Fig. 3.
CARM1 acts as an E2F-dependent coactivator on CCNE1 promoter. (A) Ectopic expression of CARM1 stimulates the CCNE1 promoter and potentiates its E2F/DP-dependent transactivation in CARM1−/− cells. CARM1−/− 3T3 cells were transfected with a luciferase reporter gene driven by the WT human CCNE1 promoter (CycE1-luc) or the corresponding DNA bearing mutations within the six E2F sites (CycE1ΔE2F-Luc), together with CMV-β-gal and combinations of expression vectors encoding E2F1, E2F3a, DP1, or CARM1, as indicated. Results are expressed in relative luciferase units (RLU), normalized to β-gal. (B) CARM1 ectopic expression potentiates the transactivation of some, but not all, E2F1/DP1-responsive promoters. NIH 3T3 cells were transfected with luciferase reporter genes driven by human CCNE1 promoter, human DHFR promoter, or human CCND1 promoter, together with CMV-β-gal and vectors encoding E2F1, DP1, CARM1, or an enzymatically inactive CARM1-E/Q mutant, as indicated. (C) siRNA-mediated depletion of CARM1 inhibits the E2F1/DP1-dependent transactivation of the human CCNE1 and DHFR promoters but not of the CCND1 promoter. U2Os cells were transfected with the indicated luciferase reporters, CMV-β-gal and E2F1/DP1, together either with scrambled control siRNA or increasing amounts of siRNA directed against human hCARM1 and 48 h later were analyzed for luciferase activity normalized to β-gal (RLU). (D) pRb-associated E2Fs (E2F1, E2F2, and E2F3) are required for CARM1 recruitment and H3R17 methylation at the CCNE1 gene in vivo. ChIP analysis of the mouse CCNE1 promoter with antibodies to CARM1 and arginine-methylated histone H3 (H3R17me) was performed on chromatin samples prepared from exponentially growing E2F1−/−E2F2−/−E2F3flox/flox 48 h after infection either with Cre recombinase (+Cre) or empty control (−Cre) retroviruses. Immunoprecipitated chromatin samples were analyzed by PCR for mouse CCNE1 promoter fragment as in Fig. 1.