The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

The tumour-suppressor gene CDKN1A (encoding p21Waf/Cip1) is thought to be epigenetically repressed in cancer cells. FBI-1 (ZBTB7A) is a proto-oncogenic transcription factor repressing the alternative reading frame and p21WAF/CDKN1A genes of the p53 pathway. FBI-1 interacts directly with MBD3 (methyl-CpG–binding domain protein 3) in the nucleus. We demonstrated that FBI-1 binds both non-methylated and methylated DNA and that MBD3 is recruited to the CDKN1A promoter through its interaction with FBI-1, where it enhances transcriptional repression by FBI-1. FBI-1 also interacts with the co-repressors nuclear receptor corepressor (NCoR), silencing mediator for retinoid and thyroid receptors (SMRT) and BCL-6 corepressor (BCoR) to repress transcription. MBD3 regulates a molecular interaction between the co-repressor and FBI-1. MBD3 decreases the interaction between FBI-1 and NCoR/SMRT but increases the interaction between FBI-1 and BCoR. Because MBD3 is a subunit of the Mi-2 autoantigen (Mi-2)/nucleosome remodelling and histone deacetylase (NuRD)-HDAC complex, FBI-1 recruits the Mi-2/NuRD-HDAC complex via MBD3. BCoR interacts with the Mi-2/NuRD-HDAC complex, DNMTs and HP1. MBD3 and BCoR play a significant role in the recruitment of the Mi-2/NuRD-HDAC complex– and the NuRD complex–associated proteins, DNMTs and HP. By recruiting DNMTs and HP1, Mi-2/NuRD-HDAC complex appears to play key roles in epigenetic repression of CDKN1A by DNA methylation.

To prepare stable cell, FLAG tagged FBI-1 in the pcDNA5/FRT/TO expression vector and pOG44 were co-transfected into the Flp-In TM T-RE X TM 293T cells (Invitrogen, Carlsbad, CA). T-RE X -FBI-1 cells were selected by blasticidin and hygromycin and FBI-1 expression was induced by doxycycline.
To isolate nuclear protein complexes that interact with FBI-1 from doxycycline inducible stable HEK293-TREx-FLAG FBI-1 cells, FBI-1 expression was induced overnight and the nuclei were separated by glycerol gradient centrifugation.

GST fusion protein purification, in vitro transcription and translation
GST, GST-POZFBI-1, and GST-ZFFBI-1 proteins were prepared from E. coli BL21 (DE3) transformed with the pGEX4T series GST, GST-POZFBI-1, and GST-ZFFBI-1 protein expression plasmids. E. coli cultures were induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) for 4 h at 37°C. The cells were lysed in a buffer containing 1× PBS, 1 mM PMSF, 2 mM EDTA, and 0.2 mg/ml lysozyme, and sonicated 3-5 times at 0.5 cycle and 50% amplitude (Dr. Hielscher GmbH, Germany) to prepare lysates. The recombinant proteins were purified with glutathione-agarose 4 beads by affinity chromatography (Peptron, Daejeon, Korea). The purified proteins were resolved with 12% SDS-PAGE to quantitate and assess purity. Aliquots of the protein-agarose bead complex were used in GST-fusion protein pull down assays. Polypeptide expression level was analysed by running 3 μl of the total mixture on a 10% SDS-PAGE.

GST fusion protein pull-down assays
The purified GST fusion proteins (5 μg) were incubated with GSH-agarose (Sigma, St. Louis, MO) for 1 h in HEMG buffer [40 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 5 mM MgCl 2 , 0.1% Nonidet P-40, 10% glycerol, 1.5 mM dithiothreitol, and 1 tablet/50 ml of a protease inhibitor mixture (Roche, Mannheim, Germany)] at 4°C for 1 h. After the agarose-GST protein complexes were washed -4 -three times with 1 ml of cold HEMG buffer, 10 μl of the in vitro translated MBD3 was added and incubated in HEMG buffer at 4°C for 4 h. The reaction mixtures were centrifuged at 3,000 × g at 4°C, the supernatants were removed, and the pellets were washed five times with cold HEMG buffer. The bound proteins were separated by a 10% SDS-PAGE. The SDS-PAGE gel was dried and exposed to X-ray film using an image-intensifying screen (Kodak, Rochester, NY).

Immunofluorescence assay
HeLa cells grown on coverslips ( HKMG buffer, and precipitated by centrifugation. The precipitates were separated by SDS-PAGE and was analyzed by Western blotting assay using anti-FBI-1, anti-MBD3 and anti-GAPDH antibodies.

EMSA (electro-mobility shift assay)
EMSAs were carried out as described previously (1). Oligonucleotide sequences used are as follows; with 4% non-denaturing PAGE at room temperature in 0.5X TBE (89mM TBE, pH 8.3) at 150 V for 2 h. The gels were exposed to X-ray film with a Kodak intensifying screen.

Mammalian two-hybrid reporter assays
Monkey kidney cells (CV-1) were grown in DMEM supplemented with 10% FBS (Invitrogen, Carlsbad, CA). The cells were inoculated into 6-well culture dishes at a density of 5×10 5 cells/well.  Table I.

Transcriptional analysis of the CDKN1A-Luc promoter
Two CDKN1A-Luc promoter reporter fusion plasmids (with -2.4 kb or -133 bp upstream regulatory sequence), pcDNA3-FBI-1 and pCMV-LacZ, were transiently transfected into HEK293 cells using Lipofectamine Plus reagent. After 48 h of incubation, cells were harvested and analysed for luciferase activity. The assays were repeated 3 times. The reporter activity was normalised with co-transfected βgalactosidase activity or protein concentration.

Two-dimensional gel electrophoresis
Isoelectric focusing (IEF) was performed using pre-cast immobilised pH gradient strips (24 cm, pH 3-10, linear, Amersham Biosciences, Uppsala, Sweden). 500 μg of proteins were solubilised in rehydration buffer (9 M urea, 2% CHAPS, 60 mM DTT, 0.5% pharmalyte, pH 3-10, 0.002% bromophenol blue) and protein samples were loaded on IPG strips and rehydrated overnight. The IEF gel was run for a total of 36 kVh during which the voltage was increased linearly from 100 to 8000 V over 6 h and then, maintained for 3 h at 8000 V. After IEF, strips were first equilibrated for 15 min in a reducing solution (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 1% (w/v) DTT), and then for a further 15 min in an alkylating solution, which was identical in make-up to the reducing solution except that 2.5% (w/v) iodoacetamide was substituted for DTT. A second gel electrophoresis was performed using a standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protocol, using the Ettan DALT 6 System (Amersham Biosciences, Uppsala, Sweden). SDS-PAGE was run on 12% polyacrylamide gel, and gels were visualised by silver staining.

In-gel digestion and mass spectrometry
Sliver-stained gel spots were excised, destained by reduction using a solution of 30 mM potassium  cells. Nuclear lysates were immunoprecipitated using the anti-FLAG M2-Agarose antibody and the immunoprecipitates were separated by 2D IEF (pH 3-10)/12% SDS-PAGE and stained with a silver staining solution. The spots that showed differential expression were isolated, digested with