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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
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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 MALDITOF MS and MS sequencing using MALDI-TOF/TOF MS/MS 3,4.
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-
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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.
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