SRC-3 is methylated by CARM1 in vivo. (A) Cell extracts of CARM1 wild-type and knockout MEFs were immunoprecipitated with an antibody against mono- or dimethylated arginine (α-mArg), followed by Western blotting with specific antibodies against p300 or SRC-3 (upper panel). Input lanes (lower panel) represent 5% of the total amount of SRC-3 used for immunoprecipitation (IP). (B) Cell extracts of MCF7 or CARM1 wild-type and knockout MEFs were immunoprecipitated with antibody against SRC-3 (α-SRC-3), followed by Western blotting with an antibody against mono- or dimethylated arginine. The amount of immunoprecipitated SRC-3 is also shown (upper panel). The lower panel shows the amount of CARM1 protein in 5% of the immunoprecipitation input from the three different cell line extracts. (C) SRC-3 methylation is induced by estrogen signaling. MCF7 cells were cultured in medium containing 5% charcoal-dextran-stripped FCS for 3 days before the addition of estradiol (10 nM), followed by different incubation times prior to harvesting. Cell lysates were immunoprecipitated with an antibody against methylated arginine, followed by Western blotting (IB) with antibody against SRC-3. The amount of ERα, CARM1, and SRC-3 in different samples was determined by Western blotting. In all panels, β-actin serves as a loading control. (D) CARM1 is responsible for SRC-3 methylation in response to estrogen signaling. The same experiment was done as for panel C except that the MCF7 cells were transfected with 20 nM scramble siRNA or siRNA against CARM1 for 3 days prior to hormone induction.

Mutation of SRC-3 methylation sites increases its coactivator function in vivo. (A) SRC-3 methylation site mutants, when coexpressed with CARM1, display higher coactivation activity for ERα-mediated transcription. CV-1 cells were transiently transfected with 200 ng of ERE-Luc reporter, 6 ng of pCR3.1-ERα, 100 ng of pSG5-HA-CARM1, and 100 ng of pSG5-Flag-SRC3 wild type (WT) or indicated mutant in each well of a 12-well plate. Ten nanomolar estradiol (E2) was added 24 h after transfection, and luciferase activity was measured at 48 h posttransfection. Luciferase activity was normalized for protein content. t test was performed, and the statistical significance compared with wild-type SRC-3 (n = 4) was shown as follows: *, P value of <0.05; **, P value of <0.01. (B) A similar experiment was performed as for panel A, except that SRC-3^−/− MEFs were used. (C and D) The same experiments were carried out as for panel B, except that a mouse mammary tumor virus-luciferase reporter was used, and PR-B or GR was cotransfected instead of ERα. (E) SRC-3 methylation mutants display higher coactivation activity on an endogenous promoter. HEK293T cells were transiently transfected with 100 ng of pCR3.1-ERα and 400 ng of pSG5-Flag-SRC-3 wild type or indicated mutants. Forty-eight hours after transfection, cells were treated with 10 nM E2 overnight to induce endogenous gene transcription. The amount of pS2 gene expression was determined by real-time RT-PCR, and the relative induction is shown. The expression of cyclophilin was used for normalization. +, present; −, absent.

SRC-3 methylation dissociates CARM1 from the coactivator complex. (A) HEK293T cells were transfected with the indicated Flag-SRC-3 constructs. Following immunoprecipitation (IP) with anti-Flag-M2 (α-Flag) affinity gel, the protein samples were separated on a 4 to 15% SDS-PAGE gel. Endogenous CARM1 associated with Flag-SRC-3 was examined by Western blotting (upper panel). The same membrane was also used to probe for SRC-3 (lower panel). (B) R1171 is a bona fide methylation site on SRC-3. Two peptides, one with an asymmetric dimethylated arginine at R1171 and the other one without any methyl group, were incubated with recombinant SRC-3 protein in the CARM1-mediated methylation assay. Triangles indicate three different peptide concentrations used (1.5 μM, 15 μM, and 150 μM). (C) Peptide with unmodified R1171 competes with SRC-3 for binding CARM1. Peptide P1 or P2 (150 μM) was incubated together with recombinant Flag-SRC-3 and CARM1 for 1 h at 30°C. SRC-3 protein was immunoprecipitated with anti-Flag-M2 affinity gel, and the presence of associated CARM1 was determined by Western blotting. (D) The methylation region of SRC-3 overlaps with the CARM1 docking region. Wild-type (WT) or methylation site-mutated SRC-3 proteins were incubated with GST or GST-CARM1 for 2 h at 4°C. Glutathione beads were used to pull down GST control and GST-CARM1 proteins. Associated SRC-3 proteins were determined by Western blot analysis. (E) A similar experiment was performed as for panel A, except that ERα was also transfected. The levels of p300 and ERα were determined by Western blotting as well.

SRC family proteins are methylated by CARM1 in vitro. (A) Recombinant p300, SRC-1, SRC-3, ERα, and PR-A were incubated with recombinant CARM1 in the presence of [³H]AdoMet for 1 h at 30°C. Products were analyzed by Coomassie blue staining and fluorography. Arrowheads indicate the positions of full-length proteins. (B) SRC-2 is methylated by CARM1 in vitro. Recombinant SRC-2 and SRC-3 (0.5 μg) were methylated by recombinant CARM1. (C) SRC-3 is a preferred substrate for CARM1 in vitro. Four micrograms of HeLa core histone protein was incubated in the absence (−) or presence (+) of 0.2 μg of SRC-3 protein in the methylation assay. (D) CARM1 is responsible for methylation of SRC-3 in vitro. SRC-3 protein (0.5 μg) was incubated with either wild-type CARM1 or an inactive mutant (E267Q). The positions of protein size standards (in kilodaltons) are indicated beside each panel.

Identification of SRC-3 methylation sites. (A) Mapping regions on SRC-3 that are methylated by CARM1. Five fragments of SRC-3 representing different functional domains were generated as GST fusion proteins. The amount of each protein (1 μg) used in the methylation assay was determined by Coomassie blue staining (middle panel). Arrowheads indicate the positions of full-length proteins. The incorporation of [³H]methyl into the proteins is shown on the bottom panel. (B) Deletion mapping further defines the region of SRC-3 that is methylated. The C-terminal region of SRC-3 from 1041 to 1417 was further divided into three fragments and fused with GST. These protein fragments contain 5, 8, and 3 arginine residues, respectively (upper panel). Full-length proteins are indicated by arrowheads, and the incorporation of [³H]methyl into the proteins is shown on the bottom panel. (C) Primary sequence alignment of the methylation region of SRC-3 compared to the other SRC family proteins. Highly conserved regions are shadowed. (D) Site-directed point mutagenesis (arginine-to-alanine) of the SRC-3 methylation region further refines which arginine residues serve as methylation acceptor sites. Eight methylation mutants were generated, and their activities were compared with the wild-type protein in the CARM1 methylation assay. Arrowheads indicate the positions of full-length proteins. (E) Confirmation of methylation sites in the content of full-length SRC-3 protein. Wild-type and methylation mutants of SRC-3 were expressed and purified from a baculoviral system and tested in the CARM1 methylation assay. The amount of protein (0.3 μg) used in each assay was determined by Coomassie blue staining.

Mutation of SRC-3 methylation sites increases its coactivator function in cell-free transcription assays. (A) Analysis of in vitro-assembled chromatin by partial digestion with MNase. Twenty-five microliters of assembled chromatin was digested with 3 U, 1 U, and 0.3 U of MNase (lanes from left to right, respectively). Ladders of nucleosomal bands were visualized by ethidium bromide staining in a 1% agarose gel. (B) Recombinant proteins used in the in vitro transcription assay. Purity was determined by Coomassie blue staining. (C) The schematic in the upper right corner shows the plasmid pERE-E4 that was assembled into chromatin and transcribed. Triangles indicate 20 or 60 ng of SRC-3 protein and 50 or 150 ng of CARM1 protein employed in the different transcription reactions for which results are shown in the bottom panel. The relative amount of E4 transcript was determined by real-time RT-PCR. Error bars shown throughout Fig. 5 represent standard deviations. (D) A similar experiment was performed as for panel C, except that 20 ng of SRC-3 protein and 50 ng of wild-type or mutated CARM1 was added to the mixture individually or sequentially. +, present; −, absent.

Model for coactivator assembly and disassembly on estrogen-responsive promoters. SRC-3 is phosphorylated at distinct sites by different kinases induced by various signaling pathways. Dependent upon the differential phosphorylation patterns, SRC-3 may assemble different coactivator complexes at regulated target gene promoters. Estrogen signaling results in estrogen-bound receptors interacting with both promoter DNA and SRC-3 coactivator complexes. Enzymatic activities enriched in the complex modify core histones and thereby facilitate transcription initiation. After one (or limited) round(s) of transcription initiation and to terminate signaling, p300/CBP and CARM1 modify components within the coactivator complex, leading to disassembly of the complex and probably also dissociation of receptor from the promoter DNA. We propose that CARM1, and likely also p300/CBP, is a dual-function coactivator: it not only activates transcriptional initiation by modifying core histone tails but also terminates hormone signaling by disassembling the coactivator complex. It remains unknown as to how many of the transcriptional coactivators in the complex are reutilized for another round of transcription initiation or instead subjected to transcription-linked protein degradation.