Ubch5c

H2A ubiquitylation assays were carried out as previously (Rose et al., 2016 ). Briefly, UBE1 (Boston Biochem), UbcH5c (Enzo), methylated ubiquitin (Boston Biochem) and ATP (Life technologies) were pre-incubated for 20 min at 37°C prior to addition of reconstituted PRC1 and nucleosomes. Reactions were allowed to proceed for 1 h at 37°C then quenched with 30 mM EDTA and subject to SDS-PAGE for western blot analysis. Western blots were probed with antibodies which recognize Histone H2A in both ubiquitylated and unmodified form (Millipore 07-146) and Histone H3 (CST, 96C10), followed by incubation with LiCOR IRDye secondary antibodies (800CW goat anti-rabbit and 680RD goat anti-mouse). Western blots were imaged using the LiCOR Odyssey Fc imaging system and band intensities were quantified using ImageStudio. H2A band intensities were normalized to H3 and the fraction of ubiquitylated H2A relative to total H2A was quantified. Data were visualized and dose-response curves fitted using GraphPad Prism 7.

For in vitro ubiquitination assay, FLAG-tagged plasmid of each CRL4 components (CUL4A, CUL4B, DDB1), DCAFs (RepID FL and its mutants or RBBP7), and BUB3 was transfected to HCT116 cells using Lipofectamine 2000 (Invitrogen) and purified using FLAG-Sepharose beads and FLAG-peptide (Sigma, A2220, F4799). Purified proteins were mixed with 1 μg ubiquitin (Sigma, U6253), 60 ng human recombinant E1, 300 ng UbcH5c, Mg-ATP solution, and ubiquitination buffer provided from Enzo Life Science (BML-UW9920-0001). After 60-min incubation at 30 °C, the reaction was denatured by adding SDS-containing loading buffer, boiled at 100 °C for 5 min, separated by SDS-PAGE, transferred to a PVDF membrane, and detected ubiquitinated BUB3 with anti-BUB3 antibody. For in vitro binding assay, purified proteins as indicated in the figure were mixed in lysis buffer and BUB3 was precipitated using anti-BUB3 antibody. Co-precipitated proteins were detected with anti-FLAG antibody. All experiments report representative results of at least three independent repetitions.