Extended Data Fig. 10: Regulation of chromatin and transcription by APC/C–WDR5.

a, Comparison of genes co-occupied by Flag–CDC20 and Flag–WDR5 from mitotic HEK293T cells with known gene-expression profiles reveals a strong overlap with ES cell and medulloblastoma cancer cell lines. n = 1,628 genes were analysed (P values represent a one-sided Fisher’s exact test with Bonferroni correction). b, Loss of APC/C–WDR5 function interferes with the expression of genes marked with K11-linked ubiquitin chains in H1 human ES cells. Poly(A)-selected RNA was purified from asynchronous H1 human ES cells transfected with control siRNA or siRNA against WDR5 for 48 h, and subjected to RNA-sequencing analysis (a biological replicate of Fig. 4h). c, Transcript analysis of WDR5 depletion on APC/C–WDR5-dependent genes (from Fig. 4h and b). Box plots include the median TPM value (n = 90 genes) with quartile ranges Q1–Q3; top whiskers represent the 3rd quartile + 1.5× interquartile range; bottom whiskers represent the 1st quartile − 1.5× interquartile range. P values were calculated from comparing individual TPM values of APC/C–WDR5-regulated genes (n = 90) versus all transcripts (n = 18,791) using a two-sided Student’s t-test (unpaired). d, Real-time qPCR analysis of nascent RNA reveals APC/C–WDR5 target genes are reactivated upon mitotic exit and gene reactivation is dependent on WDR5. Initial screening from a single experiment. e, The RNA levels of genes regulated by APC/C–WDR5 do not change upon mitotic exit. RNA-sequencing analysis was performed on poly(A)-selected RNA purified from H1 human ES cells at the indicated cell-cycle stages. Box plots were derived as described in c (n = 90 genes). f, Ubiquitylated H2B preferentially associates with p97–UBXN7 in vitro. H2B was preubiquitylated by APC/C in vitro, and incubated with immobilized p97 or p97–UBXN7 complexes. Bound histone H2B was detected by western blotting. This experiment was performed three independent times with similar results. g, Flag–UBXN7 associates with polyubiquitylated H2B, p97 and K11/K48-linked branched ubiquitin chains in mitosis. Native Flag–UBXN7 immunoprecipitations were performed on mitotic HEK293T cells and bound proteins were detected by western blotting or Ponceau staining. This experiment was performed three independent times with similar results. h, H2B ubiquitylation is stabilized by p97 inhibition in cells. Denaturing K11/K48 immunoprecipitations were performed on H1 human ES cells synchronized in prometaphase or released into 10 μM NMS-873 for 2 h. This experiment was performed four independent times with similar results. i, p97 inhibition restores K11 deposition at sites regulated by APC/C–WDR5 upon mitotic exit. Anti-K11 MNase ChIP–seq was performed from H1 human ES cells synchronized in mitosis (0 h) or released into fresh medium without (2 h + DMSO) or with p97 inhibition (2 h + 10 μM NMS-873). j, Anti-K11 MNase ChIP–qPCR of candidate targets from mitotic H1 human ES cells (mean of n = 3 independent replicates ± s.d.). H1 human ES cells were synchronized in mitosis (0 h) and released into fresh medium for 2 h with the indicated drugs. k, Model of APC/C-dependent gene activation upon mitotic exit.