Fig 2: BCLAF1–TET2 mediated Ascl2 promoter demethylation inhibited stemness, and TET2–BCLAF1 coexpression patterns predicted CRC patient survival.A and B, GSEA of the downstream genes of Ascl2 (Oikonomou et al. cell reports. 2015) between CRC patients with high coexpression of TET2 and BCLAF1 versus those with low coexpression of TET2 and BCLAF1. C and D, the mRNA and protein expression levels of Cdca7, c-myc, Lgr5, and Sox9 in stable SW620 cells overexpressing the TET2 catalytic domain, which were then transiently transfected with a BCLAF1-overexpressing construct or BCLAF1-interfered siRNA for 48 h E, FACS for CD133 in stable SW620 cells described as (C and D). F, tumorsphere cells were derived from stable SW620 cells described as (C and D), and spheres were formed on the fifth day after transfection. G, survival analysis of 444 CRC patients with coexpression of TET2 and BCLAF1. ∗p < 0.05, ∗∗p < 0.01, unpaired two-tailed Student’s t test. CRC, colorectal cancer; FACS, fluorescence-activated cell sorter.

Fig 3: BCLAF1–TET2 repressed Ascl2 expression by mediating hydroxymethylation and methylation levels of the Ascl2 TSS1500 promoter.A and B, TET2, BCLAF1, and Ascl2 expression (mRNA in A, protein in B) in SW620 cells with TET2 catalytic domain overexpression or BCLAF1 overexpression or BCLAF1 interference for 48 h. C, stable SW620 cells transfected with lentiviral particles expressing the TET2 catalytic domain were transiently transfected with HA-BCLAF1 construct for 48 h. The occupancy of TET2 and BCLAF1 at the P1-P4 sites on the TSS1500 promoter regions of Ascl2 was determined by ChIP-qPCR with specific primers. Rabbit IgG was included as a negative control. D, site-specific levels of 5hmC and 5mC at TSS1500 of the Ascl2 promoter in SW620 cells described in (C) were determined by using GluMS-qPCR. E, stable SW620 cells transfected with lentiviral particles expressing the TET2 catalytic domain were transfected with BCLAF1-interfered siRNA for 48 h. The occupancy of TET2 at P1-P4 sites on the TSS1500 promoter regions of Ascl2 was determined by ChIP-qPCR with specific primers. Rabbit IgG was included as a negative control. F, site-specific levels of 5hmC and 5mC at TSS1500 of the Ascl2 promoter in SW620 cells described in (E) were determined by using GluMS-qPCR. nsp > 0.05, ∗p < 0.05, ∗∗p < 0.01. ∗∗∗p < 0.001, unpaired two-tailed Student’s t test. ChIP, chromatin immunoprecipitation; GluMS, glucosylated hydroxymethyl–sensitive; qPCR, quatitative PCR; TSS, transcription start site.