Total RNA was prepared from soybean (Glycine max L. Merr. cv. Union) and reverse-transcribed to make the cDNA samples for molecular cloning as reported previously . Gene specific primers for making cloning and the details of PCR settings were given in Additional File 9, Table S3. For sub-cloning into expression vectors, a BamHI (GGATCC) and a SalI (GTCGAC) site were added to the 5'-ends of forward and reverse primers, respectively.
The PCR products were separated on 1% agarose gels, purified, digested with specific restriction enzymes BamHI or SalI (New England Biolabs), and sub-cloned into the expression plasmid vectors (GST: pGEX-4T-1, GE healthcare, Wisconsin, USA, Product number 28-9545-49; MBP: pMAL-C2, New England Biolabs, MA, USA) pre-digested with the same restriction enzymes. GmPHD5 was inserted into the GST expression vectors, while other cloned genes, GmISWI1, GmISWI2, GmGNAT, and GmElongin, were ligated into the MBP expression vector. All clones were confirmed by sequencing using the ABI PRISM™ dRhodamine Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer, Connecticut, USA; Product number: 402078) as described in the manufacturer's manual.
Expression of recombinant proteins
Recombinant plasmids containing the target clones were transformed into the bacteria strain DE3. The transformed bacteria were inoculated into Luria-Bertani (LB) broth supplemented with 100 μg/ml of ampicillin and incubated at 37°C for 2.5-3 h until the optical density at 600 nm reached about 0.6-0.8. IPTG was then added to reach a final concentration of 1 mmol/L to induce the expression of the recombinant proteins at 25°C. After overnight expression, the bacteria were collected, suspended in phosphate buffered saline (PBS) and lysed with 1 mg/ml lysosome on ice for at least 1 h. The supernatant was collected after centrifuged at 4°C for 15 min at 21,500 g and stored at -80°C until use.
Peptide synthesis and antibody production
Peptides (GmPHD5: GKNERKRLFQMINDLPT, residue 116 to residue 132 and TPAKAEHIKQYK, residue 230 to residue 241; GmISWI: GEEATAELDAKMKKFTEDAIK, residue 596 to residue 616) were synthesized using the standard procedures of the F-moc solid-phase peptide synthesis protocol of the Applied Biosystems 433A solid-phase peptide synthesizer. The synthesized peptides were dissolved in milli-Q water, purified by standard reversed-phase HPLC and the homogeneity of the purified peptides was determined by MALDI-TOF mass spectrometry on an ABI 4700 proteomics analyzer.
Purified peptides were conjugated to KLH (Keyhole Limpet Hemocyanin; Sigma, Missouri, USA; Product number: H8283) as described in our previous work . An equal amount of complete Freunds adjuvant (Sigma, Missouri, USA; Product number: F-5881) was mixed with purified peptide-KLH solution (contain about 100 μg peptide) and emulsified manually. Six to eight-week-old rabbits were immunized with these emulsions subcutaneously. After the priming immunization, rabbits were given a booster with 100 μg antigen emulsified in incomplete Freunds adjuvant (Sigma, Missouri, USA; Product number: F-5881) (1:1) three times in two-week intervals. Finally, the serum was collected and tested by western blotting. Control serum was collected before the priming immunization. All the rabbits were raised in the animal centre of The Chinese University of Hong Kong according to animal ethics.
Nucleic protein extraction
Soybean leaf tissue was ground into powder in liquid nitrogen, and suspended in nuclei isolation buffer (NIB) containing 20 mM Tris-HCl (pH 7.5), 10 mM KCl, 10 mM MgCl2, 6% sucrose, 0.6% Triton X-100, 0.05% β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), as described previously  with some modifications. After homogenization on ice, the tissue was passed through filter paper (pore size 30 μm). The resulting nuclei fraction was harvested by centrifugation at 4000 g for 10 min, and then washed twice with NIB.
Isolated nuclei were swelled in low salt buffer (20 mM Tris-HCl, pH 7.6, 10 mM KCl, 2.5 mM MgCl2, 2 mM DTT and 0.5 mM PMSF), and total nuclear proteins were then extracted using high salt extraction buffer (500 mM NaCl, 25% glycerol in low salt buffer) . The concentration of the NaCl in the extracted protein solution was diluted to 250 mM before use.
Histone protein extraction
Nuclei isolation method followed the above nucleic protein extraction protocol . The white nuclei were re-suspended in 40% guanidine hydrochloride. Then the core histones were extracted by 0.4 M HCl followed by centrifuging at 12000 g for 10 min. Finally, the core histones in the supernatant dried upon the speed vacuum system.
Interacting between GmPHD5 and other nuclear proteins
The GST-PHD5 fusion protein was first bound to the GST column (GE healthcare, Wisconsin, USA; Product number: 17-0756-01) by incubating the protein with GST agarose beads at room temperature for 30 min. Selected nucleic proteins were then applied to the beads and incubated at 4°C overnight. The beads were washed 10 times with wash buffer (25 mM Tris-HCl (pH 8.0), 10% glycerol, 1 mM EDTA, 200 mM NaCl, 1 mM PMSF, 1 mM DTT, 0.1% Triton X-100) and subsequently boiled with SDS-PAGE gel loading buffer at 99°C for 10 min before gel separation. SDS-PAGE gels were stained with silver  and the target protein bands were excised, destained, and digested before subjected to MALDI-TOF/TOF for identification .
For the in vitro protein-protein interaction studies, the GST-PHD5 fusion protein was independently incubated with MBP-ISWI, MBP-ISWI2, MBP-GNAT or MBP-elongin in the GST column at 4°C overnight. Each of the equilibrated column was washed twice with the buffer containing 25 mM Tris (pH 8.0), 10% glycerol, 1 mM EDTA, 500 mM NaCl, 1 mM PMSF, 1 mM DTT, 1% Triton X-100, followed by an additional six washes with buffer containing 25 mM Tris (pH 8.0), 10% glycerol, 1 mM EDTA, 150 mM NaCl, 1 mM PMSF, 1 mM DTT, 0.1% Triton X-100. Finally, the beads from each column were separately recovered and boiled with SDS page gel loading buffer at 99°C for 10 min. Western blotting was followed as described  using anti-MBP antibody.
Co-immunoprecipitation (co-IP) and peptide pull down assays
Co-immuno-precipitation (co-IP) assays were carried out using specific antibodies raised against the targeted proteins. Antibody against the biat protein was used for immuno-precipitating the complexes from the nuclear protein extracts, and the second antibody was used to detect the interacting partner via western blotting assay.
For peptide pull down assay, biotin-conjugated peptides containing H3K4me, H3K4me2 or H3K4meme3 were purchased [Millipore, Massachusetts, USA; Catalogue number: 12-563 (mono-), 12-460(di-), and 12-564(tri-)]. Biotin conjugated peptides containing H3K9 trimethylation (Millipore, Massachusetts, USA; Catalogue number: 12-568) were used as the control. The peptides were immobilized onto avidin agarose beads (Pierce, Illinois, USA; Product number: 20219). Recombinant analogs of GST-GmPHD5 was incubated with these beads at 4°C overnight. The beads were then washed twice with buffer containing 25 mM Tris buffer (pH 8.0), 10% glycerol, 1 mM EDTA, 500 mM NaCl, 1 mM PMSF, 1 mM DTT, 1% Triton X-100, followed by another six washes with buffer containing 25 mM Tris buffer (pH 8.0), 10% glycerol, 1 mM EDTA, 150 mM NaCl, 1 mM PMSF, 1 mM DTT, 0.1% Triton X-100. Finally, the beads were boiled in SDS-PAGE gel loading buffer at 99°C for 10 min and western blotting was followed using anti-GST antibody (Sigma, Missouri, USA; Product number: G7781).
Chromatin immuno-precipitation (ChIP) assays
ChIP assays were performed using the chromatin immuno-precipitation assay kit (Millipore, Massachusetts, USA; Catalogue Number: 17-295), following the instruction in the user manual. Soybean leaves were first fixed in 1% formaldehyde for 15 min. The fixation was terminated by adding glycine to a final concentration of 125 mM. Nuclei were then extracted from the fixed leaves and re-suspended in SDS lysis buffer and incubated for 10 min on ice. The lysates were sonicated to shear the genome DNA to lengths between 200-1000 bp. Thereafter, the samples were centrifuged at 21,500 g for 10 min at 4°C. The collected supernatants were then diluted 10 fold with ChIP dilution buffer and 1% of these collected supernatants were aliquoted as input samples. The rest supernatants were subsequently pre-cleared with protein A agarose/salmon sperm DNA (50% slurry) with agitation for 1 h at 4°C. Immuno-precipitating antibody was then added and the mixture was incubated overnight with rotation at 4°C. Subsequently, protein A agarose/salmon sperm DNA (50% slurry) bead was used to precipitate the antibody/protein/DNA complexes. Then, the bead/antibody/protein/DNA complexes were washed with low salt wash buffer, high salt wash buffer, LiCl wash buffer and TE buffer sequentially. Bound protein/DNA complexes were then eluted from the beads with freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3). To reverse the protein-DNA crosslinks, 5 M NaCl was applied to the eluted samples to a final concentration of 200 mM and heated at 65°C for over 4 h. The total DNA was finally recovered from the samples by phenol/chloroform extraction and ethanol precipitation.
ChIP-PCR reactions were set up as follows: 4 ul template (~ < 0.1 nmol) was mixed with 0.4 ul dNTP (10 mM), 0.4 ul forward primer (10 uM), 0.4 ul reverse primer (10 uM), 2 ul 10 × PCR buffer, 0.25 ul Taq polymerase (Promega, Wisconsin, USA), and 1 ul MgCl2 (25 mM). The final volume was adjusted to 20 ul by distilled milli-Q water. Information on primers and PCR settings were summarized in Additional File 8, Table S2. The PCR products were resolved on a 2% agarose gel.
In vitro acetyltransferase activity assay
The MBP-GNAT recombinant protein was mixed with 125 μM Acetyl-Coenzyme A (GE healthcare, Wisconsin, USA; Product number: 27-6200-01), 60 μg histone extracted from soybean (or the other tested proteins), 1.5 mM DTT, 10% glycerol, 0.15 mM EDTA, 15 mM sodium butyl, 15 mM nicotiamide, 1 mM PMSF, 1 mM protease inhibitor, and then incubated overnight at 30°C. The reaction was then concentrated and protein acetylation was determined by western blotting with an anti-acetyl-K antibody (Millipore, Massachusetts, USA; Catalogue number: 05-515).