Wang et al. 1

Role of H2A Ubiquitination in Polycomb Silencing

Hengbin Wang, Liangjun Wang, Hediye Erdjument-Bromage,

Miguel Vidal, Paul Tempst, Richard S. Jones and Yi Zhang

Supplementary Material

Supplementary Methods

Purification and identification of histone H2A ubiquitin ligase complex

HeLa nuclear proteins were separated into nuclear extract and nuclear pellet as described 1. Nuclear pellet solubilization, fractionation on DEAE52, P11 and DEAE5PW columns were performed as described 2. Proteins bound to DEAE5PW column were eluted with 12-column volume (cv) linear gradient from 50 mM to 500 mM ammonium sulfate in buffer D [20 mM Tris-HCl (pH 7.9), 0.1 mM EDTA, 2 mM DTT, 0.2 mM PMSF, and 10% glycerol]. Fractions containing the ubiquitin ligase activity for H2A were eluted out of the column between 70-150 mM ammonium sulfate. Active fractions were then combined and adjusted to 500 mM ammonium sulfate with saturated ammonium sulfate before loading onto a 22 ml Phenyl Sepharose column (Phamacia). The Phenyl Sepharose column was eluted with 20 cv linear gradient from 500mM to 0 mM ammonium sulfate in buffer D. The H2A ubiquitin ligase activity was eluted out between 345-240 mM ammonium sulfate. Active fractions were pooled and concentrated to 5 ml before loading to a 120 ml Sepharcy 300 gel filtration column (Phamacia). The ubiquitin ligase activity was eluted out between 220-443 kDa. Active fractions were combined and dialyzed against buffer P [5 mM HEPES-KOH (pH 7.5), 40 mM KCl, 0.01% Triton X-100, 0.01 mM CaCl2, 0.5 mM PMSF, 1 mM DTT, and 10% glycerol] containing 10 mM potassium phosphate (BP10) and loaded to a 5 ml hydroxyapatite column (Bio-Rad). The bound proteins were eluted with 20 cv linear gradient from BP10 to BP600. The H2A ubiquitin ligase activity was eluted out of the column between 340-550 mM potassium phosphate. Active fractions were combined and loaded to a 1 ml MonoQ column (Phamacia) after dialysis against buffer C [40 mM HEPES-KOH (pH 7.9), 0.1 mM EDTA, 2 mM DTT, 0.2 mM PMSF, and 10% glycerol] containing 150 mM KCl. Bound proteins were eluted with 20cv linear gradient from 150 mM to 500 mM KCl in buffer C. The H2A ubiquity ligase activity was eluted out between 220-320 mM KCl. To identify the proteins that coelute with the H2A ubiquitin ligase activity, active fractions between 29-32 were combined, dialyzed to BC50 and bound to a 200 l P11 column before being eluted with BC600. The eluted proteins were resolved in 8-15% gradient SDS-PAGE. After Coomassie staining and destaining, candidate polypeptides were excised and subjected to trypsin digestion and a combination of peptide mass fingerprinting using MALDI-TOF MS and MS sequencing using MALDI-TOF/TOF MS/MS3,4.

For immunopurification of Ring1 complex, affinity purified Ring1 antibodies 5 were cross-linked to protein A agarose beads as described 6 and were incubated at 4oC for 4 hrs with an aliquot of Hydroxyapatite input dialyzed against BC50. After washing with BC500 for 3 times and BC50 for two times, beads were divided into four aliquots for ubiquitin ligase assays, Western blotting, silver staining, and protein identification (5 times of other aliquots). Protein identification was performed as described above. Parallel experiments with rabbit IgG was also performed.

Generation and characterization of Ring2 knock-down cell lines

Ring2 knock-down cell lines were generated using vector-based knock-down strategy previously described 7. Briefly, vectors expressing the following hairpin RNAs were transfected into HeLa cells by Effectene (Invitrogen). Transfected cells were selected in the presence of 2 g/ml puromycin. Selected clones were amplified and the efficiency of Ring2 knock-down as well as its effect on H2A ubiquitination were analyzed. Total cell lysates used for Ring2 Western was prepared as described 7. To prepare samples for evaluation of H2A ubiquitination, cells from a 100 mm plate were collected, washed with cold PBS twice before being suspended in 200 l PBS and 2 N HCl was added to a final concentration of 0.2 N. The suspension was kept on ice for 30 min and centrifuged for 10 min at 13,000 rpm. The supernatant was mixed with 200 l 25% TCA and kept on ice for another 30 min. The precipitated proteins were collected and washed with acetone. Proteins were then dissolved in SDS loading buffer and analyzed by Western blotting. The two siRNAs that target Ring2 are: 5’-AGAACACCATGACTACAAATTCAAGAGATTTGTAGTCATGGTGTTCT-3’ and 5’-GGCTAGAGCTTGATAATAATTCAAGAGATTATTATCAAGCTCTAGCC-3’. Knock-down cell line 1 was established from the first set of siRNA targeting amino acid 58-65, while knock-down cell line 2 was established from the second set of siRNA targeting amino acid 204-210. The effect of Ring2 knock down on cell growth was analyzed as previously described 8.

Extraction of ubiquitinated histones from Drosophila SL2 cells

Ubiquitinated histone from S2 cells was extracted as previously described with minor modifications 9. Briefly, 50 ml of S2 cells (6-8 x 106 cells/ml) was collected and lysed in 5 ml buffer A [0.25 M sucrose, 10 mM sodium HEPES (pH 7.5), 3 mM CaCl2, 10 mM NaCl, 1 mM PMSF, 1 mM DTT, 0.25% Nonidet 40] on ice for 30min. The lysate was then centrifuged at 3000 rpm for 10 min to pellet the nuclei. The nuclei were then washed by buffer A one more time before being suspended in 800 l buffer B [0.25 M sucrose, 10 mM sodium HEPES (pH 7.5), 3 mM CaCl2, 10 mM NaCl, 1 mM PMSF, 1 mM DTT]. 2 N HCl was then added to a final concentration of 0.2 N. The suspension was extracted at 4oC overnight and was then centrifuged for 10 min at 13,000 rpm. The supernatant was mixed with equal volume of 50% TCA and kept on ice for 1 hr. The precipitated proteins were collected and washed with acetone. Proteins were then dissolved in SDS loading buffer and analyzed by Western blotting shown in Fig. S1.

Supplementary references

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