Gas5 is an essential lncRNA regulator for self-renewal and pluripotency of mouse embryonic stem cells and induced pluripotent stem cells
© The Author(s). 2018
Received: 22 January 2018
Accepted: 21 February 2018
Published: 21 March 2018
The regulatory role of long noncoding RNAs (lncRNAs) have been partially proved in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).
In the current study, we investigated mouse ESC (mESC) self-renewal, differentiation, and proliferation in vitro by knocking down a lncRNA, growth arrest specific 5 (Gas5). A series of related indicators were examined by cell counting kit-8 (CCK-8) assay, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), Western blot, alkaline phosphatase staining, propidium iodide (PI) staining, Annexin V staining, competition growth assay, immunofluorescence, and chromatin immunoprecipitation (ChIP)-qPCR. An in vivo teratoma formation assay was also performed to validate the in vitro results. qRT-PCR, fluorescence-activated cell sorting (FACS), alkaline phosphatase staining, and immunofluorescence were used to evaluate the role of Gas5 during mouse iPSC reprogramming. The regulatory axis of Dicer-miR291a–cMyc-Gas5 and the relationship between Gas5 and Tet/5hmC in mESCs was examined by qRT-PCR, Dot blot, and Western blot.
We identified that Gas5 was required for self-renewal and pluripotency of mESCs and iPSCs. Gas5 formed a positive feedback network with a group of key pluripotent modulators (Sox2, Oct4, Nanog, Tcl1, Esrrb, and Tet1) in mESCs. Knockdown of Gas5 promoted endodermal differentiation of mESCs and impaired the efficiency of iPSC reprogramming. In addition, Gas5 was regulated by the Dicer-miR291a–cMyc axis and was involved in the DNA demethylation process in mESCs.
Taken together, our results suggest that the lncRNA Gas5 plays an important role in modulating self-renewal and pluripotency of mESCs as well as iPSC reprogramming.
The mammalian genome encodes a vast number of long noncoding RNAs (lncRNAs) which are a class of RNAs increasingly recognized as major players in gene regulation . Like coding mRNAs, most lncRNAs are transcribed by RNA polymerase II, 5′ capped, spliced, and some polyadenylated, but they lack protein-coding potential. Recent studies indicate that some lncRNAs have versatile biological functions under different conditions [2, 3].
As expected, lncRNAs are emerging regulators in embryonic stem cells (ESCs) . Several regulatory lncRNAs for pluripotency have been identified in ESCs based on their specific expression pattern . From recent studies, lncRNAs also appear as regulators for ESC lineage differentiation . Specifically, a lncRNA, Malat1, has been shown to regulate synaptogenesis . Another lncRNA, Braveheart, is required for cardiovascular lineage commitment from mesoderm . The role of lncRNAs in endodermal differentiation from pluripotent ESCs or induced pluripotent stem cells (iPSCs) remains unknown.
Growth arrest specific 5 (Gas5) belongs to the 5’ terminal oligopyrimidine class and is a small nucleolar RNA (C/D box snoRNA genes) host gene . Multiple functions have been associated with this lncRNA, mainly including cell growth and apoptosis . Gas5 is also identified as an essential regulator in cancer . Recently, the role of Gas5 in human ESCs had been reported . However, the role of Gas5 in other pluripotent stem cells (such as mouse ESCs (mESCs) and iPSCs) is still unknown. Here, we reveal an essential role of Gas5 in mESCs and iPSCs. Gas5 is highly conserved in vertebrates. Depletion of Gas5 RNA in mESCs affected a number of genes involved in self-renewal and endodermal differentiation possibly through interaction with the pluripotent transcriptional factors and the DNA demethylation regulator. In addition, proliferation was repressed in Gas5-knockdown mESCs. Consistent with the effect on pluripotency, disruption of Gas5 expression impaired the efficiency of somatic reprogramming to iPSCs. Taken together, our results suggest that Gas5 is required for the maintenance of mESC self-renewal and proliferation by inhibiting endodermal lineage differentiation.
Mouse E14Tg2A ESCs were maintained on 0.1% gelatin-coated culture plates in Dulbecco's modified Eagle's medium (DMEM; GIBCO, New York, USA) supplemented with 15% ES-qualified fetal bovine serum (ES-FBS; GIBCO), 55 mM β-mercaptoethanol (GIBCO), 2 mM l-glutamax (GIBCO), 0.1 mM nonessential amino acid (NEAA; GIBCO), gentamycin (GIBCO), and 1000 U/ml leukemia inhibitory factor (LIF; ESGRO, Millipore, Billerica, USA) under feeder-free condition. Cells were passaged every 2–3 days by dissociation with recombinant trypsin (Sigma, Darmstadt, Germany).
Published data analysis
Related sequencing data (GSE8024, GSE36114, and GSE26833) were retrieved from the NCBI GEO database.
Plasmids and miRNA mimics/inhibitors
Gas5, Dicer, and cMyc short hairpin (sh)RNAs were designed using the on-line design program from MIT (http://sirna.wi.mit.edu/home.php). The 19-nucleotide hairpin-type shRNAs with a 9-nucleotide loop were cloned into pSUPER-puromycin (OligoEngine, Seattle, USA) and pLVTHM (Addgene, Cambridge, USA) according to the manufacturers’ protocols. Puromycin selection (pSUPER vector, for reprogramming part) and green fluorescent protein (GFP) sorting (pLVTHM vector) were used to isolate successfully transfected cells. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used for validation of RNA expression. MiR-291a mimics and inhibitors was purchased from GenePharma (Shanghai, China).
For LIF withdrawal and differentiation assays, cells were cultured by the hanging drop method in a 10-cm culture dish and LIF was removed the day after (day 0). After 2 days of embryoid body (EB) formation by the hanging drop method, EBs were transferred to a petri plate for suspension culture up to 8 days. The morphology and number of EBs was also noted. Retinoic acid (RA) or Activin A (Act A) was used to induce specific germ layer differentiation. Ectoderm was induced by RA (10−9 M, Sigma) and mesendoderm was induced by different concentrations of Act A (2.5 ng/ml for mesoderm and 50 ng/ml for endoderm inductions; R&D Systems, Minneapolis, USA).
1 × 105 cells were cultured on a cover glass in a 12-well plate with 700 μl of medium. The cells were allowed to grow to the desired morphology and density before the staining procedure. To stain the cells, cells were first washed once with phosphate-buffered saline (PBS) and fixed by 4% paraformaldehyde/4% sucrose in PBS at room temperature, followed by permeabilization and DNA denaturation by 0.2% TritonX-100 in 4 M HCl. After that, the cells were washed with PBS and blocked in 80 μL bovine serum albumin (BSA; 3%). The cells were incubated by Sox2 (SC-17320, 1:100, Santa Cruz, Dallas, USA) and Gata4 (SC-25310, 1:50, Santa Cruz) in BSA (3%) at 4 °C overnight, and then conjugated with RED-X-conjugated mouse anti-rabbit monoclonal antibody (1:500, Santa Cruz) and 4’,6-diamidino-2-phenylindole (DAPI; 1:1000, Santa Cruz). The glass slides were mounted with a cover slip before imaging.
Alkaline phosphatase (ALP) staining
ALP activity detection was carried out using the blue-color and red-color AP staining Kits (SBI, Palo Alto, USA) according to the manufacturer’s protocol.
RNA extraction, cDNA synthesis, and real-time PCR
Total RNA was extracted by Trizol reagent (Invitrogen, Carlsbad, USA) according to a standard protocol. Concentration and quality of all RNA samples were evaluated by Nanodrop 2000 (Thermo, Waltham, USA), and the 260/280 and 260/230 values of all samples were more than 1.8 and 1.9, respectively. Reverse transcription was performed with the MasterMix kit (Takara, Shiga, Japan) following the standard protocol. Quantitative PCR was performed using the Universal SYBR Green Master mix (Applied Biosystems, Waltham, USA) on a StepOnePlus real-time PCR system (Applied Biosystems). Gene expression was normalized to GAPDH unless otherwise stated.
Cells were lysed in SDS buffer. The protein concentration was measured by BCA assay kit (Thermo). Equal amounts of cell lysates were loaded, blotted onto a polyvinylidene difluoride (PVDF) membrane, and probed with the following primary antibodies: Oct4 (SC-8628, 1:1000, Santa Cruz), Sox2 (SC-17320, 1:1000, Santa Cruz), Tet1 (ab191698, 1:500, Abcam, Cambridge, UK), Tet2 (ABE364, 1: 1000, Millipore), and GAPDH (ab8245, 1:4000, Abcam). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. After incubation with the appropriate secondary antibodies, signals were visualized by enhanced chemiluminescence (GE systems, Fairfield, USA).
Chromatin immunoprecipitation (ChIP) assays were performed in accordance with the manufacturer’s instructions for the Imprint Chromatin Immunoprecipitation Kit (Sigma). qPCR was consequently performed according to a standard protocol.
mESCs were trypsinized and resuspended at a concentration of 1 × 106 cells/100 μL and injected into nude mice subcutaneously. After approximately 5–8 weeks, teratomas were harvested for qRT-PCR and histologic analysis when tumors exceeded 2.0 cm in diameter and were fixed overnight in 4% paraformaldehyde. Paraffin sections and hematoxylin and eosin (H&E) staining were performed according to a general protocol. Animal handling and maintenance were performed in accordance with institutional guidelines.
The cell proliferation rate was measured using the cell counting kit-8 (CCK-8; DOJINDO, Tabaru, Japan) according to the manufacturer’s protocol.
Cell cycle analysis
Cell cycle regulation was determined by a propidium iodide (PI; Sigma) staining assay according to a standard protocol.
Apoptosis analysis was performed by a Annexin V/PI (Invitrogen) staining assay according to a standard protocol.
Competition growth assay
GFP+ mESCs (pLVTHM-Gas5 and pLVTHM) and GFP– mESCs (wild-type (WT)) were mixed at a nearly 1:1 ratio and cultured together for two passages. The ratio of GFP+ and GFP– cells was determined before and after passaging by a flow cytometer.
Genomic DNA samples were prepared with twofold serial dilutions in Tris-EDTA (TE) buffer and then denatured in 0.4 M NaOH at 72 °C for 10 mins. Denatured DNA samples were spotted on a PVDF membrane. The membrane was baked at 80 °C for 10 mins and crosslinked by ultraviolet (UV) light for 10 mins. The membrane was then blocked with 5% blocking buffer for 1 h, incubated with 5-hmC primary antibody (39,769, 1:5000, Active Motif, Carlsbad, USA) for 1 h and, after incubation with the horseradish peroxidase (HRP)-conjugated rabbit secondary antibodies (1:10,000, GE systems), signals were visualized by enhanced chemiluminescence.
Mouse embryonic fibroblasts (MEFs) were isolated from E13.5 Oct4-GFP mouse embryos and washed in PBS. Reprogramming were performed based on the Lentiviral mediated tet-inducible reprogramming system.
The error bars represent the standard error of mean (SEM) of three independent experiments, and statistically significant differences by Student’s t test are indicated by *, **, and ***, indicating P < 0.05, P < 0.01, and P < 0.001, respectively.
Characterization of lncRNA Gas5 in ESCs
ESCs are an excellent in vitro model for studying the role of lncRNAs in pluripotent cells and cellular differentiation [13, 14]. To identify lncRNAs participating in mESC pluripotency and lineage differentiation, we analyzed the transcriptome of mESCs during differentiation . Among the differentially expressed lncRNAs, we identified a novel lncRNA, Gas5, that was highly enriched in pluripotent ESCs. The role of this lncRNA in mESCs remains unknown.
Gas5 is required for mESC self-renewal
Gas5 represses mESC endodermal differentiation
In addition, teratoma formation assay was performed to examine the role of Gas5 in modulating germ layer differentiation of mESCs in vivo. Gas5 KD mESCs and control mESCs were subcutaneously injected into nude mice. After approximately 4–6 weeks, both Gas5 KD mESCs and control mESCs formed teratomas (Fig. 3d–f). Histological analysis showed more ectodermal differentiation in teratomas from control mESCs, while Gas5 KD teratomas contained more differentiated endodermal tissues (Fig. 3g). Consistent with the in vitro results, endodermal markers were elevated in Gas5 KD teratomas (Fig. 3h and i). Together, these in vitro and in vivo results suggest that Gas5 KD promotes endodermal differentiation in mESCs.
Gas5 is required for mESC proliferation under differentiating conditions
To further verify this observation, we performed a competitive growth assay by mixing mESCs stably expressing control-GFP or Gas5 KD-GFP with the same number of WT mESCs. The percentage of GFP+ mESCs before passaging was approximately 50% of the total cells. After three passages, the percentage of mESCs expressing control-GFP remained almost the same, but the percentage of Gas5 KD mESCs was decreased to approximately 20% (Fig. 4c). These data indicated that Gas5 is important for mESC growth under differentiating conditions.
Self-renewing ESCs usually have a long S phase in a cell cycle and a low apoptotic rate. Our data showed that knockdown of Gas5 impairs mESC proliferation. We thus further hypothesized that Gas5 regulates the cell cycle and/or apoptosis in mESCs. The PI staining results indeed showed that Gas5 KD increased the proportion of cells in the G0/G1 phase, with a concomitant decrease in the S and G2/M phases (Fig. 4d). Furthermore, as shown in Fig. 4e, Gas5 KD mESCs exhibited a significantly higher apoptotic rate when compared with control mESCs under LIF withdrawal conditions for 6 days. Together, these data indicate that Gas5 prevents mESCs from apoptosis under differentiating conditions.
Gas5 is regulated by the Dicer-miR291a–cMyc pathway in ESCs
cMyc is another important transcriptional factor essential for mESC pluripotency, and its expression decreases upon Dicer loss, an effect due to loss of miR-290 cluster expression as documented previously . From our experiments, cMyc KD mESCs showed decreased Gas5 expression, similar to what we observed in Dicer KD and miR-291a KD mESCs (Fig. 5d). Conversely, forced expression of cMyc in Dicer or miR-291a KD mESCs could counteract the downregulation of Gas5 (Fig. 5e). As shown in Fig. 2h, cMyc could directly bind to the promoter of Gas5 and regulate its transcription. Overall, these results suggest that Gas5 is regulated by the Dicer-miR-291a–cMyc axis in mESCs.
Gas5 promotes iPSC reprogramming efficiency
Gas5 interacts with the Tet family and regulates 5hmC in ESCs
Embryonic stem cells (ESCs) can self-renew indefinitely in vitro and differentiate into all germ layers . Understanding the molecular mechanisms required for ESCs to maintain a balance between pluripotency and differentiation is critical for advancing stem cell-based therapies in regenerative medicine. It is evident that a number of lncRNAs plays their roles in the mammalian transcriptome  via versatile mechanisms . Some lncRNAs regulate networks that contribute to ESC self-renewal and differentiation . For example, it was shown that many lncRNAs are involved in ESC function by two RNAi screens. One study shows that knocking down 26 out of 147 lncRNAs results in decreased expressions of pluripotency markers . Another genome-scale RNAi screen of 1280 lncRNAs in ESCs revealed 20 lncRNAs that are involved in the maintenance of pluripotency . Interestingly, Gas5 was found in the candidate list from both studies, suggesting that Gas5 might be critical for ESC pluripotency. On the other hand, whilst we were preparing this manuscript, another group showed a similar function of Gas5 in human ESCs , additionally supporting our findings in the current study. Moreover, we further identified a role of Gas5 in mESC endodermal differentiation, iPSC reprogramming, and epigenetic regulation, all of which have not been reported before.
In this study, we found that KD of Gas5 resulted in loss of pluripotency. Consistent with this, proliferation was decreased in Gas5 KD mESCs. In addition, the cell cycle was altered and apoptosis was induced in Gas5 KD mESCs. These findings suggest that Gas5 may influence the cell cycle regulatory network of mESCs, a possibility consistent with the known involvement of the cell cycle machinery in the establishment and maintenance of the pluripotent state in mESCs . Apart from being indispensable for the self-renewal of mESCs, our data also imply an indispensable role of Gas5 in repression of endodermal differentiation in mESCs.
Although lncRNAs have been previously linked to stem cell pluripotency , we report for the first time that the lncRNA Gas5 could affect the “key pluripotent network” in ESCs: Sox2, Oct4, Nanog, Tcl1, Esrrb, and even the epigenetic regulator Tet family. These results suggest the master regulatory role of Gas5 in maintaining pluripotency of mESCs. Further experiments are required to understand the precise mechanism by which Gas5 acts, and identification of proteins that interact with Gas5 in ESCs may help to elucidate its function.
Many epigenetic regulators, including Polycomb group proteins and DNA methyltransferases, are critical for ESC differentiation [27, 28]. Therefore, Gas5-mediated chromatin and transcriptional regulation in lineage differentiation further highlights the importance of chromatin dynamics in cell-fate transitions. From the present study, some lncRNAs might have (biological) functions in ESCs through epigenetic regulations. More studies are required to elucidate the specific mechanisms, such as genome-wide comparison of DNA or histone modifications between specific lncRNA knockout and overexpression in ESCs. The current study first shows that a lncRNA is necessary to repress endoderm differentiation, suggesting that deregulation of Gas5 expression may contribute to endodermal-related disorders.
Many lncRNAs contribute to the epigenetic regulation of gene expression by serving as modular scaffolds for histone modification complexes. We found that Gas5 is regulated by bi-epigenetic regulations (DNA demethylation and histone methylation in ESCs). Gas5 is marked by H3K4me1, H3K4me3, and H3K36me3 modifications in both mouse and human ESCs and there is no repressive mark H3K27me3 deposit at the same genomic locus. The expression of Gas5 gradually decreases during ESC differentiation . We also found that Gas5 mutually promotes Tet family enzymes in mESCs. The diverse roles of these proteins suggest that Gas5 may regulate gene expression through multiple mechanisms in mESCs. Chromatin modification of Gas5 in undifferentiated ESCs appears to demarcate three chromatin domains containing constitutively active or developmentally regulated genes.
Therefore, like some other lncRNAs, Gas5 may contribute to the ‘fine-tuning’ scaffold of chromatin rather than acting as a regulator of gene transcription. Facile and efficient manipulation of genomic segments should help to elucidate the subtle cis- and trans-regulatory roles of lncRNAs, leading to a better understanding of the evolutionary and functional mechanisms of lncRNAs. However, during establishment of the pluripotent state, it is unclear whether Gas5 transcription might promote an open chromatin conformation to prime future activation of its target genes during differentiation. This question should be our next step to further elucidating the role of Gas5 in mESCs.
Collectively, we demonstrated the function of Gas5 in mESC self-renewal, endodermal differentiation, and iPSC reprogramming, suggesting another layer of complexity in the networks controlling stem cell biology. Gas5 interacts with key nuclear proteins and epigenetic modifications to exert their biological functions in mESCs.
This study is supported in part by funds from the Natural Science Foundation of Anhui Province for young scholars (1708085QH200) and Grants for Scientific Research of BSKY from Anhui Medical University (4501041101).
Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.
JT and TlL conceived and designed all the experiments. JT and GT performed the experiments. JT, HHC, and WW drafted and revised the article. All authors read and approved the final version.
All experiments involving animals were performed in accordance with guidelines approved by the Committee for Animal Care at the Chinese University of Hong Kong (CUHK).
Consent for publication
All authors consent to the publication of this study.
The authors declare that they have no competing interests.
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