Supplementary Figure 1

Recombinant proteins used for in vitro ubiquitination and methylation of nucleosomes.

(a) Coomassie-stained 4-12% polyacrylamide gel showing purified recombinant human ubiquitin activating enzyme UBE1 (lane1), human ubiquitin-conjugating enzyme UBCH5C (lane 2), mouse ubiquitin ligase Ring1b1-130/Bmi11-109 (lane 3) and Ubiquitin (lane 4). (b) Reconstituted recombinant Drosophila or Xenopus oligonucleosomes were subjected to ubiquitination (+ Ring1b1-

130/Bmi11-109, lane 2 in both cases) or mock (– Ring1b1-130/Bmi11-109, lane 1 in both cases) reactions and products were visualized by Coomassie staining after separation on denaturing 16% Tris-Glycine polyacrylamide gels (top) Note the size shift of the unmodified H2A band in lanes 1 and 3 to the monoubiquitinated H2A band in lanes 2 and 4, respectively; in both cases, a very small fraction appears to become di-ubiquitinated (H2Aub2). The band marked by an asterisk is a contaminant that sometimes is observed in UBE1 preparations. Bottom: Western blot analysis of the same reactions shown above with antibody against unmodified histone H2A. Reaction products were separated on 4-12% BT MES polyacrylamide gels. Note the size shift of the unmodified H2A band in lanes 1 and 3 to the monoubiquitinated H2A band in lanes 2 and 4, respectively.

Nature Structural and Molecular Biology: doi:10.1038/nsmb.2833

Supplementary Figure 2

Recombinant proteins and nucleosomes used for HMTase assays on mono- and oligonucleosomes.

(a) Coomassie stained polyacrylamide gels with purified recombinant PRC2 (left), AEBP2-PRC2 (middle) and JARID2 (right). Recombinant purified PRC2 consisting of EZH2, SUZ12, EED, RBBP4 (left). AEBP2-PRC2 additional contains AEBP2 (middle). These PRC2 complexes and JARID2 were used for the experiments shown in Fig. 2 and Figure S2. (b) Non-ubiquitinated and ubiquitinated mononucleosomes after coupling to streptavidin coated Dynabeads (M-280). (c) and (d) JARID2 and H2Aub stimulate H3-K27 methyltransferase activity of AEBP2-PRC2. (c) Western blot analysis of H3-K27me1 and -me3 formation in HMTase reactions performed with AEBP2-PRC2 (134 nM, lanes 3-6) in the absence (lanes 3, 4) or presence of JARID2 (120 nM, lanes 5,6) on recombinant Xenopus mononucleosomes (460 nM) that were unmodified (lanes 1, 3, 5) or contained H2Aub (lanes 2, 4, 6) (see Figure S1); histone H4 signal served as loading control. Histogram shows quantification of H3-K27me3 chemiluminescence signal by ImageJ; no signal is detected in lanes 1, 2 (asterisk). Note the increase of H3-K27me3 tri-methylation when H2A is monoubiquitinated and JARID2 is added to the reaction. The reduction of H3-K27me1 signal in lane 6 suggests that most H3-K27 residues became di- or tri-methylated. (d) as in (c) but with recombinant Xenopus 4-mer oligonucleosomes (460 nM) as substrate. The reaction contained 67 nM AEBP2-PRC2 and 60 nM JARID2. Supplementary Figure S5 shows original blots.

Nature Structural and Molecular Biology: doi:10.1038/nsmb.2833

Supplementary Figure 3

Original Coomassie gels for pulldown reactions.

Left: Full gel image of the triplicate pull-down reactions from Drosophila nuclear extracts. Area of the gel boxed in blue is shown in Figure 1a.

Right: Full gel image of the SILAC pull-down experiment from mouse embryonic stem cell nuclear extracts. Area of the gel boxed in blue is shown in Figure 1b.

Nature Structural and Molecular Biology: doi:10.1038/nsmb.2833

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Supplementary Figure 4

Full-size scans of western blot membranes shown in Figure 2.

Scans are arranged in the same order as shown in Figure 2a,b and cropped portions are boxed in blue.

Nature Structural and Molecular Biology: doi:10.1038/nsmb.2833

Supplementary Figure 5

Full-size scans of western blot membranes shown in Supplementary Figure 2.

Scans are arranged in the same order as shown in Figure S2c,d and cropped portions are boxed in blue.

 

Nature Structural and Molecular Biology: doi:10.1038/nsmb.2833