The protein arginine methyltransferase PMRT8 increases embryonic stem cell apoptosis and modulates cell cycle progression

CARLOS LUZZANI; CLAUDIA SOLARI; MARIA SOLEDAD COSENTINO; ARIEL WAISMAN; CAMILA VAZQUEZ ECHEGARAY C; ALINA SASSONE; GUSTAVO SEVLEVER; LINO BARAÑAO; SANTIAGO G. MIRIUKA; ALEJANDRA S. GUBERMAN

Foz de Iguazu

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

Addition of methyl groups to arginine residues is c  atalyzed by a group of enzymes called Protein  Arginine Methyltransferases (PRMT). Although PRMT1,  the most studied member of the family, is  essential for development, little it is known about  its paralogue PRMT8. Despite regulation and  cellular substrates of PRMT8 are not well understoo  d, it was reported that its expression is  restricted to adult brain neurons of mice and that  it plays a critical role in embryonic and neural  development in zebrafish.  Since the role of PRMTs in ESCs has not been studied in great detail and PRMT1 seems to be necessary for embryonic development but not for ESC  survival, we decided to investigate if  PRMT8 has a role in the maintenance of ESCs propert  ies.  Ainv15 and R1 mouse Embryonic Stem Cell (ESC) lines  , HC11 and induced pluripotent stem cells  (iPSCs), previously obtained in our lab from mouse  embryonic fibroblasts, were cultured under  standard conditions. Pluripotency was evaluated by  embryoid body and teratoma formation  assays. Lentivirus were used for stable cell line g  eneration and ectopic transcription factor  expression. The doxycicling (dox) inducible pLKO-Te  t-On system was used for short hairpin RNA  (shRNA) silencing of PRMT8. Gene expression was ana  lyzed by real-time quantitative RT-PCR  and immunofluorescence. Cell proliferation was stud  ied by Bromodeoxy Uridine (BrdU)  incorporation and crystal violet assays. Cell cycle  analysis of DNA content was performed by  propidium iodide (PI) staining and flow cytometry a  nalysis. Cell death was measured by Anexin-  V/PI and TUNEL assays.  We found that PRMT8 expression was up-regulated in  ESCs and in iPSCs, and that it decreased  during differentiation. We showed that PRMT8 expres  sion was induced by pluripotency  transcription factors Oct4, Nanog and Sox2, since e  ctopic expression of these factors affected  PRMT8 expression in a heterologous system (HC11, ma  mmary epithelial cell line). Then we  established a R1 derived ES cell line (R1 1036) tha  t enables PRMT8 silencing in an inducible  manner by a shRNA regulated by dox. PRMT8 sub-expre  ssing ESCs were ableto self-renew and  remained pluripotent. Remarkably, we found that the  se cells formed bigger colonies. Intriguingly,  the percentage of PRMT8 sub-expressing cells that w  ere in S phase diminished when assessed  by BrdU incorporation and propidium iodide DNA stai  ning. Finally, we found that PRMT8 down-  regulation increased ES cell survival as dox-treate  d R1 1036 showed a diminution of cells  undergoing early apoptosis and necrosis compared to  control cells, analyzed by anexin-V and  TUNEL assays.  Although PRMT8 down-regulation did not affect ESC s  elf-renewal or pluripotency, we found that  PRMT8 modulated ES cell cycle and apoptosis, showin  g to be relevant for stem cells? survival.  These results suggest novel roles for PRMT8 in key  cellular mechanisms in pluripotent stem cells.