SOD2 gene expression is induced by nanog

CLAUDIA SOLARI; MARIA SOLEDAD COSENTINO; CAMILA VAZQUEZ ECHEGARAY; ARIEL WAISMAN; CARLOS LUZZANI; NOELIA LOSINO; MARIA VICTORIA PETRONE; MARCOS FRANCIA; SANTIAGO G. MIRIUKA; LINO BARAÑAO; ALEJANDRA S. GUBERMAN

Foz de Iguazu

Congreso; I Latin American - VIII Brazilian and I Argentine Congress of Stem cells and Cell Therapy; 2014

Pluripotent stem cells (PSCs) have two main propert  ies: self-renewal and pluripotency. Oct4, Sox2  and Nanog are critical transcription factors (TF) t  o preserve these properties. Embryonic stem  cells (ESCs) and induced pluripotent stem cells (iP  SCs) possess a complex system that protects  them from oxidative stress and ensures genomic stab  ility, vital for their role in development. It has  been reported that antioxidant activity diminishes  along stem cell differentiation, but little is know  n  about the transcriptional regulation of the involve  d genes.  With the hypothesis that some genes involved in ant  ioxidant defense systems in PSCs could be  regulated by Oct4, Sox2 and/or Nanog, we decided to  study the expression profile of Catalase,  Glutaredoxin, Glutathione peroxidase, Glutathione r  eductase, Peroxiredoxins, Superoxide  dismutases (Sod), Thioredoxins, and Thioredoxin red  uctases.  Ainv15 and R1 mouse ESC lines, HC11 and iPSCs, prev  iously obtained in our lab from mouse  embryonic fibroblasts, were cultured under standard  conditions. PSCs were differentiated using  the in vitro embryoid body formation assay. In expe  riments 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 R  T-PCR. We then generated a reporter vector,  pSod2-Luc, cloning a 1142 kbp fragment of the promo  ter 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 so  ftware. When necessary, Tukey Test was used for  comparisons between means.  We studied the gene expression pattern of some of t  he components of the oxidative stress  defense system in ESCs and iPSCs in the undifferent  iated state and during differentiation. We  found a great diversity in their transcriptional pr  ofiles, some genes were found to be up-regulated  along the process, others highly repressed, and som  e resulted unaffected. Nevertheless, Sod2  gene was highly repressed during differentiation an  d its expression pattern was similar to Nanog  gene?s profile in both experiments: hanging drop di  fferentiation protocol and LIF?depleted culture  conditions. Moreover, Sod2 promoter activity was in  duced by Nanog when transactivation assay  was performed.  Sod2 expression is repressed during differentiation  . Its expression pattern under different culture  conditions is similar to Nanog?s, one of the main s  temness? transcription factors in pluripotent stem  cells. Performing a transactivation assay, we found  that Nanog induced Sod2 gene promoter.