CCAAT displacement protein/cut homolog recruits G9a histone lysine methyltransferase to repress transcription

Abstract

CCAAT displacement protein/cut homolog (CDP/cut) is a highly conserved homeodomain protein that contains three cut repeat sequences. CDP/cut is a transcriptional factor for many diverse cellular and viral genes that are involved in most cellular processes, including differentiation, development, and proliferation. Here, we report that CDP/cut interacts with a histone lysine methyltransferase (HKMT), G9a, in vivo and in vitro. The deletion of the cut repeats within CDP/cut abrogates the interaction with G9a. The transcriptional repressor function of CDP/cut is mediated through HKMT activity of G9a associated with CDP/cut. We show that the recruitment of G9a to the human p21(waf1/cdi1) promoter is contingent on the interaction with CDP/cut, and CDP/cut is directly associated with an increase in the methylation in vivo of Lys-9 in histone H3 within the CDP/cut-regulatory region of the p21(waf1/cdi1) promoter. The endogenous level of p21(waf1/cdi1) expression is repressed through CDP/cut and mediated by HKMT activity of G9a. Furthermore, we report the identification of G9a as a component of CDP/cut complex. G9a colocalizes with CDP/cut in the nucleus. These results indicate that G9a functions as a transcriptional corepressor in association with a CDP/cut complex. These studies now reveal the interaction of G9a with a sequence-specific transcription factor that regulates gene repression through CDP/cut.

Fig. 1.

CDP/cut interacts with hG9a in vivo and in vitro.(A) hG9a coimmunoprecipitates with FLAG-tagged CDP/cut from cell extracts of transfected 293T cells. Cells were transfected with FLAG-CDP/cut and/or EGFP-hG9a expression vectors as indicated. Whole-cell extracts were immunoprecipitated with anti-FLAG Ab, and immunoprecipitates were analyzed by Western blot with anti-GFP Ab. (B) Schematic representation of CDP/cut with evolutionarily conserved domains (cc, coiled coil; cut, cut repeat; HD, homeodomain) and truncated derivatives. (C) Association with hG9a is disrupted by C-terminal truncation in CDP/cut. Immunoprecipitates from transfected cells with expression vectors for EGFP-hG9a and FLAG-CDP/cut truncated derivatives were analyzed as in A.(D) GST-CDPC (C-terminal region of CDP/cut) fusion protein interacts with in vitro-translated hG9a. GST pull-down assays were performed with in vitro-translated [³⁵S]hG9a and GST fusion proteins as indicated. Input lane shows 10% of total.

Fig. 2.

CDP/cut represses transcription through HKMT activity of hG9a. (A) hG9a corepresses transcription with CDP/cut. The 293T cells were transfected with a Gal4-driven luciferase reporter with combinations of the expression vector for Gal4DBD-CDPC and increasing amounts of the EGFP-hG9a or EGFP-hG9a (ΔSET) expression vector. Luciferase activity was measured 48 h after transfection. The means ± SD of duplicate determinations from five separate experiments are shown. (B) hG9a (ΔSET) interacts with CDPC. The 293T cells were transfected with FLAG-CDPC and EGFP-hG9a or EGFP-hG9a (ΔSET) expression vectors. Whole-cell extracts were immunoprecipitated with anti-FLAG Ab, and immunoprecipitates were analyzed by Western blot with anti-GFP Ab. (C) Immunoprecipitated EGFP-hG9a possesses histone H3 methyltransferase activity, but EGFP-hG9a (ΔSET) does not. The 293T cells were transfected with EGFP-hG9a or EGFP-hG9a (ΔSET) expression vector, and the whole-cell extracts were immunoprecipitated with anti-GFP Ab. Immunoprecipitates were assayed for in vitro HMT activity by using histone H3 and H4 as substrates. (D) CDP/cut-mediated repression is associated with H3-K9 and H3-K27 methyltransferase activity by G9a. The 293T cells were transfected, and the whole-cell extracts were immunoprecipitated as in B. Immunoprecipitates were assayed for in vitro HMT activity by using GST-histone H3 (amino acids 1–57) fusion proteins (WT and mutants) as substrates. Also shown are Coomassie blue staining of purified GST-histone H3 fusion proteins introduced into assays as well as Western blots with Abs against CDP and GFP of inputs and immunoprecipitates with anti-FLAG Ab.

Fig. 3.

CDP/cut recruits hG9a to repress p21^waf1/cdi1. (A) hG9a recruitment to the p21_pro is CDP/cut dependent. ChIPs from transfected 293T cells with combinations of FLAG-CDPC and EGFP-hG9a expression vectors as indicated were performed with anti-FLAG Ab, anti-GFP Ab, or normal serum. Immunoprecipitates were analyzed for the presence of the p21_pro and the upstream region (p21_up) by PCR amplification. Results are representative of three independent experiments. (B) hG9a recruitment elevates dimethylated H3-Lys-9 in the p21_pro. ChIPs were performed as in A except that experiments were carried out with anti-dimethyl H3-Lys-9 Ab. (C) p21^waf1/cdi1 expression is repressed by CDP/cut mediated through hG9a. Total RNA was isolated from human diploid fibroblast IMR90 cells that were transfected with expression vectors as indicated and analyzed for the expression of p21^waf1/cdi1 by RT-PCR. Results are representative of three independent experiments.

Fig. 4.

hG9a is a component of CDP/cut complex. (A) Endogenous hG9a is a component of purified CDP/cut complex. (His)₆-Xpress-CDPC complex was purified from 293 EBNA cells stably expressing (His)₆-Xpress-CDPC by using Ni⁺-agarose, followed by immunoprecipitation with anti-Xpress Ab, and analyzed by SDS/PAGE and silver staining. Identities of protein bands were determined by MS. As a control, mock purification was performed from 293 EBNA cells generated with an empty (His)₆-Xpress-tagged protein expression vector. (B) Endogenous hG9a specifically associates with the CDP/cut complex. The eluates of (His)₆-Xpress-CDPC complex in A were analyzed by Western blots with Abs against CDP, TAF_II100, Sp1, or G9a. (C) Endogenous hG9a associates with endogenous CDP/cut. The eluates of (His)₆-Xpress-CDPC complex in A were immunoprecipitated with anti-CDP Ab, anti-G9a Ab, or normal serum, and the immunoprecipitates were analyzed by Western blot with anti-CDP Ab.

Fig. 5.

hG9a colocalizes with CDP/cut in the nucleus. The 293T cells were transfected with an expression vector for FLAG-hG9a and double-stained with anti-FLAG and anti-CDP Abs. FLAG-hG9a was detected with FITC-conjugated secondary Ab, and endogenous CDP/cut was detected with Rhodamine-conjugated secondary Ab. Representative red immunofluorescent image for endogenous CDP/cut (Left), green immunofluorescent image for FLAG-hG9a (Center), and merged image (Right) are shown. The yellow dots in the merged images indicate colocalizaion of hG9a and CDP/cut.