G9a functions as a molecular scaffold for assembly of transcriptional coactivators on a subset of glucocorticoid receptor target genes

Abstract

Histone H3 lysine-9 methyltransferase G9a/EHMT2/KMT1C is a key corepressor of gene expression. However, activation of a limited number of genes by G9a (independent of its catalytic activity) has also been observed, although the precise molecular mechanisms are unknown. By using RNAi in combination with gene expression microarray analysis, we found that G9a functions as a positive and a negative transcriptional coregulator for discrete subsets of genes that are regulated by the hormone-activated Glucocorticoid Receptor (GR). G9a was recruited to GR-binding sites (but not to the gene body) of its target genes and interacted with GR, suggesting recruitment of G9a by GR. In contrast to its corepressor function, positive regulation of gene expression by G9a involved G9a-mediated enhanced recruitment of coactivators CARM1 and p300 to GR target genes. Further supporting a role for G9a as a molecular scaffold for its coactivator function, the G9a-specific methyltransferase inhibitor UNC0646 did not affect G9a coactivator function but selectively decreased G9a corepressor function for endogenous target genes. Overall, G9a functioned as a coactivator for hormone-activated genes and as a corepressor in support of hormone-induced gene repression, suggesting that the positive or negative actions of G9a are determined by the gene-specific regulatory environment and chromatin architecture. These findings indicate distinct mechanisms of G9a coactivator vs. corepressor functions in transcriptional regulation and provide insight into the molecular mechanisms of G9a coactivator function. Our results also suggest a physiological role of G9a in fine tuning the set of genes that respond to glucocorticoids.

Fig. 1.

G9a negatively and positively regulates specific subsets of GR target genes. (A) Large Venn diagram represents the total number of dex-regulated genes from the microarray analysis: genes with significantly different expression (q ≤ 0.01 and ≥1.5-fold increase or decrease) after treatment with 100 nM dex for 24 h compared with untreated cells; cells were uninfected or infected with lentivirus encoding nonspecific shRNA (i.e., shNS). Small Venn diagram represents the number of G9a-regulated genes with significantly different expression (q ≤ 0.05) in dex-treated cells expressing shG9a vs. dex-treated control cells (uninfected cells and cells infected with the virus encoding shNS). Overlap area indicates the number of genes belonging to both sets. Complete lists of genes for each set depicted are found in Datasets S1 and S2. (B) Table summarizing results of bioinformatics analysis of Illumina microarray results for the dex-regulated gene set. (C and D) RT-qPCR quantification of mRNA (C) or pre-mRNA (D) levels for the indicated genes. Cells expressing shNS (open symbols) or shG9a (filled symbols) were treated with 100 nM dex for the indicated times (in hours). Results shown are mean ± SD for three PCR reactions performed on the same cDNA sample and are representative of three independent experiments.

Fig. 2.

G9a is recruited to GR target genes that require G9a for dex-regulated expression. ChIP was performed on A549 cells untreated or treated with 100 nM dex for 0 or 5 h. Immunoprecipitated DNA was analyzed by qPCR by using the indicated primers (sequences given in Dataset S3), and results are normalized to input chromatin and shown as the ratio of 5 h vs. 0 h of dex treatment. Results shown are mean ± SD (n = 3) of PCR reactions from a single experiment, and are representative of at least three independent experiments.

Fig. 3.

G9a interacts with GR in vitro and in vivo. (A) Right: Diagram of full-length mG9a (FL) and mG9a fragments used in this experiment showing amino acid sequence numbers and specific domains: Glu-rich (marked with “E”), Cys-rich ring finger-like (Cys), ankyrin repeat (ANK), methyltransferase (SET), and Cys-rich Pre-SET (Pre) and post-SET (Post). Left: Full-length human GR was synthesized in vitro, incubated with 1 µM dex or vehicle for 4 h, and incubated with GST or GST fused to mG9a fragments or to an SRC1a C-terminal fragment bound to glutathione-agarose beads as indicated. Bound human GR protein was detected by immunoblot with anti-GR antibody. A 10% input sample was loaded for comparison. (B) Coimmunoprecipitation of endogenous GR with G9a from lysates of A549 cells treated with 100 nM dex for the indicated times (in hours). Immunoprecipitation was performed with anti-G9a, anti-GR, or control IgG antibodies. Western blots were performed with anti-GR (Upper) and anti-G9a (Lower) antibodies. A 2% input sample was loaded for comparison. (C) Effect of G9a depletion on GR binding to target genes. ChIP was performed on A549 cells expressing shNS (open symbols) or shG9a (filled symbols) and treated with 100 nM dex for the indicated times. Immunoprecipitated DNA was analyzed by qPCR by using primers that amplify the GBS region of the indicated GR target genes. Results shown are mean ± SD of PCR reactions (n = 3) performed with DNA samples from a single experiment, and are representative of three independent experiments.

Fig. 4.

G9a facilitates recruitment of p300 and CARM1 for activation of G9a-dependent GR target genes. (A) A549 cells expressing shNS (open bars) or shG9a (filled bars) were treated with dex for 0 or 5 h. ChIP was performed with antibodies against CARM1 (Left) or p300 (Right), and immunoprecipitated DNA was analyzed by qPCR by using primers specific for the GBS of the indicated genes. Results are normalized to input chromatin and the mean ± SD of the ratio between 5 h and 0 h dex treatment for three independent experiments are shown. (B) A549 cells transfected with the indicated siRNA were treated with dex for 0 or 5 h. ChIP was performed with antibody against G9a, and immunoprecipitated DNA was analyzed by qPCR by using primers specific for the GBS of the indicated gene. Results shown are mean ± SD of triplicate PCR reactions performed on the same DNA sample, are from a single experiment, and are representative of two independent experiments. (C) COS-7 cells were transfected with pSG5-2XFLAG-hG9a (full-length) and pSG5-HA-mCARM1 (full-length) or pSG5-HA-mGRIP1 (full-length) and treated with dex for 0 or 4 h. G9a was immunoprecipitated from cell extracts with an anti-FLAG antibody or nonimmune IgG (for background estimation), and bound protein was analyzed by immunoblot with anti-G9a antibody (Lower) to control for levels of immunoprecipitated G9a or with anti-HA antibody to detect CARM1 or GRIP1. A 5% input sample was loaded for comparison. (D) Model for positive G9a-mediated regulation of GR target gene expression. First, hormone-activated GR binds to GBS. Next, G9a is recruited to GBS at least in part through interaction with GR. G9a then facilitates recruitment or stabilizes the association at the GBS of p300 and CARM1 coactivators, which acetylate histones H3 and H4 (H3/H4 Ac) and dimethylate histone H3 at R17 (H3R17me2), respectively, leading to the recruitment of basal transcription factors (TFIIB and TBP) and RNA polymerase II (Pol II) complex. (E) RT-qPCR quantification of mRNA levels for the indicated genes normalized to β-actin mRNA. Cells were treated with 100 nM dex (white and black bars) or the equivalent volume of EtOH (vehicle; gray bars) for 8 h. At 1 h before hormone or EtOH treatment, cells were treated with 2 µM UNC0646 (black bars) or equivalent volume of DMSO (vehicle; white and gray bars). Results shown are mean ± SD for at least three independent experiments (*P ≤ 0.05, paired t test).