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: CDC20 regulates p65 degradation in an APC11‐dependent manner, related to Fig 6 A, BNo interaction was found in the immunoprecipitation of p65 and APC2 in HEK293T cells.C, DThe expression of p65 remained stable under the knockdown (C) or overexpression (D) of APC2 in HEK293T cells determined by Western blot analyses.E, FNo change of p65 protein (E) and CDC20 protein (F) were seen in NC and CDH1sh HEK293T cells.GThe interaction of APC11 and p65 remained stable in NC and CDH1sh HEK293T cells.HThe knockdown efficiency of APC11 in hBMSCs was determined by qRT–PCR (n = 6). Data information: Data are displayed as mean ± SD and show one representative of n ≥ 3 independent experiments with three biological replicates. Statistical significance was calculated by one‐way ANOVA followed by a Tukey’s post hoc test and defined as ***P < 0.001.

Fig 4: SETD5 is transiently stabilized and degraded by APC/C and proteasome during the early adipogenesis.a Immunoblot showing expression of endogenous SETD5 in 3T3-L1 preadipocytes. b Immunoblot showing expression of SETD5-V5, NCoR2, HDAC3, and CDC20 in SETD5-transduced 3T3-L1 preadipocytes. The graph shows the band intensity of SETD5-V5 and CDC20. c Cell cycle analysis of empty virus-transduced 3T3-L1 preadipocytes. The ratios of the cells at G1, S, or G2/M phase are shown. d Immunoblot showing expression of SETD5-V5 in SETD5-transduced 3T3-L1 preadipocytes treated with MDI and MG132. The graph shows band intensity of SETD5-V5. e Immunoblot showing ubiquitinated SETD5-V5. SETD5-transduced 3T3-L1 preadipocytes were treated with MDI and then MG132 at 9 h after MDI induction. Preadipocytes were subjected to immunoprecipitation using anti-V5 antibody and immunoblotting with anti-ubiquitin and anti-V5 antibodies. f Heatmap representing the abundance of APC/C subunits and RNF213 in SETD5-V5 immunoprecipitates. Spectrum count for each protein in proteomics analysis (Fig. 1j) was normalized by spectrum count for common region of SETD5 (a.a. 1 to a.a. 272 and a.a. 919 to a.a. 1441). g Expression of mRNAs of APC/C subunits during the early adipogenesis. mRNA expression of Cdc20 and Anapc2 in SETD5-V5-transduced preadipocytes were quantified by qPCR. h Immunoblot showing SETD5-V5 stability by the cycloheximide chase assay. 3T3-L1 preadipocytes transduced with SETD5-V5 were transfected with control siRNA or siRNA targeted to Anapc11 and induced for differentiation with MDI mixture. Preadipocytes were treated with cycloheximide (CHX) at 9 h after MDI induction and subjected to immunoblot analysis with anti-V5. Representative of three independent experiments. The graph shows regression analysis of SETD5-V5 protein stability after CHX treatment. Data are mean ± SEM of three independent experiments. Protein half-life (T1/2) was calculated based on exponential decay curve fit to the average data at each time point. a, b, d Equal loading of the protein was confirmed by blotting with anti-HDAC1 (a) or anti-actin β (b, d) antibody. a, b, d, e, g Representative of three (a, b) or two (d, e, g) independent experiments. c, g Data are mean ± SD of three technical replicates. Source data are provided as a Source data file.

Fig 5: The ubiquitination of p65 by APC11CDC20 AImmunoblot of Flag‐p65‐linked ubiquitination promoted by Myc‐CDC20. HEK293T cells were co‐transfected with Flag‐p65, HA‐Ubiquitin, with or without Myc‐CDC20 or vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection.BImmunoblot of Flag‐p65‐linked ubiquitination promoted by HA‐APC11. HEK293T cells were transfected with Flag‐p65, His‐Ubiquitin, with or without HA‐APC11 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection.C, DImmunoblot of Flag‐p65‐linked in vitro ubiquitination promoted by GST‐CDC20 (C) or GST‐APC11 (D). Bacterially expressed and purified GST‐CDC20 or GST‐APC11 were incubated with purified proteins, including HA‐Ubiquitin, E1, E2, and Flag‐p65 as indicated at 32°C for 1 h.EImmunoblot of Flag‐p65‐linked ubiquitination promoted by truncated Myc‐CDC20 fragments. HEK293T cells were transfected with Flag‐p65, HA‐Ubiquitin, with or without Myc‐CDC20 171‐499 fragment (containing WD40 domain), Myc‐CDC20 1–170 fragment (WD40 domain deficient), and vector plasmids. Transfected cells were then treated with 10 μM MG132 for 6 h before collection.FImmunoblot of Flag‐p65‐linked ubiquitination promoted by HA‐APC11 in NC and CDC20sh cells. NC or CDC20sh HEK293T cells were co‐transfected with Flag‐p65, His‐Ubiquitin, with or without HA‐APC11 and vector plasmids. Transfected cells were treated with 10 μM MG132 for 6 h before collection.