Fig. 4.

CARM1 and the p160 coactivator member ACTR/SRC3/AIB1 cooperate at the CCNE1 promoter. (A) Endogenous CARM1 coimmunoprecipitate with E2F1 and ACTR. A fraction of the input HeLa cellular extract (Input) and the proteins immunoprecipitated from this extract either by α-CARM1 or control Ab (IgG) were probed for the presence of E2F1 (Lower) or ACTR (Upper) by immunoblotting. (B) ACTR is required to detect an association between CARM1 and E2F1 in vitro. Equivalent amounts of GST-E2F1 proteins bound to beads (Bottom) were incubated with in vitro translated (IVT) ³⁵S-labeled CARM1 in the presence (+) or absence (−) of a mixture of IVT unlabeled and ³⁵S-labeled ACTR protein. GST-E2F1-pulled-down radiolabeled CARM1 (Top) and ACTR (Middle) proteins (P), and unbound proteins in the supernatant (S) were visualized by autoradiography. (C) ACTR is expressed in U20S and is required for CCNE1 mRNA full expression. Shown is Q-RTPCR analysis of the human hACTR, hCCNE1, and hActin mRNA levels in exponentially growing U2Os cells expressing either shRNA directed against hACTR (15) or scrambled control shRNA. (D) ACTR is required to CARM1 recruitment on the CCNE1 gene in vivo. ChIP analysis of the human CCNE1 promoter with antibodies to CARM1, ACTR, or modified histones (H3R17me and H4R3me) was performed on chromatin samples prepared from U2Os cells expressing either shRNA directed against hACTR or scrambled control shRNA. Immunoprecipitated chromatin samples were analyzed by PCR for human a CCNE1 promoter fragment (hCE1). (E) CARM1 and ACTR cooperatively stimulate the E2F1/DP1-mediated transactivation of the CCNE1 promoter. 3T3 CARM1^−/− fibroblasts were transfected with hCCNE1-luc and CMV-β-gal reporters, together with combinations of E2F1, DP1, CARM1, or ACTR, as indicated. Results are expressed in RLU normalized to β-gal activity. (F) Cooverexpression of CARM1 and ACTR leads to an up-regulation of the endogenous CCNE1 mRNA level. NIH 3T3 cells were transfected with a plasmid encoding a puromycin-resistance gene (pBABEpuro), together with combinations of expression vectors encoding CARM1 or ACTR, as indicated. After 4 days of selection in the presence of puromycin, transfected cells were analyzed for mCCNE1 mRNA contents by Q-RTPCR.