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Control of CBP co-activating activity by arginine methylation - PubMed

  • ️Tue Jan 01 2002

Control of CBP co-activating activity by arginine methylation

Martine Chevillard-Briet et al. EMBO J. 2002.

Abstract

The histone acetyltransferases CREB binding protein (CBP) and the related p300 protein function as key transcriptional co-activators in multiple pathways. In the case of transcriptional activation by nuclear receptors, ligand promotes the recruitment of co-activators of the p160 family, such as GRIP-1. Subsequently, the p160 co-activators recruit other co-activators via two activation domains, AD1 and AD2. AD1 binds CBP or p300, whereas AD2 has been shown to activate transcription through the recruitment of the arginine methyltransferase CARM1. Recently, the KIX domain of CBP has been shown to be methylated by CARM1 in vitro. Here, we report that another domain of CBP is specifically methylated by CARM1 on conserved arginine residues in vitro and in vivo. We also provide functional evidence that arginine residues methylated by CARM1 play a critical role in GRIP-1-dependent transcriptional activation and in hormone-induced gene activation. Altogether, our data provide strong evidence that arginine methylation represents an important mechanism for modulating co-activator transcriptional activity.

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Figures

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Fig. 1. Methylation of CBP by CARM1. (A) Left panels: 1 µg of purified core histones (hist.), bacterially expressed recombinant GST–CBP FL (CBP FL), control GST (GST), GST–CBP-C or GST–N-CBP, where indicated, were assayed for methylation by 500 ng of bacterially produced recombinant GST–CARM1. Reaction products were then analysed by SDS–PAGE followed by fluorography. The asterisks (*) indicate the position of the various full-length GST fusion proteins. The right panels show western blots allowing the detection of recombinant GST–CBP FL or mutants used in the left panels. The N-terminus of CBP was detected using the A22 antibody (Santa Cruz; α CBP NT), and the C-terminal half using the NM11 antibody (PharMingen; α CBP CT). Note that using the A22 antibody, many shorter bands could be detected, which probably reflects premature termination and/or protein degradation that occur during the production of large proteins in bacteria. (B) GST control, GST–CBP, GST–P/CAF, GST–MyoD, GST–p53 and GST–E2F-1 were assayed for methylation by GST–CARM1. Reaction products were then analysed by SDS–PAGE followed by fluorography. The asterisks (*) indicate the position of the various full-length GST fusion proteins. The right panel shows the corresponding Coomassie staining. Note the presence in CBP and P/CAF preparation of prominent truncated protein products, which most likely reflects premature termination and/or protein degradation that occur during the production of large proteins in bacteria. (C) One microgram of histones (left panel) or 10 ng of GST–CBP FL (right panel) were assayed for methylation by 500 ng of GST–CARM1, GST–PRMT1, GST–HMT1 or control GST protein as indicated. Reaction products were resolved on an 18% SDS–PAGE (left panel) or on an 8% SDS–PAGE (right panel). (D) HeLa cells and NIH 3T3 cells (30 × 106) were radiolabelled with SAM. Total cell extracts were then immunoprecipitated with either an anti-CBP antibody (A22; Santa Cruz) or with an irrelevant anti-HA antibody (Santa Cruz). Immunoprecipitates were then analysed by SDS–PAGE. Gels were fixed and fluorographed for 8 weeks. (E) U2OS cells (2 × 106) were transfected as indicated with 40 µg of pCMV-HA-CBP (1–1098) (N-CBP) and/or 20 µg of pSG5-CARM1, in the presence of 10 µg of pSG5-GRIP-1. Transfected cells were radiolabelled with SAM, and total cells extracts were immunoprecipitated using a monoclonal anti-HA antibody (12CA5; Roche Diagnostics). Immunoprecipitates were analysed by SDS–PAGE followed by fluorography (4 weeks) (upper panel). 1/200th of the immunoprecipitates was subjected to an anti-HA western blot (lower panel).

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Fig. 1. Methylation of CBP by CARM1. (A) Left panels: 1 µg of purified core histones (hist.), bacterially expressed recombinant GST–CBP FL (CBP FL), control GST (GST), GST–CBP-C or GST–N-CBP, where indicated, were assayed for methylation by 500 ng of bacterially produced recombinant GST–CARM1. Reaction products were then analysed by SDS–PAGE followed by fluorography. The asterisks (*) indicate the position of the various full-length GST fusion proteins. The right panels show western blots allowing the detection of recombinant GST–CBP FL or mutants used in the left panels. The N-terminus of CBP was detected using the A22 antibody (Santa Cruz; α CBP NT), and the C-terminal half using the NM11 antibody (PharMingen; α CBP CT). Note that using the A22 antibody, many shorter bands could be detected, which probably reflects premature termination and/or protein degradation that occur during the production of large proteins in bacteria. (B) GST control, GST–CBP, GST–P/CAF, GST–MyoD, GST–p53 and GST–E2F-1 were assayed for methylation by GST–CARM1. Reaction products were then analysed by SDS–PAGE followed by fluorography. The asterisks (*) indicate the position of the various full-length GST fusion proteins. The right panel shows the corresponding Coomassie staining. Note the presence in CBP and P/CAF preparation of prominent truncated protein products, which most likely reflects premature termination and/or protein degradation that occur during the production of large proteins in bacteria. (C) One microgram of histones (left panel) or 10 ng of GST–CBP FL (right panel) were assayed for methylation by 500 ng of GST–CARM1, GST–PRMT1, GST–HMT1 or control GST protein as indicated. Reaction products were resolved on an 18% SDS–PAGE (left panel) or on an 8% SDS–PAGE (right panel). (D) HeLa cells and NIH 3T3 cells (30 × 106) were radiolabelled with SAM. Total cell extracts were then immunoprecipitated with either an anti-CBP antibody (A22; Santa Cruz) or with an irrelevant anti-HA antibody (Santa Cruz). Immunoprecipitates were then analysed by SDS–PAGE. Gels were fixed and fluorographed for 8 weeks. (E) U2OS cells (2 × 106) were transfected as indicated with 40 µg of pCMV-HA-CBP (1–1098) (N-CBP) and/or 20 µg of pSG5-CARM1, in the presence of 10 µg of pSG5-GRIP-1. Transfected cells were radiolabelled with SAM, and total cells extracts were immunoprecipitated using a monoclonal anti-HA antibody (12CA5; Roche Diagnostics). Immunoprecipitates were analysed by SDS–PAGE followed by fluorography (4 weeks) (upper panel). 1/200th of the immunoprecipitates was subjected to an anti-HA western blot (lower panel).

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Fig. 2. The region methylated by CARM1 maps onto a 90 amino acid region within the N-terminal part of CBP. (A) One microgram of control GST, 100 ng of GST–N CBP or CBP mutants A, B, C and D were assayed for methylation by GST–CARM1 and analysed by SDS–PAGE followed by fluorography. The asterisks (*) indicate the position of the various full-length GST fusion proteins. Note that fusion protein D migrates at a larger molecular weight than expected for an unknown reason. The open circle indicates a band reflecting CARM1 weak automethylation. The right panel shows the corresponding Coomassie staining. (B) Ten nanograms of GST–CBP FL and of GST–CBP Δ685–774 (which has been deleted of 90 amino acids encompassing the methylated region) were assayed for methylation by GST–CARM1 and were analysed by SDS–PAGE followed by fluorography. Lower panel: The amounts of GST–CBP FL and of GST–CBPΔ685–774 were assayed by western blot using the NM11 antibody.

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Fig. 3. CBP is methylated on three arginine residues conserved in p300. (A) GST–p300 FL, GST–p300 1–744 and GST–p300 1–595, as indicated, were assayed for methylation by GST–CARM1 and were analysed by SDS–PAGE followed by fluorography. The asterisks (*) indicate the position of the various full-length GST fusion proteins. Note that the major band of methylated GST–p300 FL (#) migrates below the 180 kDa marker: this band corresponds to a major truncated product of p300. In the right panel, the expression of p300 and of the deletion mutants were assayed by western blot using the NM11 antibody (α p300 CT) or by Coomassie Blue staining, as indicated. Note the presence in the p300 FL preparation of prominent truncated protein products, which most likely reflects premature termination and/or protein degradation that occur during the production of large proteins in bacteria. (B) Sequence alignment of the CBP methylated region with the corresponding sequence of p300. Conserved residues are indicated in bold. The # indicate the three conserved arginines (R). Sequences and names of the peptides used in (C) are indicated on the top of the sequence alignment. (C) Biotinylated peptides (6 µM final concentration) were assayed for methylation using either 1 µg of GST, 50 ng of GST–CARM1 or 50 ng of GST–HMT1 as indicated. Peptide methylation was measured using a scintillation counter. (D) Upper panel: GST–CBP FL and GST–CBP 3R→A (in which R714, R742 and R768 were replaced by alanines) were assayed for methylation by GST–CARM1 and were analysed on an 8% SDS–PAGE followed by fluorography. Lower panel: the amount of GST–CBP FL and GST–CBP 3R→A was assayed by western blot using the NM11 antibody.

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Fig. 3. CBP is methylated on three arginine residues conserved in p300. (A) GST–p300 FL, GST–p300 1–744 and GST–p300 1–595, as indicated, were assayed for methylation by GST–CARM1 and were analysed by SDS–PAGE followed by fluorography. The asterisks (*) indicate the position of the various full-length GST fusion proteins. Note that the major band of methylated GST–p300 FL (#) migrates below the 180 kDa marker: this band corresponds to a major truncated product of p300. In the right panel, the expression of p300 and of the deletion mutants were assayed by western blot using the NM11 antibody (α p300 CT) or by Coomassie Blue staining, as indicated. Note the presence in the p300 FL preparation of prominent truncated protein products, which most likely reflects premature termination and/or protein degradation that occur during the production of large proteins in bacteria. (B) Sequence alignment of the CBP methylated region with the corresponding sequence of p300. Conserved residues are indicated in bold. The # indicate the three conserved arginines (R). Sequences and names of the peptides used in (C) are indicated on the top of the sequence alignment. (C) Biotinylated peptides (6 µM final concentration) were assayed for methylation using either 1 µg of GST, 50 ng of GST–CARM1 or 50 ng of GST–HMT1 as indicated. Peptide methylation was measured using a scintillation counter. (D) Upper panel: GST–CBP FL and GST–CBP 3R→A (in which R714, R742 and R768 were replaced by alanines) were assayed for methylation by GST–CARM1 and were analysed on an 8% SDS–PAGE followed by fluorography. Lower panel: the amount of GST–CBP FL and GST–CBP 3R→A was assayed by western blot using the NM11 antibody.

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Fig. 4. CBP is methylated by CARM1 on R742 in vivo. (A) Left panel: the anti-methylated CBP antibody was tested for its specificity by a slot blot experiment with the indicated amount of various peptides. Right panel: 10 ng of GST–CBP FL and of GST–CBP 3R→A were methylated by GST–CARM1 (lanes 3 and 2, respectively) or not (lane 1). Reaction products were resolved onto an 8% SDS–PAGE followed by a western blot using the purified anti-methylated CBP antibody. (B) Immunoprecipitations from HeLa nuclear extracts (100 µl) were performed using an anti-HA polyclonal antibody (Irr.), an anti-CBP antibody and the affinity purified anti-methylated CBP antibody. Immunoprecipitates and the input (1 µl of HeLa nuclear extracts) were analysed by western blot using the anti-methylated CBP antibody. (C) Lysates of HeLa cells transfected either with control siRNA duplexes (CTR) or with specific CARM1 siRNA duplexes (CARM1) were tested by western blot for the expression level of CARM1, of HDAC-1, -2 and -3 (anti-HDAC3; Transduction Laboratories), of CBP and of CBP specifically methylated on R742.

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Fig. 5. CBP methylation is required for GRIP1- and steroid hormone-induced transcriptional activation. (A) Upper panel: U2OS cells were transiently transfected with 2 µg of reporter vector pBS-Gal4-luciferase, 2.5 µg of pCMV-Gal4-HA-GRIP1 and 50 ng of pCMV LacZ to monitor transfection efficiency and with increasing amounts of pCMV-HA-CBPΔ685–774, of pCMV-HA-CBP 3R→A or of pCMV-HA-CBP (1, 2 or 5 µg, as indicated by the height of the triangle). Luciferase and β-galactosidase activities were measured 48 h later. The luciferase data shown here are representative of at least four independent transfection experiments (the mean and error bars from the four experiments can be seen in the Supplementary data). Only points where β-galactosidase activity was equivalent were taken into consideration. Middle and lower panels: lysates used in upper panel were tested by western blot for the expression of HA-CBPΔ685–774, HA-CBP 3R→A or HA-CBP, and for the expression of HA-Gal4-GRIP-1 (12CA5 anti-HA antibody; Roche Diagnostics). (B) U2OS cells were transfected with 2 µg of reporter vector pBS-Gal4-luciferase, 50 ng of pCMV LacZ, 2 µg of pHK-Gal4-CREB, 1 µg of PKA+ (an expression vector for a constitutively active PKA) and increasing amounts of pCMV-HA-CBP 3R→A or pCMV-HA-CBP (100 ng, 500 ng or 2 µg as indicated by the height of the triangle). Luciferase and β-galactosidase activities were measured as in (A). (C) MCF7 cells were transiently transfected with 2 µg of reporter vector p17ERE-β-globin-luciferase, 50 ng of pCMV LacZ to monitor transfection efficiency and with increasing amounts of pCMV-HA-CBP 3R→A or pCMV-HA-CBP (1, 2 or 5 µg, as indicated by the height of the triangle). 10–8 M of 17 β estradiol (E2) was added as indicated 24 h before harvesting the cells. Luciferase and β-galactosidase activities were measured as in (A). (D) HeLa cells were transiently transfected with 2 µg of reporter vector pRARE-luciferase, 50 ng of pCMV LacZ to monitor transfection efficiency and with increasing amounts of pCMV-HA-CBP 3R→A (1, 2 or 5 µg, as indicated by the height of the triangle). All-trans retinoic acid (10–7 M final concentration) (RA) was added 24 h before harvesting the cells. Luciferase and β-galactosidase activities were measured as in (A).

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Fig. 5. CBP methylation is required for GRIP1- and steroid hormone-induced transcriptional activation. (A) Upper panel: U2OS cells were transiently transfected with 2 µg of reporter vector pBS-Gal4-luciferase, 2.5 µg of pCMV-Gal4-HA-GRIP1 and 50 ng of pCMV LacZ to monitor transfection efficiency and with increasing amounts of pCMV-HA-CBPΔ685–774, of pCMV-HA-CBP 3R→A or of pCMV-HA-CBP (1, 2 or 5 µg, as indicated by the height of the triangle). Luciferase and β-galactosidase activities were measured 48 h later. The luciferase data shown here are representative of at least four independent transfection experiments (the mean and error bars from the four experiments can be seen in the Supplementary data). Only points where β-galactosidase activity was equivalent were taken into consideration. Middle and lower panels: lysates used in upper panel were tested by western blot for the expression of HA-CBPΔ685–774, HA-CBP 3R→A or HA-CBP, and for the expression of HA-Gal4-GRIP-1 (12CA5 anti-HA antibody; Roche Diagnostics). (B) U2OS cells were transfected with 2 µg of reporter vector pBS-Gal4-luciferase, 50 ng of pCMV LacZ, 2 µg of pHK-Gal4-CREB, 1 µg of PKA+ (an expression vector for a constitutively active PKA) and increasing amounts of pCMV-HA-CBP 3R→A or pCMV-HA-CBP (100 ng, 500 ng or 2 µg as indicated by the height of the triangle). Luciferase and β-galactosidase activities were measured as in (A). (C) MCF7 cells were transiently transfected with 2 µg of reporter vector p17ERE-β-globin-luciferase, 50 ng of pCMV LacZ to monitor transfection efficiency and with increasing amounts of pCMV-HA-CBP 3R→A or pCMV-HA-CBP (1, 2 or 5 µg, as indicated by the height of the triangle). 10–8 M of 17 β estradiol (E2) was added as indicated 24 h before harvesting the cells. Luciferase and β-galactosidase activities were measured as in (A). (D) HeLa cells were transiently transfected with 2 µg of reporter vector pRARE-luciferase, 50 ng of pCMV LacZ to monitor transfection efficiency and with increasing amounts of pCMV-HA-CBP 3R→A (1, 2 or 5 µg, as indicated by the height of the triangle). All-trans retinoic acid (10–7 M final concentration) (RA) was added 24 h before harvesting the cells. Luciferase and β-galactosidase activities were measured as in (A).

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