Extended Data Figure 10 ∣. Chromatin- and transcription-regulation by APC/C^WDR5.

a, Comparison of genes co-occupied by ^FLAGCDC20 and ^FLAGWDR5 from mitotic HEK 293T cells with known gene expression profiles reveals strong overlap with embryonic stem cell and medulloblastoma cancer cell lines. n=1628 genes were analyzed (p-values represent a one-sided, Fisher’s exact test, Bonferroni correction).

b, Loss of APC/C^WDR5 function interferes with the expression of genes marked with K11-linked ubiquitin chains in H1 hESCs. Poly(A)-selected RNA was purified from asynchronous H1 hESCs transfected with siCTRL or siWDR5 for 48 h and subjected to RNAseq analysis (a biological replicate of Fig. 4h).

c, Transcript analysis of WDR5 depletion on APC^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=Q3+1.5*IQR, bottom whiskers=Q1−1.5*IQR). P-values were calculated from comparing individual TPM values of APC/C^WDR5-regulated genes (n=90) versus all transcripts (n=18791) using a two-sided, Student’s t-test (unpaired).

c, RT-qPCR analysis of nascent RNA reveals APC/C^WDR5 target genes are re-activated upon mitotic exit and is dependent on WDR5. Initial screening from a single experiment.

d, RNA levels of genes regulated by APC/C^WDR5 do not change upon mitotic exit. RNAseq analysis was performed on poly(A)-selected RNA purified from H1 hESCs at the indicated cell cycle stages. Box plots were derived as described in c (n=90 genes).

f, Ubiquitylated H2B preferentially associates with p97/VCP^UBXN7 in vitro. H2B was pre-ubiquitylated by APC/C in vitro, and incubated with immobilized p97/VCP or p97/VCP^UBXN7 complexes. Bound histone H2B was detected by Western blotting. This experiment was performed three independent times with similar results.

g, ^FLAGUBXN7 associates with polyubiquitylated H2B, p97/VCP, and K11/K48-linked branched ubiquitin chains in mitosis. Native ^FLAGUBXN7 IPs were performed on mitotic HEK 293T cells and bound proteins were detected by Western blotting or Ponceau. This experiment was performed three independent times with similar results.

h, H2B ubiquitylation is stabilized by p97/VCP inhibition in cells. Denaturing K11/K48 IPs were performed on H1 hESCs synchronized in prometaphase or released into 10 μM NMS-873 for 2h. This experiment was performed four independent times with similar results.

i, p97/VCP inhibition restores K11 deposition at sites regulated by APC/C^WDR5 upon mitotic exit. αK11-MNChIPseq was performed from H1 hESCs 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, αK11-MNChIP-qPCR of candidate targets from mitotic H1 hESCs (mean of n=3 independent replicates ± SD). H1 hESCs were synchronized in mitosis (0 h) and released into fresh medium for 2 h with indicated drugs.

k, Model of APC/C-dependent gene activation upon mitotic exit.