MCAF mediates MBD1-dependent transcriptional repression

Abstract

DNA methylation is involved in a variety of genome functions, including gene control and chromatin dynamics. MBD1 is a transcriptional regulator through the cooperation of a methyl-CpG binding domain, cysteine-rich CXXC domains, and a transcriptional repression domain. A yeast two-hybrid screen was performed to investigate the role of MBD1 in methylation-based transcriptional repression. We report a mediator, MBD1-containing chromatin-associated factor (MCAF), that interacts with the transcriptional repression domain of MBD1. MCAF harbors two conserved domains that allow it to interact with MBD1 and enhancer-like transactivator Sp1. MCAF possesses a coactivator-like activity, and it seems to facilitate Sp1-mediated transcription. In contrast, the MBD1-MCAF complex blocks transcription through affecting Sp1 on methylated promoter regions. These data provide a mechanistic basis for direct inhibition of gene expression via methylation-dependent and histone deacetylation-resistant processes.

FIG. 1.

MBD1-mediated transcriptional repression is resistant to HDAC inhibitors. (A) Effect of HDAC inhibitors on transcriptional repression by MBD proteins. The reporter constructs contain both the yeast GAL4 DNA binding site (5xGAL) and sequences from the human promoter-associated CpG island upstream of the luciferase cDNA. Effectors express the TRDs of MeCP2 and MBD1 and the MBD2a and MBD3 in a fusion to the GAL4 DNA binding domain. An effector (0.3, 0.6, and 1.2 μg) and the insertless pCMV-GAL4 (0.9, 0.6, and 0 μg) were introduced into HeLa and U2OS cells together with the reporter containing VHL promoter (1.0 μg) and an internal control pRL-CMV (0.02 μg). At 30 h after transfection, the cells were treated for 12 h with the HDAC inhibitors, TSA and sodium butyrate (SB), and with the solvent alone (black). The luciferase activities in combination with pCMV-GAL4 (mock) were normalized to 100 (as indicated by a line). (B) Little influence of HDAC inhibitors on repression by TRD of MBD1 from a distance. GAL4 binding motifs were inserted more than 3 kb upstream of the transcription start site in the reporter constructs. Four gene promoters were used: p16 (black), VHL (hatched), E-cadherin (gray), and SNRPN (white). An effector (0 to 1.0 μg) was transfected into HeLa cells. (C) Induction of acetylated histones H3 and H4 by HDAC inhibitors. Lane C, the solvent alone.

FIG. 2.

MBD1 interacts with a transcriptional modulator, MCAF. (A) Structure of MBD1. MBD1 contains an MBD, three cysteine-rich CXXC domains, and a TRD. A yeast two-hybrid screen using the TRD identified MCAF. Two amino acid residues within the TRD (indicated by capital letters) are important for MBD1-dependent repression (Fig. 4). (B) Expression of MCAF. Anti-MCAF polyclonal antibodies and a vector expressing FLAG-tagged MCAF were utilized. (C) Complex formation of MBD1 and MCAF. Endogenous MCAF and HA-tagged MBD1 were immunoprecipitated from HeLa cells. IP, immunoprecipitation; Ab, antibody. (D) Interaction between endogenous MBD1 and MCAF. Mouse anti-MBD1 monoclonal antibodies and rabbit anti-MCAF polyclonal antibodies were used for immunoprecipitation. (E) Biochemical binding assay. MCAF possesses two distinct conserved domains, named domain 1 (Δ18) and domain 2 (Δ8). GST and GST-MCAF (2 μg) were immobilized on glutathione-agarose beads and incubated with His-tagged TRD of MBD1 (2 μg). The input indicates 10% of the indicated proteins in the reaction mixture.

FIG. 3.

MCAF is a transcriptional regulator. (A) Effect of MCAF on promoter activities. The full length of MCAF (0 to 3 μg) and the insertless pcDNA3 (3 to 0 μg) were introduced into HeLa cells together with the luciferase reporter (1.0 μg) and pRL-CMV (0.02 μg). The luciferase activities in combination with mock vector (1.0 μg) were normalized to 10. (B) Inhibition of an Sp1-activated transcription by TRD of MBD1 and MCAF. Full-length MCAF and Δ2 mutant deficient in association with MBD1 (Fig. 2E) were expressed in D. melanogaster SL2 cells, together with GAL4-TRD of MBD1 and Sp1. GAL4 motif-containing reporter vector (1.0 μg), pPacSp1 (1.0 μg), pAc5.1-MCAF, pAc5.1-MCAF Δ2 and its insertless mock version (1.0 μg), and pAc5.1-GAL4-TRD of MBD1 (0.5 or 1.0 μg) and its mock version (0.5 or 0 μg) were utilized. The repression level in combination with the mock (1.0 μg) was normalized to 100 (black bars), and the relative luciferase activities were corrected by an internal control, pAc5.1-pRL (0.02 μg). (C) MCAF did not enhance an E2F1-activated transcription. Full-length MCAF (0 to 3 μg) and the insertless pcDNA3 (3 to 0 μg) were introduced into the cells together with the SNRPN or VHL luciferase reporter (1.0 μg) and E2F1-expressing vector (1.0 μg).

FIG. 4.

Interaction between MBD1 and MCAF is required for transcriptional repression. (A) TRD mutants of MBD1 reduce the repressive activity. Wild-type (wt) and mutant TRDs of MBD1 in a GAL4 fusion were expressed in HeLa cells. The repression level in combination with wild-type GAL4-TRD of MBD1 was normalized to 100. The amino acid residues in Fig. 2A were subjected to mutagenesis (I576R, L579R, and I576R/L579R). (B) Loss of ability of TRD mutants to bind MCAF. Wild-type and mutant TRDs fused to GST were immobilized and incubated with His-tagged MCAF (Δ8). The input indicates 10% of the protein in the reaction mixture. (C and D) Localization of MBD1 and MBD1 (I576R) mutant in the nucleus. An immunofluorescence analysis of DsRed- or EGFP-fused MBD1 was performed in HeLa cells. MBD1 (MBD+NLS) expresses only the MBD and the nuclear localization signal. (E) Colocalization of MBD1 and native MCAF. (F) Dissociation of MCAF from MBD1 (I576R) mutant.

FIG. 5.

Association of MBD1 and MCAF on endogenous gene promoter. (A) Methylation-specific PCR. The promoter region of the p16 tumor suppressor gene was studied in human lung cancer cell lines NCI-H1299 and SBC-5. W, U, and M indicate specifically amplified fragments corresponding to unmodified, unmethylated, and methylated sequences, respectively. (B) Link of MCAF by MBD1 to methylated promoter. The cells were treated with a protein-protein cross-linker (DTBP) and formaldehyde. Specific fragments of p16 promoter were detected by PCR amplification by using a set of W primers in the immunoprecipitates. HA-tagged MBD1 was expressed for a chromatin immunoprecipitation with anti-HA antibodies (upper). Wild-type or mutant (I576R) HA-MBD1 was coexpressed together with FLAG-MCAF, followed by the chromatin immunoprecipitation with anti-FLAG antibodies (lower). (C) Competition of MBD1 with Sp1 for binding to MCAF. His-MCAF Δ3 (2 μg) (Fig. 2E) on nickel-chelating resin was incubated with GST-TRD of MBD1 and GST-Sp1 Δ1 containing the residues 90 to 785 of Sp1 (0 to 2 μg each). Bound proteins on the resin were eluted by imidazole. The input indicates 10% of the protein in the reaction mixture.