Histone H3 lysine 9 methyltransferase G9a is a transcriptional coactivator for nuclear receptors

Abstract

Methylation of Lys-9 of histone H3 has been associated with repression of transcription. G9a is a histone H3 Lys-9 methyltransferase localized in euchromatin and acts as a corepressor for specific transcription factors. Here we demonstrate that G9a also functions as a coactivator for nuclear receptors, cooperating synergistically with nuclear receptor coactivators glucocorticoid receptor interacting protein 1, coactivator-associated arginine methyltransferase 1 (CARM1), and p300 in transient transfection assays. This synergy depends strongly on the arginine-specific protein methyltransferase activity of CARM1 but does not absolutely require the enzymatic activity of G9a and is specific to CARM1 and G9a among various protein methyltransferases. Reduction of endogenous G9a diminished hormonal activation of an endogenous target gene by the androgen receptor, and G9a associated with regulatory regions of this same gene. G9a fused to Gal4 DNA binding domain can repress transcription in a lysine methyltransferase-dependent manner; however, the histone modifications associated with transcriptional activation can inhibit the methyltransferase activity of G9a. These findings suggest a link between histone arginine and lysine methylation and a mechanism for controlling whether G9a functions as a corepressor or coactivator.

Fig. 1

G9a is a coactivator for AR and ERα. CV-1 cells were transfected with MMTV-LUC (A) or MMTV(ERE)-LUC (B and C) reporter plasmids (125 ng) and expression vectors encoding AR (10 ng), ERα (1 ng), GRIP1 (50 ng), CARM1 (200 ng), G9a ( 50, 100, 200 (+) and 400 ng) and p300 (200 ng) as indicated. Cells were grown with or without 20 nM DHT or 20 nM E2 as indicated and assayed for luciferase activity.

Fig. 2

The methyltransferase activity of CARM1 but not that of G9a is required for coactivator synergy. (A and B) CV-1 cells were transfected with MMTV-LUC reporter plasmid (125 ng) and with expression vectors encoding AR (10 ng), GRIP1 (50 ng), CARM1 wild type or mutant (200 ng), and G9a wild type or mutant (50, 100, 200 and 400 ng). Cells were treated with 20 nM DHT prior to luciferase assays. The schematic diagram shows full-length G9a and the residue numbering of fragments used in this study. E, polyglutamate; Cys, cysteine rich region; ANK, six ankyrin repeats; Pre- and Post-SET domains; SET, H3 Lys-9 methyltransferase domain; asterisk, location of H1166K mutation in the SET domain. (C) Cos-7 cells were transfected with the G9a expression vectors used in panel B above. Whole-cell extracts were analyzed for G9a expression by immunoblotting with anti-HA antibody. (D) CV-1 cells were transfected with GK1 reporter plasmid (250 ng) and expression vectors encoding Gal4 DBD (vector) or Gal4 DBD fused to various G9a fragments (250 ng each). After 48 hrs cell extracts were analyzed for luciferase activity. Inset graph shows the same results on a different scale without assay 3.

Fig. 3

Specificity of coactivator synergy among protein arginine and lysine methyltransferases. (A and B) CV-1 cells were transfected with MMTV-LUC reporter plasmid (125 ng) and with plasmids encoding AR (10 ng); GRIP1 (50 ng); CARM1, PRMT1, PRMT2, PRMT3, or RMT1 (200 ng); and HA-tagged G9a or PR-SET7 (50, 100, 200, and 400 ng). Cells were treated with 20 nM DHT and luciferase assays were performed. (C) Cos-7 cells were transfected with the G9a and PR-SET7 vectors used in panel B or mock transfected. Whole-cell extracts were analyzed for protein expression by immunoblotting with anti-HA antibody.

Fig. 4

Binding of G9a to GRIP1. (A) Cos-7 cells were transfected with expression vectors (2.5 μg each) for Flag-G9a and one of the following: HA-GRIP1.N, HA-GRIP1.M or HA-GRIP1.C. Cell extracts were subjected to immunoprecipitation (IP) with anti-Flag antibody or normal IgG and then immunoblotted (W) with anti-HA antibody. The positions of coimmunoprecipitated GRIP1.N and GRIP1.C and of immunoglobulin heavy chains (IgG) are indicated. (B) ³⁵S-labelled G9a was incubated with GST, GST-GRIP1.N, GST-GRIP1.M or GST-GRIP1.C immobilized on glutathione-agarose beads. Bound proteins were eluted and analyzed by SDS-PAGE and autoradiography. (C) Cos-7 cells were transfected as in panel A with Flag-G9a full-length (FL), either wild type (WT) or H1166K (H/K) mutant, N-terminal truncations (residues 464–1263, 685–1263 or 936–1263/ΔANK) or no G9a (none) and with HA-GRIP1.N. Extracts were processed as in panel A; coimmunoprecipitated GRIP1.N is indicated. (D) Cos-7 cells were transfected with G9a N-terminal truncations and either with or without HA-GRIP1.N. Cell extracts were subjected to immunoprecipitation and immunoblotting as indicated. (E) Extracts from Cos-7 cells with transfected HA-GRIP1.N in panel D were subjected to immunoprecipitation and immunoblotting as indicated. Arrows in panels D and E indicate positions of G9a N-terminally truncated proteins.

Fig. 5

G9a is necessary for efficient transcriptional activation by AR. siRNA against G9a or control siRNA was transfected into LNCaP cells. After growth with or without DHT, cDNA was synthesized from total RNA and analyzed by quantitative real-time PCR. The level of G9a mRNA (A) or PSA mRNA (B) was normalized to that of β-actin; all ratios are expressed relative to the DHT-only samples (assay 2). Compared with DHT treatment alone, siRNA against G9a reduced G9a mRNA by 75% (p<0.0001 and 95% confidence interval of ±3%) and reduced PSA mRNA by 48% (p<0.0001 and 95% confidence interval of ±9%). Statistical significance was calculated using a paired 2-tailed t-test on eight independent experiments. (C) LNCaP cells were treated with siRNA and DHT as above, and cell extracts were analyzed by immunoblot, using antibodies against the indicated proteins.

Fig. 6

The SET methyltransferase activity of G9a can repress transcription but can also be inhibited by histone modifications associated with active transcription. (A) CV-1 cells were transfected with UAS-tk-LUC reporter plasmid (500 ng) and expression vectors (250 ng) encoding Gal4 DBD (vector) or Gal4 DBD fused to either full-length wild-type (WT) or active site deleted (ΔNHLC), or fragments of G9a with wild type or H1166K (H/K) mutant sequence. (B) Cos-7 cells were transfected with 2.5 μg of the wild type or H/K mutant G9a-Gal4 DBD fusion expression vectors used in panel A. Whole cell extracts were analyzed by immunoblotting with anti-G9a antibody. The positions of the fusion proteins are indicated; arrow indicates position of endogenous G9a. (C) Histone H3 peptides (residues 1–21) were methylated with GST-G9a (730–1263) or GST-CARM1. Reaction products were analyzed by SDS-PAGE and autoradiography. Equal amounts of each peptide (determined by staining) were loaded on the gel (data not shown).

Fig. 7

ChIP assay for the androgen receptor and G9a at the PSA gene. (Top) schematic diagram of the PSA gene showing the positions of major elements including AREs I, II and III (black boxes). E-P, intervening region between the enhancer and promoter regions. Arrow indicates the transcription start site and direction. Thick lines below the gene diagram indicate positions of PCR amplified fragments. (Bottom) real-time PCR analysis using primer pairs as described in Experimental Procedures. The results are shown as a percentage of the input chromatin and are the mean and standard deviation of triplicate reactions. The immunoprecipitating antibody (antiAR, anti-G9a or IgG) is indicated at left.

Sup. Fig. S1

G9a is a coactivator for GR. CV-1 cells were transfected with MMTV-LUC reporter plasmid (125 ng) and expression vectors encoding GR (10 ng), GRIP1 (50 ng), CARM1 (200 ng), and G9a (50, 100, 200 (+) and 400 ng). Cells were grown with or without 20 nM dexamethasone as indicated and assayed for luciferase activity.

Sup. Fig. S2

GRIP1 functional domains involved in CARM1 and G9a synergy. CV-1 cells were transfected with MMTV-LUC reporter plasmid (125 ng) and expression vectors encoding AR (10 ng), wild-type or mutant GRIP1 (50 ng), CARM1 (200 ng), and G9a (50, 100, 200, and 400 ng). Transfected cells were grown in medium with 20 nM DHT for 48 hrs before assay for luciferase activity.