Fig 1: m6A-modified SREBF2-AS1 induced DNA demethylation at SREBF2 promoter through recruiting FXR1 and TET1. (A) The binding of SREBF2-AS1 to SREBF2 promoter, gene body, or 3’UTR in HuH-7 cells was measured using ChIRP assays with SREBF2-AS1 antisense probes or control probes. GAPDH promoter was employed as negative control. (B) ChIRP assays with SREBF2-AS1 antisense probes were performed in HuH-7 cells with overexpression of wild-type or m6A modification sites mutated SREBF2-AS1 to measure the binding of SREBF2-AS1 to SREBF2 promoter. (C) Schematic structure of SREBF2-AS1 and SREBF2 genomic locus. (D) The binding of SREBF2-AS1 to FXR1 in HuH-7 cells was measured using RIP assays with FXR1 specific antibody. (E) RIP assays with FXR1 specific antibody were performed in HuH-7 cells with METTL3 or METTL14 overexpression to measure the binding of SREBF2-AS1 to FXR1. (F) RIP assays with FXR1 specific antibody were performed in HuH-7 cells with wild-type or m6A modification sites mutated SREBF2-AS1 overexpression to measure the binding of SREBF2-AS1 to FXR1. (G) CUT&RUN assays with FXR1 or TET1 specific antibodies were performed in HuH-7 cells with wild-type or m6A modification sites mutated SREBF2-AS1 overexpression to measure the binding of FXR1 and TET1 to SREBF2 promoter. (H) CUT&RUN assays with FXR1 or TET1 specific antibodies were performed in HuH-7 cells with METTL3 or METTL14 overexpression to measure the binding of FXR1 and TET1 to SREBF2 promoter. (I) CUT&RUN assays with FXR1 or TET1 specific antibodies were performed in HuH-7 cells with SREBF2-AS1 depletion to measure the binding of FXR1 and TET1 to SREBF2 promoter. (J) Bisulfate DNA sequencing of CpG157 from HuH-7 cells with wild type or m6A modification sites mutated SREBF2-AS1 overexpression. (K) Bisulfate DNA sequencing of CpG157 from HuH-7 cells with SREBF2-AS1 depletion. (L) SREBF2 expression was measured by RT-qPCR in HuH-7 cells with SREBF2-AS1 overexpression and concurrent FXR1 depletion. (M) SREBF2 expression was measured by RT-qPCR in HuH-7 cells with SREBF2-AS1 overexpression and concurrent TET1 depletion. (N) The correlation between SREBF2 and FXR1 expression level in 371 HCC tissues, derived from TCGA LIHC dataset. r = 0.3412, p < 0.0001 by Spearman correlation analysis. (O) The correlation between SREBF2 and TET1 expression level in 371 HCC tissues, derived from TCGA LIHC dataset. r = 0.3415, p < 0.0001 by Spearman correlation analysis. For (A,B), and (D–M), results are shown as mean ± SD of 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, by one-way ANOVA followed by Dunnett's multiple comparisons test (A,B,E,G–K) or Student’s t test (D,F,L,M).