Stable Transfection of E2F6 Variants in ESCs

For stable transfection experiments in ESCs, the sequences coding for E2F6 WT, as well as E68 and MB4 mutants, were inserted into the pCAG-EGFP-IB vector^{60 (link)}. To generate the E68 DNA-binding mutant, the amino acids in positions 68 and 69 were changed from Leu-Val to Glu-Ser^{18 (link)}. To generate the MB4 mutant, the E2F6 marked box domain (aa 180–240) was replaced by the marked box domain of E2F4 (aa 138–198). These constructs were transfected into ESCs by electroporation with the Amaxa Nucleofector II device using the Amaxa Cell Line Nucleofector kit R and stable transfectants were selected with blasticidin (7.5 μg/ml). For the DNA-binding domain swap experiment, the sequences coding for E2F6 WT, DBD1, and MB4 mutants were inserted in the lentiviral expression vector pCDH-MSCV-MCS-EF1α-GFP + Puro (System Biosciences). The DBD1 mutant was generated by replacing the DNA-binding domain of E2F6 (aa 61–129) with the DNA-binding domain of E2F1 (aa 122–187). Viral particles were produced in HEK293T cells and ESCs were infected with 8 μg/ml polybrene. Twenty-four hours after transduction, puromycin (2 μg/ml) was added for 72 h before collecting the RNA samples.