Fig 2: SETD5 is co-recruited to primed enhancers with NCoR-HDAC3 complex and diminishes prior to enhancer activation.a Venn diagram (left) and heatmap (right) representation of genomic regions occupied by SETD5-V5, NCoR, and HDAC3. Approximately 60% of NCoR and 71% of HDAC3 genomic binding regions were co-occupied by SETD5-V5. Heatmap analysis showing ChIP-seq data for SETD5-V5, NCoR, and HDAC3 at a 20-kb region centered on SETD5-V5 binding region. b Genome browser representation for SETD5-V5, NCoR, HDAC3, and H3K27ac on Cebpa and Pparg genes in 3T3-L1 preadipocytes. Tracks 1-2 and 7-9 are the same data as Fig. 4c. c, d ChIP-qPCR analyses of NCoR2 (c) and HDAC3 (d) on Cebpa and Pparg genes during the early adipogenesis. 3T3-L1 preadipocytes transduced with control empty virus were subjected to ChIP-qPCR analyses using anti-NCoR2 or anti-HDAC3 antibody. Data are mean ± SEM (0, 48 h n = 5; 6 h of NCoR2 n = 4; 6 h of HDAC3 n = 5; 12, 24 h n = 3; independent experiments). e ChIP-qPCR analysis of SETD5-V5 on Cebpa and Pparg genes under knockdown of ANAPC11 or CDC20. 3T3-L1 preadipocytes transduced with SETD5-V5 were transfected with control siRNA or siRNA targeting to Anapc11 or Cdc20 and subjected to ChIP-qPCR analysis at 6 h and 24 h of differentiation using anti-V5 antibody. ChIP signal was presented as relative SETD5 recruitment. Data are mean ± SD of four technical replicates. f Schematic model of SETD5-NCoR-HDAC3 complex formation and the loss of SETD5 on enhancers during the transition from primed to active states. Heatmap shows the degree of recruitment of SETD5, NCoR2, and HDAC3 to Cebpa and Pparg enhancers (based on Figs. 4d, 5c, and 5d). Darker color indicates more recruitment. a, b ChIP-seq for NCoR and HDAC3 was from the previously published paper32. c–e One-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01. Source data are provided as a Source data file.

Fig 3: Increased expression of CDC20 in cSCC tumors and cell lines. (A) Bioinformatics analysis results for GSE32628 and GSE45216. *P<0.05; ***P<0.001. (B) Immunohistochemical analysis of CDC20 in pairs of tumor (left) and adjacent normal tissue (right) samples from patients with cSCC (magnification, ×100). (C) Semiquantitative analysis of CDC20 staining in 21 paired samples. ***P<0.001 vs. tumor. (D) Western blot analysis of CDC20 protein expression in primary NHEK cells and three cSCC cell lines: A431, SCC13 and SCL-1. CDC20, cell division cycle 20; cSCC, cutaneous squamous cell carcinoma; NHEK, normal human epidermal keratinocytes; AK, actinic keratosis; wd, well-differentiated; md/pd, moderately or poorly differentiated.

Fig 4: CDC20 interacts with and destabilizes CDKN1B.A The interaction of endogenous CDC20 with CDKN1B was evaluated by Co-IP assay in U87 cells. B The interaction of exogenous CDC20 with CDKN1B was assessed by Co-IP assay in HEK293T cells. C U87 cells with or without CDC20 depletion were transfected with His-Ubi and CDKN1B-FLAG plasmids and then were treated with MG132 (20 μM) for 6 h. Cell lysates were immunoprecipitated with an anti-FLAG antibody and CDKN1B ubiquitination was detected by immunoblotting. D HEK293T cells with or without CDC20 overexpression were transfected with His-Ubi and CDKN1B-FLAG plasmids and then were treated with MG132 (20 μM) for 6 h. Cell lysates were immunoprecipitated with an anti-FLAG antibody and CDKN1B ubiquitination was examined by immunoblotting. E HEK293T cells were transfected with CDC20-HA WT or CDC20-HA-N1-171 or CDC20-HA-WD40, CDKN1B-FLAG, and His-Ubi expressing plasmids, and then were treated with MG132 (20 μM) for 6 h. Cell lysates were immunoprecipitated with an anti-FLAG antibody and CDKN1B ubiquitination was determined by immunoblotting.

Fig 5: Effects of the mutations on CDC20 transcript, CDC20 and cyclin B1 protein levels. (A) Agarose gel electrophoresis illustrating the effect of the CDC20 c.330 + 1G > A mutation. Compared with the wild-type lane, the c.330 + 1G > A lane showed a larger size band. (B) The CDC20 c.330 + 1G > A splicing mutation missed the canonical donor splice site, resulting in retention of intron 2 and the premature termination of the CDC20 protein. (C) The effects of the mutations on CDC20 and cyclin B1 protein level by western blotting in CHO cells transfected with wild type or mutant vectors. (D) Quantification of wild type and mutant CDC20 protein levels and cyclin B1 protein levels in CHO cells. Quantification was performed by measuring the band intensity of CDC20 and cyclin B1 relative to that of vinculin. The data are shown as means and SEM. *p < 0.05, **p < 0.01, ns, not significant.