Fig 2: JAZF1-SUZ12 decreases H3K27me3 and increases H4Kac at PRC2 target genes(A) Metagene plots (above) and heatmaps (below) of H3K27me3 (left) and H4Kac (right) around sites shared by SUZ12, SUZ12?93, and JAZF1-SUZ12 in ESCs (n = 1,894) or EBs (n = 998) (averages of two independent ChIPs). In the heatmaps, occupancy (normalized reads) is indicated by color, according to the scales on the right. At each time point, sites are ordered by H3K27me3 in cells expressing SUZ12, from high to low.(B) H3K27me3 and H4Kac (reads per 10-bp window) at the regions shown in Figure 2D in ESCs expressing SUZ12, SUZ12?93, or JAZF1-SUZ12 at days 0 and 8 during differentiation.See also Figure S3.

Fig 3: Bach1 interacts with PRC2 in hESCs.(A) WT hESCs were immunofluorescently stained for Bach1 (red) and EZH2 (green) expression; nuclei were counterstained with DAPI (blue). Scale bars, 50 µm. (B) Bach1 was immunoprecipitated from WT hESCs; then, the amount of EZH2, EED, and SUZ12 present in the precipitate was evaluated via Western blot. (C) EZH2, EED, and SUZ12 were immunoprecipitated from WT hESCs and then, the amount of EZH2, EED, SUZ12, and Bach1 present in the precipitate was evaluated via Western blot. (D) Co-IP between Bach1 and EZH2 was performed in WT hESCs in the presence and absence of DNAse and RNase A. (E) HEK293 cells were transfected with a vector coding for Flag-tagged Bach1 and an empty vector or with vectors coding for Flag-tagged Bach1 and HA-tagged EZH2 and lysed; then, Bach1 was immunoprecipitated with an anti-Flag antibody, and Bach1 was detected in the immunoprecipitate and cell lysate via Western blot with an anti-Flag antibody, while EZH2 was detected in the precipitate via Western blot with an anti-HA antibody. ß-Actin was evaluated in the lysate to confirm equal loading. (F) Bacterially expressed His-tagged EZH2 was incubated with GST-tagged versions of the full Bach1 protein (Bach1-Full-GST) or with mutated versions of Bach1 that lacked either the C-terminal bZip domain (Bach1-N-GST) or the N-terminal BTB domain (Bach1-C-GST) then, the reaction products were precipitated with glutathione-Sepharose 4B beads, and EZH2 was detected in the precipitate via Western blot with anti-His antibodies.

Fig 4: Alterations of histone modifications in the KRT13 promoter in OSCC cells. (A) Schematic of the promoter region of the KRT13 gene. The arrow represents the transcription start site, and arrowheads indicate the primer set used for quantitative PCR. The methylation patterns of histone H3 (H3K4me3 and H3K27me3) in the KRT13 promoter in the three OSCC cell lines and HaCaT cells were examined by chromatin immunoprecipitation (ChIP) assay. Fold enrichment of histone H3 modifications was normalized to total histone H3 and calculated relative to that of HaCaT cells. The value of H3K4me3 in the HaCaT cells was set to 1. The means ± SEM for each group (n = 3–5) are shown. Statistical analysis was performed using Student’s t-test. *p < 0.05, **p < 0.01. (B) The levels of PRC2 proteins (Ezh2 and Suz12) were analyzed in the three OSCC cell lines and HaCaT cells by western blotting. Representative images are shown. Fold change in Ezh2 and Suz12 proteins were normalized to β-actin and calculated relative to band intensity of the HaCaT cells. The means ± SEM for each group (n = 3) are shown. Statistical analysis was performed by a one-way analysis of variance followed by Dunnett’s post hoc test. N.S. (not significant).

Fig 5: METTL5 regulates the translation efficiency of SUZ12. (A,B) The genomic view of SUZ12 mRNA reads of RNA-seq of si-METTL5 vs. si-Ctrl and AdMETTL5 vs. AdGFP under the stimulation of PE in the IGV browser. (C) Detection of the expression of SUZ12 in si-METTL5 and si-Ctrl treated NRVCs under the stimulation of PE. GAPDH serves as control. (D) Quantification of SUZ12 protein level and qRT-PCR detection of the relative gene expression of SUZ12 in si-METTL5 and si-Ctrl treated NRVCs under the stimulation of PE. (E) The translation efficiency (TE) is presented by calculating the ratio between transcript level and protein level. n = 3 for each group. (F) Detection of the expression of SUZ12 in Ad-METTL5 and Ad-Ctrl treated NRVCs under the stimulation of PE. GAPDH serves as control. (G) Quantification of SUZ12 protein level and qRT-PCR detection of the relative gene expression of SUZ12 in Ad-METTL5 and Ad-Ctrl treated NRVCs under the stimulation of PE. (H) The translation efficiency (TE) is presented by calculating the ratio between transcript level and protein level. n = 3 for each group. (I) Detection and quantification of the knockdown of SUZ12 in Ad-METTL5 treated NRVCs under the stimulation of PE. GAPDH serves as control. n = 3 for each group. (J,K) Relative gene expression of hypertrophy markers in NRVMs treated with (H) si-SUZ12 or (I) SUZ12 inhibitors under Ad-METTL5 or Ad-GFP transfection with or without PE treatment. n = 3 in each group. (L) Relative gene expression of Mef2a and Mef2d in METTL5 knockdown and overexpression NRVCs and control cells under the stimulation of PE by RT-PCR. n = 3 in each group. The unpaired T-test was used for 2-group comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.