Genome-wide repression of NF-κB target genes by transcription factor MIBP1 and its modulation by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase

Abstract

The transcription factor c-MYC intron binding protein 1 (MIBP1) binds to various genomic regulatory regions, including intron 1 of c-MYC. This factor is highly expressed in postmitotic neurons in the fetal brain and may be involved in various biological steps, such as neurological and immunological processes. In this study, we globally characterized the transcriptional targets of MIBP1 and proteins that interact with MIBP1. Microarray hybridization followed by gene set enrichment analysis revealed that genes involved in the pathways downstream of MYC, NF-κB, and TGF-β were down-regulated when HEK293 cells stably overexpressed MIBP1. In silico transcription factor binding site analysis of the promoter regions of these down-regulated genes showed that the NF-κB binding site was the most overrepresented. The up-regulation of genes known to be in the NF-κB pathway after the knockdown of endogenous MIBP1 in HT1080 cells supports the view that MIBP1 is a down-regulator of the NF-κB pathway. We also confirmed the binding of the MIBP1 to the NF-κB site. By immunoprecipitation and mass spectrometry, we detected O-linked β-N-acetylglucosamine (O-GlcNAc) transferase as a prominent binding partner of MIBP1. Analyses using deletion mutants revealed that a 154-amino acid region of MIBP1 was necessary for its O-GlcNAc transferase binding and O-GlcNAcylation. A luciferase reporter assay showed that NF-κB-responsive expression was repressed by MIBP1, and stronger repression by MIBP1 lacking the 154-amino acid region was observed. Our results indicate that the primary effect of MIBP1 expression is the down-regulation of the NF-κB pathway and that this effect is attenuated by O-GlcNAc signaling.

FIGURE 1.

DNA binding of MIBP1 protein. A, HEK293 cells stably overexpressing MIBP1 (lanes 4 and 5) or control HEK293 cells (lanes 2 and 3) were treated with vehicle (lanes 2 and 4) or 10 ng/ml TNF-α (lanes 3 and 5). Nuclear extracts of these cells were incubated with biotinylated oligonucleotide containing an NF-κB binding sequence in the binding condition and separated in a 3.6% gel as described under “Experimental Procedures.” Nuclear extracts were also immunoblotted (IB) using 5% SDS-PAGE and detected by anti-FLAG antibody to confirm the overexpression of MIBP1 protein. B, nuclear extracts from HEK293 cells treated with TNF-α were incubated with biotinylated NF-κB-binding oligonucleotide. Excess unlabeled NF-κB (lane 4) or EBNA-binding oligonucleotide (lane 5) was added to the DNA-protein mixture. Anti-rat MIBP1 antibody (lane 6) and specific antigen peptide for the antibody (lane 7) were also added to confirm the specificity of shifted band.

FIGURE 2.

Preparative immunoprecipitation of MIBP1-binding proteins for mass spectrometry. A, lysates from HEK293 cells transfected with pCIX-FM (lane F) or pCAH-MIBP16 (lane H) were immunoprecipitated with the anti-FLAG M2 affinity gel. The precipitated proteins were resolved by 7.5% SDS-PAGE and silver-stained. The arrows with numbers indicate the positions of the bands that were excised for enzymatic digestion and subsequent analysis using an LCQ Deca mass spectrometer. The numbered bands were identified as follows: 1, MIBP1; 2, MIBP1; 3, not identifiable; 4, OGT; and 5, XRCC5. Detailed data from the mass spectrometry analyses are shown in supplemental Table S13. Recombinant human fibronectin (lane FN) was used as a positive control for protein identification by mass spectrometry. B, nuclear (NE) and cytoplasmic (CP) fractions were prepared from HEK293 cells stably transfected with pCI-neo-FHM (lane 31) or untransfected HEK293 cells (lane 293) and immunoprecipitated with the anti-FLAG M2 affinity gel. Precipitated proteins were resolved by 7.5% SDS-PAGE and silver-stained. Protein identification was performed using an LTQ mass spectrometer. The numbered bands were identified as follows: 6, EML6, MIBP1, ubiquitin, and DSP; 7, MIBP1 and DSP; 8, FBXW11; 9, WDR77, TTN, STK38, and TRIM21; 10, TRIM21; 21, MIBP1 and ubiquitin; 22, OGT, MIBP1, and ubiquitin; 23, GLDC, MIBP1, and S100A8; 24, MIBP1 and ubiquitin; 25, MIBP1, HRNR, DSG1, and DSP; 26, OGT, IPO8, CRYAA, ubiquitin, and MIBP1; 27, OGT, POLG2, MIBP1, and LIMA1; 28, CUL1 and MIBP1; and 29, OGT and PRMT5. Detailed data from the mass spectrometry analyses are shown in supplemental Table S14.

FIGURE 3.

Binding of MIBP1 with OGT. A, schematic diagram of full-length MIBP1 and its deletion mutants. The names of the respective expression plasmids are indicated in brackets. Black boxes indicate C₂H₂-type zinc fingers. The results of an OGT binding test using immunoblots are shown in the right column. B and C, lysates from HEK293 cells transfected with full-length MIBP1 or deletion mutants were immunoprecipitated (IP) by anti-FLAG M2 affinity gel (B) or anti-rat MIBP1 antibody (C) and immunoblotted (IB) with anti-OGT antibody, anti-FLAG antibody, or anti-MIBP1 antibody, respectively, as indicated in the figures. WCL, whole-cell lysates.

FIGURE 4.

154-amino acid region of MIBP1 required for OGT binding/O-GlcNAcylation. A–C, lysates from HEK293 cells transfected with full-length MIBP1, Δ154, or 154 were immunoprecipitated (IP) by anti-FLAG M2 affinity gel and immunoblotted (IB) with anti-OGT antibody, anti-FLAG antibody, or anti-O-GlcNAc antibody as indicated in the figures. The reactivity of anti-O-GlcNAc antibody was blocked by preincubating with 100 mm GlcNAc (B and C). WCL, whole-cell lysates.

FIGURE 5.

Transcriptional activity of MIBP1 and its modulation by OGT binding/O-GlcNAcylation. A, HEK293 cells were co-transfected with pGL4.23-NF-κBx5, pRL-TK, and full-length MIBP1 or the deletion mutants and incubated for 42 h. The cells were treated with the vehicle (left) or 10 ng/ml TNF-α (right) for 6 h before the cells were lysed. B, HEK293 cells were co-transfected with p3TP-LUX, pRL-TK, and full-length MIBP1 or the deletion mutants and cultured for 24 h with serum starvation. The cells were treated with the vehicle (left) or 400 pm TGF-β (right) for 24 h before the cells were lysed. In both A and B, luciferase expression was measured using the Dual-Luciferase Reporter Assay System. All experiments were performed in triplicate transfections. The values were normalized to Renilla luciferase expression and are presented as relative luciferase activity. The error bars indicate S.E. (n = 3). The effect of expression of MIBP1 or the deletion mutants on luciferase activities was evaluated by Welch's t test between the mock transfectant and the transfectant of each expression plasmid. Cells transfected with MIBP1 were also compared with cells transfected with Δ154. The asterisks indicate a p value less than 0.05.