a, APC/C–WDR5 ubiquitylates human H3 in H3–H4 tetramers in vitro. APC/C was affinity-purified from mitotic HeLa cells and incubated with E1, UBE2C, UBE2S, ubiquitin and human H3–H4 tetramers, as indicated. Reaction products were detected by western blotting using antibodies against H3 and H4. This experiment was performed once. b, APC/C ubiquitylation of H2B in X. laevis H2A–H2B histone dimers. APC/C–WDR5 was purified from mitotic HeLa cells by Flag–WDR5 affinity purification and incubated with E1, UBE2C, UBE2S, ubiquitin and X. laevis histone octamers. Ubiquitylation was detected by western blotting against ubiquitylated H2B. This experiment was performed three independent times with similar results. c, APC/C–WDR5 ubiquitylation of H2B in X. laevis H2A–H2B–H3–H4 histone octamers. Reactions were performed as described in b. This experiment was performed two independent times with similar results. d, APC/C purified from H1 human ES cells is competent to ubiquitylate human H2B. This experiment was performed two independent times with similar results. e, APC/C purified from mitotic, but not S-phase, extracts can ubiquitylate H2B in vitro. APC/C was purified from HeLa cells synchronized at the indicated cell-cycle stages and incubated with E1, UBE2C, UBE2S, ubiquitin and X. laevis H2A–H2B dimers. Histone ubiquitylation was detected by western blotting using antibodies against ubiquitylated H2B. This experiment was performed once. f, APC/C-dependent ubiquitylation of H2B requires K11 residue on ubiquitin for chain elongation. Ubiquitylation of H2A–H2B dimers by APC/C–WDR5 was performed as described in e, but with ubiquitin variants. This experiment was performed once. g, APC/C-dependent ubiquitylation of H2B requires both K11 and K48 on ubiquitin for synthesis of branched chains. This experiment was performed two independent times with similar results. h, Securin, a canonical APC/C substrate, outcompetes H2A–H2B dimers for APC/C-dependent ubiquitylation. The D-box motif (an APC/C–CDC20-specific degron) is required for full competition, whereas the KEN motif (an APC/C–CDH1-specific degron) is not. This experiment was performed two independent times with similar results. i, Polyubiquitylated H2B is degraded by the proteasome. K11/K48-branched chains were purified under denaturing conditions from mitotic HeLa cells either in the presence or absence of MG132, and modified H2B was detected using western blotting. Proteasome inhibition with MG132 was found to stabilize endogenous polyubiquitylated H2B. This experiment was performed four independent times with similar results.