Results
Glutathione reductase gene is upregulated throughout differentiation
Based on the growing evidence that antioxidant defense system is modulated when pluripotent stem cells are induced to differentiate, and that glutathione reductase is an important enzyme involved in the cellular response to oxidative stress, we decided to study Gsr gene modulation. In a previous work, we have in silico analyzed the upstream region of the coding sequence of diverse genes involved in antioxidant system, including Gsr and found the presence of multiple predicted binding sites for the transcription factors Oct4 and Nanog in a 5 kbp region upstream the transcription start site of this gene [16]. In this work, we first studied Gsr gene modulation in undifferentiated ESCs and along differentiation. To this aim, we performed an in vitro differentiation protocol culturing R1 ESCs in standard stem cells culture medium supplemented with LIF, as control condition, and in absence of this cytokine for 4 days. We observed the expected change in cell morphology throughout the differentiation, as shown in Fig. 1a. Whereas ESC grew in compact and refringent colonies and showed high nucleus/cytoplasm ratio, differentiated cells grew as a monolayer and increased their cytoplasmic proportion. As shown in Fig. 1b, although Oct4 mRNA levels remained constant, Nanog mRNA levels diminished, confirming that cells left behind the undifferentiated state displaying the expected Nanog repression. The reduction in Nanog mRNA, quantified by RT-qPCR, reflects Nanog protein levels visualized by immunofluorescence (Fig. 1c). Notably, showing a reciprocal kinetics, when Nanog expression was reduced, Gsr mRNA levels were upregulated.
Next, to further investigate Gsr gene modulation we analyzed its expression pattern along a differentiation protocol to neural progenitor. We used the 46C ESC line for this approach, which is a reporter cell line that expresses GFP driven by Sox1 promoter, a specific marker of neuroectoderm [18]. We verified the success of the differentiation process by GFP fluorescence detection (Fig. 2a) and by the analysis of the expression of the pluripotency markers Oct4 and Nanog, and the neural markers Blbp and Nestin by RT-qPCR, at days 0, 1, 3 and 6, which behaved as expected (Fig. 2). In agreement with the previous result, we found that along with Nanog downregulation, visualized both at mRNA and protein levels (Fig. 2b, c), Gsr mRNA levels increased at days 1 and 3 of this neural precursor differentiation protocol.
Nanog transcription factor modulates Gsr gene expression
Taking into account the induction of Gsr observed when Nanog was repressed along both differentiation protocols and the presence of six putative consensus sites for Nanog in the 3000 bp region upstream Gsr coding sequence, we decided to study whether Nanog modulates the endogenous Gsr gene expression by an shRNA approach. For this purpose, we downregulated this transcription factor’s mRNA levels using a shRNA targeting Nanog (shNanog). We transfected R1 ESCs with a vector encoding shNanog or targeting eGFP (shGFP) as control, and then analyzed Gsr mRNA levels by RT-qPCR. As outlined in Fig. 3, Nanog expression was downregulated by its specific shRNA, evidenced by RT-qPCR and immunofluorescence. Interestingly and according with our previous results, Gsr mRNA levels were greatly increased in Nanog-downregulated ESCs, suggesting a role in Gsr transcriptional regulation by this pluripotency transcription factor.
Discussion
There is growing evidence showing that when pluripotent stem cells are induced to differentiate, antioxidant defense system is modulated [7, 10, 19, 20]. A key enzyme involved in the response to oxidative stress is glutathione reductase (Gsr), contributing to the preservation of this antioxidant molecule in precise levels. In a previous work, by in silico analysis, we found the presence of putative binding sites for the transcriptions factors Oct4 and Nanog in a 3000 bp region upstream Gsr coding sequence [16]. Considering this evidence and the hypothesis that ESCs’ specific transcription factors critical for pluripotency maintenance regulate genes differentially expressed along the differentiation process, we studied Gsr gene modulation. As mentioned before, regarding the differentiation process, it has been shown that ROS levels [7] and mitochondrial proliferation and activity increase during ESCs differentiation [9]. These reports propose that ROS are involved in the balance between self-renewal and differentiation. The antioxidant system is essential to maintain the adequate levels of these species and GSH is a key component for redox homeostasis [21]. The enzymes glutathione synthase and Gsr are responsible to keep GSH/GSSG at accurate levels. It was previously reported that Gsr gene expression decreased since day 7 of differentiation, in human pluripotent stem cells [7, 20]. On the other hand, in this work we found that Gsr mRNA levels were upregulated in mouse ESCs, at earlier time points of two distinct differentiation protocols. In accordance, it was reported that Gsr mRNA levels in E7 mouse embryos were high, and then they fall at later days reaching similar levels as in adult mouse [22].
As aforementioned, we found that Gsr gene was expressed in pluripotent stem cells and increased throughout differentiation, showing an expression pattern opposed to Nanog’s when these cells were induced to differentiate by two distinct protocols. We have previously found by in silico analysis, multiple putative binding sites for Nanog in Gsr promoter region [16]. Specifically, there are eight sequences similar to Nanog consensus preserving the AATG core sequence since positions − 523 to − 3612 from transcription start site (+ 1). Moreover, this transcription factor was found to be bound to the promoter region of Gsr in data from genome wide chromatin immunoprecipitation approaches in ESCs [23,24,25,26,27]. Furthermore, we used ChIP Atlas platform [28] to analyze ChIP-Seq experiments data and found evidences of functional Nanog regulatory regions in GSR genomic locus. The analysis revealed peaks indicating that Nanog was bound to Gsr gene, both upstream the transcription start site and in the first intron, in multiple experiments performed in ESCs (Additional file 1: Figure S1). In accordance to these results, we found an increase in Gsr mRNA levels when Nanog was downregulated using a specific shRNA targeting this transcription factor in ESCs. It was previously reported that Nanog represses at transcriptional level genes related to the differentiation process [29,30,31]. In this work, we found that this transcription factor modulates negatively Gsr expression, a gene involved in the antioxidant system. We have previously reported that Sod1 and Sod2, both genes from this system, are modulated by pluripotency transcription factors [16, 17]. As a whole, these results suggest that transcription factors essential for pluripotency maintenance such as Nanog, play a role in the homeostasis of redox status in ESCs.
Conclusion
We found that Gsr, which is critical for maintaining GSH levels and cellular redox status, is modulated by the stemness transcription factor Nanog evidencing a link between pluripotency transcription factors and redox homeostasis. Deep understanding of the antioxidant system in pluripotent stem cells and the relationship between ROS and the differentiation process is crucial for future applications of these promising cells.
Methods
Cell culture conditions and differentiation
R1 ESC line (ATCC) were cultured and differentiated as previously described [32,33,34]. 46C Sox1-GFP ESC line [18] (a kind gift from Austin Smith) was cultured and induced to differentiate to neural progenitor as previously described [16, 35, 36]. Cells were cultured until day 6 and efficacy of the differentiation protocol was analyzed by fluorescence microscopy.
Gene expression analysis
Gene expression was analyzed by RT-qPCR and immunofluorescence. A detailed description of the methodology, the antibodies and the sequence of the primers used is included in Additional file 2: Additional methods.
Nanog downregulation by shRNA transfection
R1 ESCs were transfected in p60 plates with 3 µg pLKO.1-puro derived vectors (Sigma), expressing shRNA targeting Nanog (shNanog, SHCLND-XM_132755) or eGFP (SHC005). Transfection, selection and mRNA analysis were carried out as previously described [16, 17]. For immunofluorescence, ESCs were co-transfected with shNanog and an expression vector encoding H2B-mCherry.
Statistics and data analysis
Results were presented as mean ± Standard error mean (SEM). Statistical comparisons were performed using Student’s t test, except for Fig. 2c, where data was analyzed by a linear mixed model and DGC Test was used for comparison between means. Residuals fitted normal distribution and homogeneity of variance; p values < 0.05 were considered significant. Analysis was performed with Infostat statistical software [37].
