Methylation at arginine 17 of histone H3 is linked to gene activation

Abstract

The nuclear hormone receptor co-activator CARM1 has the potential to methylate histone H3 at arginine residues in vitro. The methyltransferase activity of CARM1 is necessary for its co-activator functions in transient transfection assays. However, the role of this methyltransferase in vivo is unclear, given that methylation of arginines is not easily detectable on histones. We have raised an antibody that specifically recognizes methylated arginine 17 (R17) of histone H3, the major site of methylation by CARM1. Using this antibody we show that methylated R17 exists in vivo. Chromatin immunoprecipitation analysis shows that R17 methylation on histone H3 is dramatically upregulated when the estrogen receptor-regulated pS2 gene is activated. Coincident with the appearance of methylated R17, CARM1 is found associated with the histones on the pS2 gene. Together these results demonstrate that CARM1 is recruited to an active promoter and that CARM1-mediated R17 methylation on histone H3 takes place in vivo during this active state.

Fig. 1. Methylation of histone H3 at R17 occurs in vivo. (A) Recombinant Drosophila histone H3 was in vitro methylated by GST–CARM1 in the presence of [³H]SAM and subjected to microsequencing of residues 1–30. Amino acid fractions were analysed for the presence of tritium by scintillation counting. Additionally non-radioactively methylated recombinant H3 was sequenced and the amino acid concentration in each fraction was determined by HPLC. The raw data of the radiosequencing were then corrected by calculation of the [³H]methyl incorporation per pmol amino acid. The result of this refinement is shown for the four arginines (R2, R7, R17 and R26) in the H3 N-terminus which are the putative methylation sites of CARM1. The amino acid sequence of residues 1–30 of histone H3 is shown above and the positions of the four arginines are indicated. (B) The antiMe-R17H3 antibody (from www.abcam.com) was raised against a histone H3 peptide (aa 11–24) asymmetrically dimethylated at R17, which represents the major site of CARM1 methylation. Western blot analysis was performed using Me-R17H3 and 2 µg of unmodified recombinant Drosophila histone H3 (lane 1), recombinant H3 after in vitro methylation with GST–CARM1 protein (lane 2), purified calf thymus histone H3 (lane 3), unmodified recombinant Drosophila histone H4 (lane 4), recombinant H4 after in vitro methylation with GST–PRMT1 protein (lane 5) and purified histone H4 (lane 6). Coomassie Blue staining (lower panel) revealed the presence of approximately equal amounts of histones in each lane. The enzymatic activity of GST–CARM1 and GST–PRMT1 on histones was confirmed by in vitro methylation of bulk histones in the presence of [³H-Me]S-adenosyl methionine, SDS–PAGE and autoradiography (data not shown). (C) Peptide competition experiment was performed by western blot analysis using 1 µg of purified calf thymus histone H3 and antiMe-R17H3 antibody in the presence of 1 µg/ml none methylated H3 peptide (aa 11–24, lane 2), R17 methylated H3 peptide (aa 11–24, lane 3) or R3 methylated H4 peptide (aa 1–14, lane 4). (D) Total U2OS cell extract was western blotted using the antiMe-R17H3 antibody. The asterisk indicates methylated histone H3. The left panel shows presence of core histones (indicated on the left) by Coomassie Blue staining. Molecular weights are indicated on the right.

Fig. 2. CARM1 co-activates the pS2 promoter in a methylation dependent manner. (A) 293T cells were transiently transfected with the indicated expression plasmids. Whole-cell extracts were used in CAT assays and the results were quantified on a PhosphorImager. The basal promoter activity of the pS2 CAT reporter in the presence of empty expression vector was normalized to 1.0, and the activities of the remaining transfection reactions were expressed relative to this. The graph shows the result of one experiment which was reproduced independently several times. Equal protein expression of wild-type full-length CARM1 (CARM1 fl) or mutant CARM1 (CARM1 284–608) was confirmed by western blot analysis (data not shown). (B) GST alone or GST fusion protein of SRC1 (984–1441) was expressed in E. coli, purified and used in GST pull-down experiments with in vitro translated (IVT), ³⁵S-labelled CARM1 fl or mutant CARM1 284–608. Interaction between the proteins was analysed by SDS–PAGE and fluorography.

Fig. 3. Activation of the pS2 gene promoter coincides with CARM1 recruitment and methylation of histone H3 at R17 in vivo. MCF-7 cells were stimulated with DMSO/ethanol (no induction control) or with 200 nM 17β-estradiol (E2), or 100 ng/ml tetradecanoyl phorbol acetate (TPA), or both (E2+TPA) for 3 h and analysed by northern blot and ChIP assay. (A) mRNA steady state levels of pS2 and GAPDH were determined by northern blot analysis. Total RNA was prepared from unstimulated and stimulated MCF-7 cells, and 10 µg RNA per sample separated through a 1.2% formaldehyde-agarose gel and blotted onto a nylon membrane. The pS2 and GAPDH RNA was detected by hybridization with ³²P-labelled pS2 or GAPDH gene probe. The GAPDH northern blot shows that equal RNA amounts were loaded. (B) For ChIP, genomic chromatin fragments from stimulated MCF-7 cells were immunoprecipitated either with antiMe-R17H3 or antiCARM1 (Upstate) antibodies. Immunoprecipitated chromatin was analysed by quantitative PCR for CARM1 binding and histone H3 R17 methylation in the proximal pS2 promoter region (nt –159 to –463). AntiMe-R17H3 immunoprecipitates were analysed by quantitative PCR with primers for the cdc25 gene promoter (nt –15 to –186). PCR analysis on input chromatin confirmed that equal chromatin amounts were used for ChIPs.