The protein arginine methyltransferase Prmt8 increases embryonic stem cell apoptosis and modulates cell cycle progression

LUZZANI, CARLOS; SOLARI, CLAUDIA; COSENTINO, SOLEDAD; WAISMAN, ARIEL; VÁZQUEZ ECHEGARAY, CAMILA; SASSONE, ALINA; SEVLEVER, GUSTAVO; BARAÑAO, LINO; MIRIUKA, SANTIAGO; GUBERMAN, ALEJANDRA

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

Introduction: Addition of methyl groups to arginine residues is catalyzed by a group of enzymes called Protein Arginine Methyltransferases (PRMT). Although PRMT1, the most studied member of the familly, is essential for development, little it is known about its paralogue PRMT8. Despite regulation and cellular substrates of PRMT8 are not well understood, 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. Objectives: 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 properties. Methods: 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 generation and ectopic transcription factor expression. The doxyciclin (dox) inducible pLKO-Tet-On system was used for short hairpin RNA (shRNA) silencing of PRMT8. Gene expression was analyzed by real-time quantitative RT-PCR and immunofluorescence. Cell proliferation was studied 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 analysis. Cell death was measured by Anexin-V/PI and TUNEL assays. Results: We found that PRMT8 expression was up-regulated in ESCs and in iPSCs, and that it decreased during differentiation. We showed that PRMT8 expression was induced by pluripotency transcription factors Oct4, Nanog and Sox2, since ectopic expression of these factors affected PRMT8 expression in a heterologous system (HC11, mammary epithelial cell line). We then established a R1 derived ES cell line (R1 1036) that enables PRMT8 silencing in an inducible manner by a shRNA regulated by dox. PRMT8 sub-expressing ESCs were able to self-renew and remained pluripotent. Remarkably, we found that these cells formed bigger colonies. Intriguingly, the percentage of PRMT8 sub-expressing cells that were in S phase diminished when assessed by BrdU incorporation and propidium iodide DNA staining. Finally, we found that PRMT8 down-regulation increased ES cell survival as dox-treated R1 1036 showed a diminution of cells undergoing early apoptosis and necrosis compared to control cells, analyzed by anexin-V and TUNEL assays. Conclusions: Although PRMT8 down-regulation did not affect ESC self-renewal or pluripotency, we found that PRMT8 modulated ES cell cycle and apoptosis, showing to be relevant for stem cells? survival. These results suggest novel roles for PRMT8 in key cellular mechanisms in pluripotent stem cells.