini of Oct4P4 lncRNA. Deleting the Oct4P4-5′UTR (Oct4P4Δ5′UTR) or Oct4P4-3′UTR (Oct4P4Δ3′UTR) resulted a reduced efficiency of Oct4 repression when compared with overexpression of full-length Oct4P4 lncRNA (Supplementary Fig. 3a,b). Ectopic expression of the 5′UTR or 3′UTR homology elements of the Oct4P4 lncRNA (Oct4P4-5′UTR or Oct4P4-3′UTR, respectively) did not impact on the expression of the ancestral Oct4 gene (Supplementary Fig. 3c,d). Together these data indicate that the 5′UTR and 3′UTR homology region of Oct4P4 collaborate to repress the expression of the ancestral Oct4 gene. Subcellular fractionation experiments revealed that ectopic Oct4P4Δ1 RNA maintains nuclear localization (Fig. 2h). In contrast, deletion of both, 5′ and 3′ UTR homology regions resulted in a non-specific subcellular distribution of Oct4P4Δ2 RNA in mESCs (Fig. 2h). Finally, individually deleting the 5′UTR or 3′UTR homology regions of Oct4P4 caused a reduced enrichment of Oct4P4Δ5′UTR or Oct4P4Δ3′UTR RNAs in the nucleus (Supplementary Fig. 3e). This indicates that the 5′UTR or 3′UTR are important to mediate nuclear localization of Oct4P4. Of note, overexpression of Oct4P4 did not cause a subcellular de-localization of ancestral Oct4 or gapdh mRNAs, excluding that Oct4P4 impairs mESC self-renewal by de-localizing the ancestral Oct4 mRNA (Supplementary Fig. 3f). We conclude that the Oct4P4 pseudogene gives rise to a sense lncRNA that employs nuclear-restricted molecular mechanisms to reduce the expression of pluripotency transcription factors, finally leading to impaired mESC self-renewal.

Oct4P4 lncRNA orchestrates silencing of the Oct4 promoter

Based on the fact that nuclear localization is essential for Oct4P4 lncRNA function, we speculated that Oct4P4 might directly interact with the promoter of the ancestral Oct4 gene. To test this hypothesis, we performed Oct4 promoter luciferase reporter experiments in NIH-3T3 cells that lack expression of endogenous Oct4 (Fig. 1c, Fig. 3a). Co-transfection of the reporter with a plasmid-encoding haemagglutanin (HA)-epitope-tagged murine OCT4 under the control of the CMV promoter was able to increase Oct4 promoter luciferase reporter activity (Fig. 3b, left panel). Importantly, Oct4P4 overexpression significantly attenuated this effect without affecting expression levels of ectopically expressed OCT4 protein (Fig. 3b, right panel). This suggests that Oct4P4-derived lncRNAs specifically reduced the activity of the Oct4 promoter used in the reporter construct. To provide evidence for a cross-talk between Oct4P4 ncRNA and the promoter of the ancestral Oct4 gene in trans, we established an ‘Oct4P4-MS2 RNA stem loop’ tethering system in mESC⁴⁹. We generated mESCs stably co-expressing a flag-tagged version of MS2 phage coat protein (MS2-flag), as well as Oct4P4 lncRNA fused to 24 repeats of the MS2 RNA stem loop (24 × MS2 stem loop), that binds with high affinity to MS2 phage coat protein (Fig. 3c; Supplementary Material and Methods). This model system permits the use of anti-flag antibodies to detect Oct4P4-24 × MS2 stem loop RNA at gene loci by chromatin immunoprecipitation (ChIP) or to identify Oct4P4 interacting proteins by RNA immunoprecipitation (RIP) (Fig. 3c). mESCs exclusively overexpressing MS2-flag were used as a control cells. In line with previous data, overexpression of MS2 stem loop-tagged Oct4P4 (Oct4P4-24 × MS2 stem loop) results in reduced OCT4 protein and mRNA expression (Fig. 3d). Importantly, we were able to efficiently amplify Oct4P4-24 × MS2 stem loop RNA by RT–PCR in anti-flag RIP experiments using mESCs stably co-expressing MS2-flag and Oct4P4-24 × MS2 stem loop constructs. This demonstrates that MS2-flag forms a complex with the Oct4P4-24 × MS2 stem loop RNA in vivo (Fig. 3e). To demonstrate that Oct4P4-24 × MS2 stem loop RNA localizes to the endogenous Oct4 promoter region in MS2-flag expressing mESCs, we performed ChIP experiments using a set of primer pairs that specifically amplify the promoter of the ancestral Oct4 gene (Fig. 3a; primer pairs A, B)⁵⁰. Performing ChIP using anti-flag antibodies, we were able to demonstrate the presence of MS2-Flag at the conserved Oct4 promoter/enhancer region CR2 in MS2-flag/Oct4P4-24 × MS2 stem loop RNA-expressing mESCs (Fig. 3f). Importantly, MS2-flag control mESCs that lack Oct4P4-24 × MS2 stem loop RNA expression did not show association of MS2-flag with the Oct4 promoter (Fig. 3f). Anti-flag ChIP did not immunoprecipitate the promoter region of the unrelated Dkk gene in control cells (Fig. 3f, right panel).

Figure 3: ***Oct4P4*** **mediates the imposition of H3K9me3 and HP1α at the** ***Oct4*** **gene.**

Together, our data demonstrate that the Oct4P4 lncRNA is required to localize MS2-flag to the Oct4 promoter in MS2-flag/Oct4P4-24 × MS2 stem loop RNA-expressing mESCs. This data support a model where a sense-oriented Oct4P4 lncRNA can localize in trans to the promoter of the Oct4 gene.

Nuclear, lncRNAs such as XIST, HOTAIR or pericentric satellite repeats plays a key role in recruiting epigenetic repressors such as the H3K9-specific SUV39H Histone HMTases or H3K27-specific HMTase containing Polycomb repressive complexes^51,52,53. We consequently speculated that upregulation of Oct4P4 ncRNA expression during mESC differentiation (Fig. 1c) might direct epigenetic silencing complexes to the endogenous Oct4 promoter. Taking advantage of our Oct4P4-MS2 RNA tethering mESC model system, we found that Oct4P4-24 × MS2 stem loop RNA overexpression in MS2-flag mESCs result in a fivefold increase of H3K9me3 at conserved Oct4 promoter/enhancer regions (CR1, CR2; detected by primer pairs A and B), as determined by ChIP (Fig. 3a,g). Consistent with the reported high binding affinity of HP1 to H3K9me3 (ref. 54), we found a sixfold increase of HP1α at the Oct4 promoter in our model mESC line (Fig. 3g). Of note, Oct4P4-24 × MS2 stem loop RNA overexpression in MS2-flag mESCs does not impact on the abundance of H4K20me3 or H3K27me3 at the Oct4 promoter (Fig. 3g). In line with increased H3K9me3 levels, we found a fourfold increase of the H3K9-specific SUV39H1 HMTase at the Oct4 promoter of MS2-flag/Oct4P4-MS2-24 × MS2 stem loop mESCs (Fig. 3h). To individuate Oct4P4 lncRNA regions that are important for SUV39H1 interaction and SUV39H1 deposition on the Oct4 promoter, we performed anti-SUV39H1 ChIP and RIP experiments using mESCs stably overexpressing full-length Oct4P4, Oct4P4Δ5′UTR, Oct4P4Δ3′UTR or Oct4P4Δ2 lncRNAs. Importantly, we found that SUV39H1 deposition on the Oct4 promoter was reduced in Oct4P4Δ5′UTR and Oct4P4Δ3′UTR cells when compared with full-length Oct4P4 overexpressing cells (Supplementary Fig. 4a). Deletion of the Oct4 5′ and 3′ UTR homology regions in Oct4P4 reduced efficiency of Oct4P4 lncRNA mediated repression of Oct4 (Fig. 2f). In line with this, mESCs overexpressing Oct4P4 lncRNAs lacking the 5′UTR (Oct4P4Δ5′UTR) or 3′UTR (Oct4P4Δ3′UTR) homology show reduced SUV39H1 abundance at the Oct4 promoter when compared with cells overexpressing the full-length Oct4P4 lncRNA (Supplementary Fig. 4a). Finally, overexpression of an Oct4P4 version lacking both the 3′ and 5′ UTR homology region (Oct4P4Δ2) did not result in the accumulation of SUV39H1 at the Oct4 promoter. This indicates that the 5′ and 3′UTR homology regions are important to localize SUV39H1 to the Oct4 promoter. Accordingly, Oct4P4Δ2 was no longer able to physically interact with SUV39H1 (Supplementary Fig. 4b), further underlying the importance of 5′ UTR and 3′UTR homology regions for Oct4P4 function.

Altogether, our data show that the Oct4P4 lncRNA, expressed in sense orientation from X-linked Oct4P4 locus, mediates the enrichment of H3K9me3, HP1α and SUV39H1 at the Oct4 promoter, located on chromosome 17. This trans-acting silencing mechanism leads to reduced Oct4 expression that finally results in impaired self-renewal potential of mESCs (Fig. 3d,g,h).

An Oct4P4–SUV39H1 complex targets the Oct4 promoter

We showed that Oct4P4 lncRNA overexpression leads to chromatin compaction at the promoter of the ancestral Oct4 gene. We next wished to test whether (i) Oct4P4 physically interacts with SUV39H1 and (ii) both players are required to induce epigenetic gene silencing at the promoter of the ancestral Oct4 gene.

Using MS2-flag/Oct4P4-24 × MS2 stem loop model mESC, we found that anti-SUV39H1-specific antibodies were able to immunoprecipitate MS2-flag but also Oct4P4-24 × MS2 stem loop lncRNA (Fig. 4a; Supplementary Fig. 5a). Importantly, also endogenous Oct4P4 lncRNA was detectable by RT–PCR in anti-SUV39H1 RIP experiments using pMEFs, that are characterized by high Oct4P4 lncRNA expression levels (Figs 1c and 4b). Of note, Oct4P4 lncRNA abundance was found to be strongly reduced in anti-SUV39H1 RNA immunoprecipitates after RNAi-mediated reduction of endogenous Oct4P4, thus confirming the specificity of our experiments (Fig. 4b).

**Figure 4: ***Oct4P4*** **interacts with SUV39H1 to silence ancestral** ***Oct4*** in ***trans***.**

Next, we wished to validate that SUV39H1 is required to reduce Oct4 expression by mediating the enrichment of H3K9me3 and its high-affinity binding partner HP1α at the Oct4 promoter.

RNAi-mediated knockdown of SUV39H1 resulted in significantly increased Oct4 mRNA and OCT4 protein levels in wild-type mESCs and Oct4P4 lncRNA-overexpressing mESCs. This provided first evidence that the SUV39H1 HMTase controls Oct4 expression in mESCs (Fig. 4c,d). To understand the relevance of SUV39H1 for the establishment of a repressive chromatin structure at the endogenous Oct4 promoter, we performed anti-flag and anti-SUV39H1 ChIP experiments in MS2-flag/Oct4P4-24 × MS2 stem loop mESCs. As expected, SUV39H1 RNAi resulted in reduced abundance of SUV39H1 and H3K9me3 at the Oct4 promoter in MS2-flag/Oct4P4-24 × MS2 stem loop mESCs (Fig. 4e, Supplementary Fig. 5b). Importantly, this effect was paralleled by reduced MS2-flag levels at the Oct4 promoter, indicative for reduced abundance of Oct4P4-24 × MS2 stem loop RNA at the ancestral Oct4 promoter. Together, this indicates that both SUV39H1 and Oct4P4 lncRNA are required to mediate localization of the silencing complex to the endogenous Oct4 promoter.

Oct4P4 blocks aberrant acquisition of self-renewal features

High Oct4P4 lncRNA expression levels in pMEFs (Fig. 1c) suggest that the Oct4P4–SUV39H1 lncRNA–protein complex has an important role in silencing the endogenous Oct4 promoter in differentiated cells. In line with this hypothesis, we found that siRNA-mediated knockdown of Oct4P4 lncRNA in pMEFs results in a fourfold or twofold reduction of SUV39H1 or H3K9me3 at the Oct4 promoter, respectively (Fig. 5a,b). Importantly, reduced chromatin compaction at the Oct4 promoter was paralleled by a significant increase in Oct4 mRNA expression (Fig. 5c). This data was confirmed in experiments using an independent Oct4P4 lncRNA-specific siRNA oligonucleotide (Supplementary Fig. 6a,b). This indicates that the Oct4P4–SUV39H1 complex plays an important role in maintaining silencing of the Oct4 promoter in pMEFs. Interestingly, we found that Oct4P4 is efficiently repressed during the conversion of pMEFs to iPS (Fig. 5d). This suggests that Oct4P4 might represent a barrier to the de-differentiation of pMEFs. To further test this idea, we performed Oct4P4 loss-of-function experiments in pMEFs. Oct4P4 knockdown caused a change of the characteristic flat and single-cell morphology of pMEFs to round-shaped cells but also the formation of multi-cellular spheres that grow in suspension or attached on top of pMEFs (Fig. 5e). The formation of spheres or round-shaped cells was inhibited when pMEFs were co-transfected with a mix of siOct4P4 and siOct4 oligonucleotides, indicating that changes in cell morphology are Oct4 dependent (Fig. 5e,f). Importantly, siOct4P4-induced changes in cell morphology were paralleled by a significantly increased expression of mESC pluripotency transcription factors Oct4 (threefold), Sox2 (threefold), Nanog (threefold) and Klf4 (twofold) (Fig. 5f). This data was confirmed in experiments using an independent Oct4P4-specific siRNA oligonucleotide (Supplementary Fig. 6c). Remarkably, siOct4P4-dependent increase of pluripotency transcription factor expression was attenuated by contemporary knockdown of Oct4 (Fig. 5f). Together, this indicates that depletion of Oct4P4 from pMEFs results in a loss of heterochromatin at the Oct4 promoter, leading to the re-activation of endogenous Oct4 that promotes the expression of additional key transcription factors of mESC self-renewal. To find evidence that loss of Oct4P4 activates other biological pathways linked to mESC self-renewal programmes, we studied the impact of Oct4P4 on telomere maintenance in pMEFs. pMEFs are characterized by progressive telomere shortening due to lack of sufficient telomerase activity. In line with this, re-activation of telomerase activity is a key step for the acquisition of pluripotency during iPS reprogramming of pMEFs⁵⁵. Importantly, we found that 2 consecutive cycles of RNAi-mediated depletion of Oct4P4 from pMEFs resulted in a 2.6-fold upregulation of the transcript for the catalytic subunit of telomerase, TERT. Importantly, Tert mRNA upregulation was paralleled by a concomitant increase in telomeric DNA repeat content, as determined by real-time PCR-based quantification of telomeric DNA repeats in experimental cells (Fig. 5g,h). This data was confirmed in experiments using independent Oct4P4-specific siRNA oligonucleotides (Supplementary Fig. 6c,d). This indicates that Oct4P4 depletion re-activates telomerase expression to drive telomere elongation. Remarkably, these effects were attenuated when pMEFs where transfected with a mix of siRNAs that target Oct4P4 lncRNA and the ancestral Oct4 mRNA (Fig. 5g,h).

Figure 5: ***Oct4P4*** **prevents the activation of features of mESC self-renewal in pMEFs.**

Together, our data demonstrate that depleting Oct4P4 from differentiated pMEFs results in the re-activation of basic features of mESC pluripotency, such as self-renewal transcription factor expression and activation of telomerase-dependent mechanisms of telomere maintenance. This further indicates that Oct4P4 has an important role in preventing the aberrant expression of self-renewal promoting transcription factors in differentiated cells.

Discussion

Pseudogene-derived transcripts have been reported to impact on the expression of ancestral protein-coding gene using molecular pathways based on RNA sequence homology. Pseudogenes derived transcripts were demonstrated to control ancestral gene by producing endogenous siRNAs, sponging miRNAs by acting as ceRNAs or altering the stability of the ancestral mRNA^{28,29,30,31,32,56}. In addition, anti-sense pseudogene transcripts can impact on the promoter activity of ancestral genes^30,36,57,58. In this study, we used mESCs to identify a set of novel transcripts derived from putative Oct4 pseudogenes (Oct4P1, Oct4P2, Oct4P3 Oct4P4 and Oct4P5). Tight regulation of subcellular localization and expression during mESC differentiation anticipate a defined role for a subset of these transcripts in mESC biology. Here we focused our studies on a nuclear-restricted non-coding RNA (ncRNA) that is transcribed in sense orientation from the processed, X-linked Oct4P4. Dramatic upregulation of sense Oct4P4 transcription during mESC differentiation gives rise to a long, non-coding RNA that forms a complex with the repressive H3K9-specific HMTase SUV39H1. This complex translocates to the promoter of the ancestral Oct4 protein-coding gene, located on chromosome 17, to impose H3K9 tri-methylation leading to a consecutive recruitment of HP1α and silencing of the ancestral Oct4 gene (Fig. 6). This is in a remarkable contrast to the ceRNA function of the human cytoplasmatic lncRNA OCT4-pg4 in hepatocellular carcinoma⁴⁵. Thus murine Oct4P4 and human OCT4-pg4 are not mechanistically conserved and act via completely different cellular pathways. A recent study reported that anti-sense transcripts originating from the human OCT4-pg5 can mediate silencing of the human OCT4 gene in cancer cells⁴⁶. Importantly, we were not able to detect anti-sense transcripts originating from murine ancestral Oct4 or Oct4P4 genes (Supplementary Fig. 2a). We also did not find a significant homology between the Oct4P4 lncRNA and the Oct4 promoter (Supplementary Fig. 7c). Finally, Oct4P4 lncRNA did not impact on Oct4P4 promoter activity (Supplementary Fig. 7a,b). Thus, these findings do not support a model that involves the expression of an anti-sense Oct4P4 transcript or direct interaction of Oct4P4 lncRNA with the Oct4 promoter that is based on sequence homology. We rather propose that sense-oriented Oct4P4 lncRNAs represents the functionally relevant pseudogene-derived lncRNA that forms a complex with SUV39H1 and then translocates to the promoter of the ancestral Oct4 gene. In line with this, deleting Oct4P4 5′ or 3′ sequences that show high homology to the 5′UTR or 3′UTR regions, respectively, of ancestral Oct4 significantly reduce the ability of Oct4P4 lncRNA to deposit SUV39H1 at the Oct4 promoter, thus leading to reduced Oct4 silencing. This highlights a possible importance of 5′UTR and 3′UTR homology regions of pseudogene-derived sense transcripts for epigenetic gene regulation.

Figure 6: **A model for** ***Oct4P4*** **lncRNA function.**

Remarkably, the human OCT4-pg3 (here OCT4P3, NR_036440.1), OCT4-pg4 (here OCT4P4, NR_034180.1) and OCT4-pg1 (here OCT4P1b NM_001159542.1) show homology to the entire OCT4 cDNA and share high structural and sequence homology to murine Oct4P4. Similar to Oct4P4 in mESCs, we found that human OCT4P3 lncRNA is enriched in the nuclei of OVCAR-3 and SKOV-3 ovarian cancer cells (Supplementary Fig. 8). This provides a first indication for a possible conservation of Oct4P4/OCT4P3 function.

Interfering with the murine Oct4P4–SUV39H1 silencing complex in differentiated cells leads to the re-activation of mESC self-renewal transcription factor expression and telomerase-dependent telomere maintenance mechanism (Fig. 6). This represents a partial re-acqusition of mESC stem cell features in short term (6 days) Oct4P4 lncRNA knock-down experiments. It will be interesting to test whether loss of Oct4P4 function can increase the efficiency of iPS cell generation protocols. Together, our data indicates that Oct4P4 and SUV39H1 are important to prevent the aberrant activation of mESCs self-renewal pathways in differentiated cells. Future experiments will aim to better define structural Oct4P4 lncRNA motifs involved in SUV39H1 recruitment and elucidate pathways that direct Oct4P4 to the ancestral Oct4 gene.

Several lncRNAs such as XIST or HOTAIR have been described to act as scaffold for the formation of chromatin-modifying complexes^51,52. To our knowledge, the Oct4P4 lncRNA represents the first example for a crucial role of a vertebrate pseudogene-derived sense lncRNAs in directing chromatin-modifying activities to the promoter of its ancestral gene in trans.

Given the vast repertoire of vertebrate pseudogenes, we anticipate the existence of a series of pseudogene-derived lncRNAs that use mechanisms analogous to Oct4P4 to control the expression of ancestral genes on the epigenetic level. Understanding pseudogene lncRNA-dependent mechanism of epigenetic gene regulation and a precise categorization of pseudogene-derived lncRNAs based on subcellular localization, gene expression regulation and deposition at gene promoters will provide important insights into the global role of pseudogene-derived lncRNAs in regulating higher order chromatin structures.

Methods

Cell culture and generation of stable cell lines

Feeder-independent mouse ESCs⁵⁹ were cultured on gelatin (0.2%) coated plates in mESC self-renewal medium (DMEM with 15% knockout serum replacement (Gibco), 1% nonessential amino acids (Gibco), 1 mM sodium pyruvate (Invitrogen), 1% L-glutamine (Invitrogen), 0.1 mM ß-mercaptoethanol, 1% penicillin/streptomycin (Invitrogen) and 1,000 U ml⁻¹ mouse leukaemia inhibitory factor. pMEFs were generated from 13.5 d.p.c C57BL/6 mouse embryos. NIH-3T3 cells were obtained from ATCC. pMEFs and NIH-3T3 cells were maintained in culture in DMEM supplemented with 10% foetal bovine serum (Lonza) and 1% penicillin/streptomycin. iPS were generated and cultivated according to standard conditions⁶⁰. OVCAR-3 cells (ATCC) were cultured in RPMI-1640 medium supplemented with 20% foetal bovine serum (Lonza), L-glutamine (2 mM), insulin (10 μg ml⁻¹; I9278, Sigma) and 1% penicillin/streptomycin. SKOV-3 cells (ATCC) and U2OS cells (ATCC) were maintained in DMEM supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin. To generate stable mESC lines, cells were transfected (TransIT-LT1; Mirus Bio LCC) with respective vectors and subjected to selection using G418 (Sigma, 300 μg μl⁻¹) or puromycin (Sigma, 3 μg ml⁻¹).

Oct4 pseudogenes analysis and sequencing

Genome blast analysis ( http://blast.ncbi.nlm.nih.gov/) was performed to identify mouse genomic regions with high similarity to Oct4 (mus musculus Pou5f1, transcript variant 1; NM_013633.3) mRNA. Genomic coordinates of identified pseudogenes were obtained using the University of California Santa Cruz (UCSC) genome browser. All candidate pseudogenes were PCR amplified from mESC cDNA using specific primers (Supplementary table 1). PCR amplicons were cloned into pCR2.1-TOPO vector (Life Technologies) and subjected to bi-directional sequencing.

Oct4P4-MS2 tethering model system

To generate the Oct4P4-24 × MS2 stem loop construct, 24 repeats of the MS2 stem loop RNA motif (obtained from pSL-MS2-24 × , ref. 61) were cloned downstream of the Oct4P4 cDNA into pLPC. Oct4P4 cDNA was PCR amplified using the following oligonucleotides: forward: 5′-GGGAATTCAAGCTTGTCCCTAGGTGACCAACTCCT3′-; reverse: 5′-GGGAATTCAGATCTTGTGTCCCAGGCTTTTTAAA3′-). pCMV-FLAG-MS2nls was provided by A. Marcello (ICGEB, Trieste, Italy).

RNA immunoprecipitation

Cells were cross-linked in culture medium with 1% formaldehyde for 10 min. After addition of 125 mM glycine (PBS), cells were washed twice in ice cold PBS. Cross-linked cells were scraped in RIPA buffer (50 mM Tris-Cl, pH 7.5, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA,150 mM NaCl) and supplemented with protease inhibitors (Complete, Roche) and RNaseOUT (Invitrogen). After incubation at 4 °C for 20 min, the cell lysate was sonicated and centrifuged. The supernatant was pre-cleared for 1 h at 4 °C with protein A/G PLUS-Agarose (sc-2003; Santa Cruz Biotechnology), supplemented with yeast transfer RNA (0.1 mg ml⁻¹, Invitrogen) and incubated overnight at 4 °C with the following antibodies: mouse monoclonal anti-FLAG M2, clone M2 (2.5 μg ml⁻¹; F1804; Sigma); mouse monoclonal anti-KMT1A/SUV39H1 (2.5 μg ml⁻¹; ab12405; Abcam); mouse monoclonal anti-HA, (2.5 μg ml⁻¹; clone HA-7, H9658; Sigma). RNA–protein complexes were recovered with protein A/G PLUS-Agarose beads and were washed six times in high-stringency RIPA buffer (50 mM Tris-Cl, pH 7.5, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 M NaCl, 1 M urea and 0.2 mM phenylmethylsulfonyl fluoride). An aliquot of beads containing immunoprecipitatated samples were saved for western blot analysis. Remaining beads were resuspended in 50 mM Tris-Cl, pH 7.0, 5 mM EDTA, 10 mM DTT and 1% SDS and incubated at 70 °C for 45 min to reverse cross-linking. The immunoprecipitated RNA and the total RNA were extracted with the Qiazol lysis reagent (Qiagen), subjected to DNase treatment (Qiagen) and subjected to reverse transcription (Quantitect reverse transcription kit; Qiagen). The obtained cDNA used for quantitative real-time PCR (SYBR Green Universal PCR Master Mix, Applied Biosystems) on a StepOnePlus real-time PCR machine (Applied Biosystems). The oligos used for quantitative RT–PCR are reported below:

Oct4P4: forward: 5′-TGGCACCTGGCTTTAGACTTT-3′; reverse: 5′-CCAGGCCAACTTAGGGCATT-3′. 24 × MS2-Oct4P4: forward: 5′-CACCACGGCTTTGGAGTTAAG-3′; reverse: 5′-CATTAGATCTTGTGTCCCAG-3′.

Analysis of immunocomplexes by western blotting: input and immunocomplexes proteins were resuspended in SDS–PAGE sample buffer, boiled for 15 min and analysed by western blotting. Primary antibodies: mouse monoclonal anti-FLAG (1:1,000; clone M2, F1804); mouse monoclonal anti-KMT1A/SUV39H1 (1:500; ab12405); mouse monoclonal anti-HA, clone (1:1,000; HA-7, H9658) rabbit polyclonal anti-Oct4 (1:1,000; ab19857; Abcam); anti-actin (1:1,000; A2066; Sigma). Secondary antibodies coupled to horseradish peroxidase (1:1,000; A-6154; A-4416; Sigma) were used for signal detection.

Chromatin immunoprecipitation

Cells were cross-linked in culture medium with 1% formaldehyde for 10 min, neutralized using 125 mM glycine in PBS and washed in PBS. Nuclei were obtained by lysing scraped cells in hypotonic buffer (5 mM Pipes pH 6.8, 85 mM KCl, 0.5% NP-40 and protease inhibitors), followed by centrifugation. Nuclei were resuspended in RIPA 100 mM buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.5% Na-Deoxycholate, 0.1% SDS supplemented with protease inhibitors). Chromatin was sonicated to 500—800 bp average fragment size and pre-cleared for 1 h at 4 °C with protein A/G PLUS-Agarose (sc-2003). Agarose was removed by centrifugation and an aliquot of supernatant was taken as input. Chromatin was immunoprecipitated overnight at 4 °C with the following antibodies: mouse monoclonal anti-FLAG M2, clone M2 (2.5 μg ml⁻¹; F1804); mouse monoclonal anti-KMT1A/SUV39H1 (44.1; 2.5 μg ml⁻¹; ab12405); rabbit polyclonal anti-trimethyl-Histone H3 (Lys9) (2.5 μg ml⁻¹; 07-442; Millipore); mouse monoclonal anti-HP1a, clone 15.19s2 (2.5 μg ml⁻¹; 05-689; Millipore); rabbit polyclonal anti-trimethyl-Histone H4 (Lys20) (2.5 μg ml⁻¹; 07-749; Millipore); rabbit polyclonal anti-trimethyl-Histone H3 (Lys27) (2.5 μg ml⁻¹; 07-449; Millipore). As a negative control for immunoprecipitation, mouse monoclonal anti-HA, clone HA-7 (2.5 μg ml⁻¹; H9658) was used. DNA–protein complexes were recovered with protein A/G PLUS-Agarose beads and washed with RIPA 100 mM buffer, RIPA 250 mM buffer (20 mM Tris-HCl pH 7.5, 250 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.5% Na-Deoxycholate and 0.1% SDS), LiCl solution (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40 and 0.5% Na-Deoxycholate) and 1 × TE. RNase treatment was performed in 1 × Tris-EDTA (TE) for 30 min at 37 °C. Cross-linking was reversed by overnight incubation at 68 °C after adding an equal volume of proteinase K solution (200 mM NaCl, 1% SDS and 0.3 mg ml⁻¹ proteinase K). Samples were resuspended in ddH₂O after phenol/chloroform extraction and ethanol precipitation. Co-immunoprecipitated DNA was analysed by RT–PCR on a StepOnePlus real-time PCR machine, using SYBR Green Universal PCR Master Mix.

The oligos used for quantitative RT–PCR are reported below.

Dkk: forward: 5′-GGGAACCAGGGAAAGAGGA-3′; reverse: 5′-GGGAAATAGGCACCCGATAA-3′. Oct4 (primer A): forward: 5′-TGCACCCCCTCCTCCTAATCC-3′; reverse: 5′-CCCTAAACAAGTACTCAACCC-3′.

Oct4 (primer B): forward: 5′-GTTGGGGGGTGGTTAGTGTCT-3′; reverse: 5′-CCACTCCTCAGTTCTTGCTTA-3′. ChIP data of Oct4 promoter regions were normalized against input and the Oct4 unrelated DKK gene.

Transient transfection

mESCs and pMEFs were transfected with siRNAs (final concentration, 30 nM) using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions. siRNA oligonucleotide: mouse Oct4 (Thermo Scientific Dharmacon; sense sequence: 5′-CGGAAGAGAAAGCGAACUAUU-3′), mouse Suv39h1 (Thermo Scientific Dharmacon; sense sequence: 5′-CCAAUUACCUGGUGCAGAA-3′) mouse Oct4P4 (Thermo Scientific Dharmacon; sense sequence: 5′-GAGCAUGAGUGGAGAGGAA-3′) and mouse Oct4P4#2 (Eurofins Genomics; sense sequence: 5′-GCCUCUCUUAAGCACUGUA-3′). siGENOME Non-Targeting siRNA#1 (D-001210-01-20, Thermo Scientific Dharmacon) and AllStars Negative Control siRNA (1027281; Qiagen; siControl#2) were used as a negative controls. Transient transfections of plasmids for pMEFs and U2OS were performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions.

Luciferase reporter assay

The mouse Oct4 promoter, corresponding to 2.9 kb of genomic region upstream of the Oct4 ATG, was PCR amplified from mESC genomic DNA; forward 5′-GGGAATTCCTCGAGATTGTACGTAAGTACTTCAGA-3′; reverse, 5′-GGGAATTCAGATCTGGGGAAGGTGGGCACCCCGA-3′ oligonucleotides. For the mouse Oct4P4 promoter reporter construct, 1.8 kb fragment upstream the Oct4P4 gene was PCR amplified from mESC genomic DNA; forward: 5′-GGGAATTCCTCGAGCATATGTGTGTCAATCTTGTT-3′; reverse: 5′-GGGAATTCAGATCTGGGGAAGTTGGGCACCCCAAG-3′. Fragments were cloned into pGL3Basic (Promega) via XhoI and BglII restriction sites located upstream of the firefly luciferase gene and sequence verified. pcDNA3-HA-OCT4 is reported in ref. 22. For luciferase reporter assays, 4 × 10⁴ NIH-3T3 cells were transiently co-transfected with 400 ng of pGL3 promoter luciferase reporters and 40 ng of CMV-Renilla to normalize for transfection efficiency; when indicated, 400 ng of pCDNA3-HA-Oct4 or pCDNA3-HA vector, and 400 ng of pCDNA3-Oct4P4 or pCDNA3 vector were co-transfected. Transient transfections were performed using Lipofectamine 2000 according to manufacturer’s instructions. For luciferase reporter assays involving siRNAs, plasmid DNA was transfected first followed by siRNA transfection after a 16-h recovery period. Firefly/renilla luciferase activity was assayed 72 h after transfection using the Dual Luciferase Assay kit (Promega).

Statistical analysis

Each finding was confirmed by three independent biological replicates, unless specified. Error bars represent s.d. All P values were determined using two-tailed t-tests and statistical significance was set at P<0.05. The variance was similar between groups that we compared.

Uncropped scans of the most important blots are shown in Supplementary Fig. 9 in the Supplementary Information section.

Additional information

How to cite this article: Scarola, M. et al. Epigenetic silencing of Oct4 by a complex containing SUV39H1 and Oct4 pseudogene lncRNA Nat. Commun. 6:7631 doi: 10.1038/ncomms8631 (2015).

References

Boyer, L. A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).
Article CAS Google Scholar
Chen, X. et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117 (2008).
Article CAS Google Scholar
Kim, J., Chu, J., Shen, X., Wang, J. & Orkin, S. H. An extended transcriptional network for pluripotency of embryonic stem cells. Cell 132, 1049–1061 (2008).
Article CAS Google Scholar
Loh, Y.-H. et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat. Genet. 38, 431–440 (2006).
Article CAS Google Scholar
Tantin, D. Oct transcription factors in development and stem cells: insights and mechanisms. Development 140, 2857–2866 (2013).
Article CAS Google Scholar
Zhao, F.-Q. Octamer-binding transcription factors: genomics and functions. Front. Biosci. (Landmark Ed.) 18, 1051–1071 (2013).
Article CAS Google Scholar
Ben-Porath, I. et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat. Genet. 40, 499–507 (2008).
Article CAS Google Scholar
Nichols, J. et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998).
Article CAS Google Scholar
Okamoto, K. et al. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell 60, 461–472 (1990).
Article CAS Google Scholar
Schöler, H. R., Hatzopoulos, A. K., Balling, R., Suzuki, N. & Gruss, P. A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor. EMBO J. 8, 2543–2550 (1989).
Article Google Scholar
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).
Article CAS Google Scholar
Sterneckert, J., Höing, S. & Schöler, H. R. Concise review: Oct4 and more: the reprogramming expressway. Stem Cells 30, 15–21 (2012).
Article CAS Google Scholar
Wang, K. et al. Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. PLoS ONE 5, e10622 (2010).
Article ADS Google Scholar
Sato, N., Kondo, M. & Arai, K. The orphan nuclear receptor GCNF recruits DNA methyltransferase for Oct-3/4 silencing. Biochem. Biophys. Res. Commun. 344, 845–851 (2006).
Article CAS Google Scholar
Li, J.-Y. et al. Synergistic function of DNA methyltransferases Dnmt3a and Dnmt3b in the methylation of Oct4 and Nanog. Mol. Cell Biol. 27, 8748–8759 (2007).
Article CAS Google Scholar
Popowski, M. et al. Bright/Arid3A acts as a barrier to somatic cell reprogramming through direct regulation of Oct4, Sox2, and Nanog. Stem Cell Rep. 2, 26–35 (2014).
Article CAS Google Scholar
Gu, P. et al. Orphan nuclear receptor GCNF is required for the repression of pluripotency genes during retinoic acid-induced embryonic stem cell differentiation. Mol. Cell Biol. 25, 8507–8519 (2005).
Article CAS Google Scholar
Sylvester, I. & Schöler, H. R. Regulation of the Oct-4 gene by nuclear receptors. Nucleic Acids Res. 22, 901–911 (1994).
Article CAS Google Scholar
Brumbaugh, J. et al. Phosphorylation regulates human OCT4. Proc. Natl Acad. Sci. USA 109, 7162–7168 (2012).
Article ADS CAS Google Scholar
Jang, H. et al. O-GlcNAc regulates pluripotency and reprogramming by directly acting on core components of the pluripotency network. Cell Stem Cell 11, 62–74 (2012).
Article CAS Google Scholar
Saxe, J. P., Tomilin, A., Schöler, H. R., Plath, K. & Huang, J. Post-translational regulation of Oct4 transcriptional activity. PLoS ONE 4, e4467 (2009).
Article ADS Google Scholar
Schoeftner, S., Scarola, M., Comisso, E., Schneider, C. & Benetti, R. An Oct4-pRb axis, controlled by MiR-335, integrates stem cell self-renewal and cell cycle control. Stem Cells 31, 717–728 (2013).
Article CAS Google Scholar
Spelat, R., Ferro, F. & Curcio, F. Serine 111 phosphorylation regulates OCT4A protein subcellular distribution and degradation. J. Biol. Chem. 287, 38279–38288 (2012).
Article CAS Google Scholar
Tay, Y., Zhang, J., Thomson, A. M., Lim, B. & Rigoutsos, I. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature 455, 1124–1128 (2008).
Article ADS CAS Google Scholar
Xu, N., Papagiannakopoulos, T., Pan, G., Thomson, J. A. & Kosik, K. S. MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell 137, 647–658 (2009).
Article CAS Google Scholar
Groen, J. N., Capraro, D. & Morris, K. V. The emerging role of pseudogene expressed non-coding RNAs in cellular functions. Int. J. Biochem. Cell Biol. 54C, 350–355 (2014).
Article Google Scholar
Tay, Y., Rinn, J. & Pandolfi, P. P. The multilayered complexity of ceRNA crosstalk and competition. Nature 505, 344–352 (2014).
Article ADS CAS Google Scholar
Wang, J. et al. CREB upregulates long non-coding RNA, HULC expression through interaction with microRNA-372 in liver cancer. Nucleic Acids Res. 38, 5366–5383 (2010).
Article CAS Google Scholar
Poliseno, L. et al. A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature 465, 1033–1038 (2010).
Article ADS CAS Google Scholar
Johnsson, P. et al. A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells. Nat. Struct. Mol. Biol. 20, 440–446 (2013).
Article CAS Google Scholar
Tam, O. H. et al. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453, 534–538 (2008).
Article ADS CAS Google Scholar
Watanabe, T. et al. Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453, 539–543 (2008).
Article ADS CAS Google Scholar
Hayashi, H. et al. The OCT4 pseudogene POU5F1B is amplified and promotes an aggressive phenotype in gastric cancer. Oncogene 34, 199–208 (2013).
Article Google Scholar
Jez, M. et al. Expression and differentiation between OCT4A and its Pseudogenes in human ESCs and differentiated adult somatic cells. PLoS ONE 9, e89546 (2014).
Article ADS Google Scholar
Kaltz, N. et al. In vivo osteoprogenitor potency of human stromal cells from different tissues does not correlate with expression of POU5F1 or its pseudogenes. Stem Cells 26, 2419–2424 (2008).
Article CAS Google Scholar
Pain, D., Chirn, G.-W., Strassel, C. & Kemp, D. M. Multiple retropseudogenes from pluripotent cell-specific gene expression indicates a potential signature for novel gene identification. J. Biol. Chem. 280, 6265–6268 (2005).
Article CAS Google Scholar
Kastler, S. et al. POU5F1P1, a putative cancer susceptibility gene, is overexpressed in prostatic carcinoma. Prostate 70, 666–674 (2010).
CAS PubMed Google Scholar
Liedtke, S., Enczmann, J., Waclawczyk, S., Wernet, P. & Kögler, G. Oct4 and its pseudogenes confuse stem cell research. Cell Stem Cell 1, 364–366 (2007).
Article CAS Google Scholar
Lin, H., Shabbir, A., Molnar, M. & Lee, T. Stem cell regulatory function mediated by expression of a novel mouse Oct4 pseudogene. Biochem. Biophys. Res. Commun. 355, 111–116 (2007).
Article CAS Google Scholar
Panagopoulos, I., Möller, E., Collin, A. & Mertens, F. The POU5F1P1 pseudogene encodes a putative protein similar to POU5F1 isoform 1. Oncol. Rep. 20, 1029–1033 (2008).
CAS PubMed Google Scholar
Redshaw, Z. & Strain, A. J. Human haematopoietic stem cells express Oct4 pseudogenes and lack the ability to initiate Oct4 promoter-driven gene expression. J. Negat. Results Biomed. 9, 2 (2010).
Article Google Scholar
Suo, G. et al. Oct4 pseudogenes are transcribed in cancers. Biochem. Biophys. Res. Commun. 337, 1047–1051 (2005).
Article CAS Google Scholar
Wezel, F., Pearson, J., Kirkwood, L. A. & Southgate, J. Differential expression of Oct4 variants and pseudogenes in normal urothelium and urothelial cancer. Am. J. Pathol. 183, 1128–1136 (2013).
Article CAS Google Scholar
Zhao, S. et al. Expression of OCT4 pseudogenes in human tumours: lessons from glioma and breast carcinoma. J. Pathol. 223, 672–682 (2011).
Article CAS Google Scholar
Wang, L. et al. Pseudogene OCT4-pg4 functions as a natural micro RNA sponge to regulate OCT4 expression by competing for miR-145 in hepatocellular carcinoma. Carcinogenesis 34, 1773–1781 (2013).
Article CAS Google Scholar
Hawkins, P. G. & Morris, K. V. Transcriptional regulation of Oct4 by a long non-coding RNA antisense to Oct4-pseudogene 5. Transcription 1, 165–175 (2010).
Article Google Scholar
Islam, S. et al. Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res. 21, 1160–1167 (2011).
Article CAS Google Scholar
Ponting, C. P., Oliver, P. L. & Reik, W. Evolution and functions of long noncoding RNAs. Cell 136, 629–641 (2009).
Article CAS Google Scholar
Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998).
Article CAS Google Scholar
Nordhoff, V. et al. Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences. Mamm. Genome 12, 309–317 (2001).
Article CAS Google Scholar
Tsai, M.-C. et al. Long noncoding RNA as modular scaffold of histone modification complexes. Science 329, 689–693 (2010).
Article ADS CAS Google Scholar
Zhao, J., Sun, B. K., Erwin, J. A., Song, J.-J. & Lee, J. T. Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science 322, 750–756 (2008).
Article ADS CAS Google Scholar
Maison, C. et al. Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component. Nat. Genet. 30, 329–334 (2002).
Article Google Scholar
Lachner, M., O’Carroll, D., Rea, S., Mechtler, K. & Jenuwein, T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116–120 (2001).
Article ADS CAS Google Scholar
Marion, R. M. et al. Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells. Cell Stem Cell 4, 141–154 (2009).
Article CAS Google Scholar
Pink, R. C. & Carter, D. R. F. Pseudogenes as regulators of biological function. Essays Biochem. 54, 103–112 (2013).
Article CAS Google Scholar
Johnsson, P., Morris, K. V. & Grandér, D. Pseudogenes: a novel source of trans-acting antisense RNAs. Methods Mol. Biol. 1167, 213–226 (2014).
Article CAS Google Scholar
Korneev, S. A., Park, J. H. & O’Shea, M. Neuronal expression of neural nitric oxide synthase (nNOS) protein is suppressed by an antisense RNA transcribed from an NOS pseudogene. J. Neurosci. 19, 7711–7720 (1999).
Article CAS Google Scholar
Murchison, E. P., Partridge, J. F., Tam, O. H., Cheloufi, S. & Hannon, G. J. Characterization of Dicer-deficient murine embryonic stem cells. Proc. Natl Acad. Sci. USA 102, 12135–12140 (2005).
Article ADS CAS Google Scholar
Marión, R. M., Schotta, G., Ortega, S. & Blasco, M. A. Suv4-20h abrogation enhances telomere elongation during reprogramming and confers a higher tumorigenic potential to iPS cells. PLoS ONE 6, e25680 (2011).
Article ADS Google Scholar
Kula, A. et al. Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function. Retrovirology 8, 60 (2011).
Article CAS Google Scholar

Download references

Acknowledgements

We thank Alessandro Marcello (ICGEB Trieste, Italy) for providing the basic vectors for the MS2 tethering model. M.S. is supported by a AIRC post-doctoral fellowship; E.C. is enroled in the PhD program for Molecular Medicine at the University of Trieste. This work was supported by an Italian Association for Cancer Research (AIRC) grant Rif 42/08, 6352 to R.B., an Italian Association for Cancer Research (AIRC) grant cod. 10299 and a Fondazione Veronesi grant to S.S. and by a grant from the Italian Ministry of Education, Universities and Research (MIUR) CTN01_00177_817708 to C.S.

Author information

Authors and Affiliations

Cancer Epigenetic Group, Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie (LNCIB), Area Science Park, Padriciano 99, Trieste, 34149, Italy
Michele Scarola, Elisa Comisso, Rhena Pascolo, Riccardo Chiaradia, Claudio Schneider, Stefan Schoeftner & Roberta Benetti
Department of Medical and Biological Sciences, University of Udine,
Claudio Schneider & Roberta Benetti
Piazzale Massimiliano Kolbe 1, Udine, 33100, Italy
Claudio Schneider & Roberta Benetti
Telomeres and Telomerase Group, Spanish National Cancer Research Centre (CNIO), C/Melchor Fernández Almagro, 3, Madrid, 28029, Spain
Rosa Maria Marion & Maria A. Blasco
Department for Life Sciences, University of Trieste, Via Weiss 2, Trieste, 34128, Italy
Stefan Schoeftner

Authors

Michele Scarola
View author publications
You can also search for this author in PubMed Google Scholar
Elisa Comisso
View author publications
You can also search for this author in PubMed Google Scholar
Rhena Pascolo
View author publications
You can also search for this author in PubMed Google Scholar
Riccardo Chiaradia
View author publications
You can also search for this author in PubMed Google Scholar
Rosa Maria Marion
View author publications
You can also search for this author in PubMed Google Scholar
Claudio Schneider
View author publications
You can also search for this author in PubMed Google Scholar
Maria A. Blasco
View author publications
You can also search for this author in PubMed Google Scholar
Stefan Schoeftner
View author publications
You can also search for this author in PubMed Google Scholar
Roberta Benetti
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

R.B., S.S. and C.S. conceived and designed the study; M.S., R.P., E.C. and R.C. performed the experiments and analysed the data; R.M.M. and M.B. generated iPS cells; S.S. and R.B. wrote the manuscript.

Corresponding authors

Correspondence to Stefan Schoeftner or Roberta Benetti.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1-9, Supplementary Table 1, Supplementary Methods and Supplementary Reference (PDF 1888 kb)

Rights and permissions

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Reprints and permissions

About this article

Cite this article

Scarola, M., Comisso, E., Pascolo, R. et al. Epigenetic silencing of Oct4 by a complex containing SUV39H1 and Oct4 pseudogene lncRNA. Nat Commun 6, 7631 (2015). https://doi.org/10.1038/ncomms8631

Download citation

Received: 19 November 2014
Accepted: 27 May 2015
Published: 09 July 2015
DOI: https://doi.org/10.1038/ncomms8631

This article is cited by

The pluripotent factor OCT4A enhances the self-renewal of human dental pulp stem cells by targeting lncRNA FTX in an LPS-induced inflammatory microenvironment
- Hong Hong
- Kai Zeng
- Xi Wei
Stem Cell Research & Therapy (2023)
The role of non-coding RNA in lupus nephritis
- Hanzhi Liang
- Qinghua Liu
Human Cell (2023)
The roles of long noncoding RNAs in the regulation of OCT4 expression
- Rui-Ting Zhou
- Yi-Ran Ni
- Fan-Jun Zeng
Stem Cell Research & Therapy (2022)
SUV39H1 regulates corneal epithelial wound healing via H3K9me3-mediated repression of p27
- Shuai Yang
- Weiwei Chen
- Dongsheng Yan
Eye and Vision (2022)
lncRNA PVT1: a novel oncogene in multiple cancers
- Ruiming Li
- Xia Wang
- Kefeng Wang
Cellular & Molecular Biology Letters (2022)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.