Fig. 4
The transactivation and DNA binding domains of E2F4 are required for its function in mouse ES cells. a Schematic of the design of E2F4 mutant constructs. All constructs were fused C-terminal to a GFP tag. Wild-type (WT) E2F4 contains a DNA binding domain (DBD), a dual nuclear export signal (NES), a DP dimerization domain, a transactivation domain, and an RB family/pocket proteins binding domain (PPBD). GFP-DBD contains three point mutations that make contacts with the E2F consensus binding motif and the DP dimerization partners. GFP-T360 is a truncation mutant that lacks the last 50 amino acid residues, inactivating the transactivation domain. b Quantification of endogenous and exogenous E2F4 expression by immunoassay (from fluorescence units) in WT (gray) and E2F4KO (KO, green) cells (n = 2 biological replicates with 1 WT and 1 E2F4KO clone). c Quantification of colony size by AP staining (unpaired t-test was performed with all individual data points from n = 2 biological replicates with 2 WT and 2 E2F4KO clones in each replicate) and d Total number of cells per well in 6-well plates one week after plating at low density (unpaired t-test was performed with all individual data points from n = 4 biological replicates with 2 WT and 2 E2F4KO clones in each replicate). e RT-qPCR analysis of E2F4 targets and canonical cell cycle genes. Expression of genes in WT and E2F4KO cells expressing each of the constructs, was normalized to Gapdh expression and then to expression levels in WT cells expressing GFP (unpaired t-test was performed with all individual data points from n = 2 biological replicates with 2 WT and 2 E2F4KO clones in each replicate). Data shown as the mean and standard error of the mean
