Sod2 gene expression is induced by Nanog

SOLARI, CLAUDIA MARÍA; COSENTINO, SOLEDAD; VÁZQUEZ ECHEGARAY, CAMILA; WAISMAN, ARIEL; LUZZANI, CARLOS; LOSINO, NOELIA; PETRONE, MARÍA VICTORIA; FRANCIA, MARCOS; MIRIUKA, SANTIAGO; BARAÑAO, LINO; GUBERMAN, ALEJANDRA

Congreso; I Latin American, VIII Brazilian and I Argentine Congress of Stem cells and cell therapy; 2014

Pluripotent stem cells (PSCs) have two main properties: self-renewal and pluripotency. Oct4, Sox2 and Nanog are critical transcription factors (TF) to preserve these properties. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) possess a complex system that protects them from oxidative stress and ensures genomic stability, vital for their role in development. It has been reported that antioxidant activity diminishes along stem cell differentiation, but little is known about the transcriptional regulation of the involved genes. Objectives. With the hypothesis that some genes involved in antioxidant defense systems in PSCs could be regulated by Oct4, Sox2 and/or Nanog, we set to study the expression profile of Catalase, Glutaredoxin, Glutathione peroxidase, Glutathione reductase, Peroxiredoxins, Superoxide dismutases (Sod), Thioredoxins, and Thioredoxin reductases. Methods. Ainv15 and R1 mouse ESC lines, HC11 and iPSCs, previously obtained in our lab from mouse embryonic fibroblasts, were cultured under standard conditions. PSCs were differentiated using the in vitro hanging drop protocol. In experiments where TF levels were modulated, R1 ESCs were cultured in the standard conditions or in the absence of LIF for 4 days. Gene expression was analyzed by real-time quantitative RT-PCR. We generated a reporter vector, pSod2-Luc, cloning a 1142 kbp fragment of the promoter region of Sod2 into pGL3-Basic vector upstream of the Luciferase gene. A trans-activation assay was performed in HC11 cell line by co-transfection of pSod2-luc vector and an expression vector for Nanog. Statistical comparisons of data were performed using Student paired t-test or a randomized block design ANOVA for biological replicates using Infostat statistical software. When necessary, Tukey Test was used for comparisons between means. Results: We studied the gene expression pattern of some of the components of the oxidative stress defense system in ESCs and iPSCs in the undifferentiated state and during differentiation. We found a great diversity in their transcriptional profiles, some genes were found to be up-regulated along the process, others highly repressed, and finally, some resulted unaffected. Nevertheless, Sod2 gene was highly repressed during differentiation and its expression pattern was similar to Nanog gene?s profile in both experiments, hanging drop differentiation protocol and LIF?depleted culture conditions. Moreover, Sod2 promoter activity was induced by Nanog when transactivation assay was performed. Conclusions: Sod2 expression is repressed during differentiation. Its expression pattern is similar to Nanog?s, one of the main stemness? transcription factors in pluripotent stem cells, under different culture conditions. Performing a transactivation assay, we found that Nanog induced Sod2 gene promoter.