Fig. 1: PHB2 is recruited to the open chromatin to initiate the transcription of lncRNA CANT2 at the chr6p22.3 locus.

A Genomic structure of CANT2 and a schematic of the core promoter fragment deletion by CRISPR-Cas9. The rectangles indicate the exons of CANT2. The black line indicates introns. The red line indicates the core promoter of CANT2. B Chromatin accessibility status at the chr6p22.3 locus with ATAC-seq and Omni-ATAC datasets obtained from the public GEO database under accession codes GSE188398⁴², GSE134432⁴³, and GSE241445⁴⁴. C Real-time PCR of luciferase activity of the DNA fragments of CANT2 promoter. Ctrl, control group without any Firefly reporter plasmids; Mock, group with wild-type Firefly reporter plasmids; 1–5, groups with different 200 bp DNA fragment of CANT2 promoter contained in Firefly reporter plasmids. All groups had Renilla reporter plasmids. All data were calculated the ratio of firefly to Renilla luciferase activity (Fluc/Rluc) in dual luciferase reporter system. For comparison, the ratio of Fluc/Rluc of the mock was arbitrarily set as 1 in the calculation. All the experiments were performed in triplicate and data are presented as the mean ± SD using an unpaired two-tailed t test; *P < 0.05; **P < 0.01. D Schematic diagram of variant primer sets in FAIRE assay. a, b, c: primers in SOX4, CANT2, PRL promoter regions, respectively. E PCR of FAIRE assay at CANT2 core promoter region. Representative images from three independent experiments. F Quantification of FAIRE assay at Chr6p22.3 in melanoma cells (A375 and A875) and normal cells (PIG1). Input DNA was used as a positive control. All the experiments were performed in triplicate and data are presented as the mean ± SD using a Dunnett’s multiple comparisons test; ****P < 0.0001. G Real-time of CANT2 expression at RNA level in melanoma cells lines. All the experiments were performed in triplicate and data are presented as the mean ± SD using an unpaired two-tailed t test; ***P < 0.001; ****P < 0.0001. H Subcellular localization of CANT2 in melanoma cells. GAPDH and U6 RNA served as positive controls for the cytoplasmic (black) and nuclear (orange) fractions, respectively. All the experiments were performed in triplicate. I Survival curves of SKCM patients with a high or low PHB2 expression (cutoff = 0.5, P < 0.05) was analyzed in GEPIA using a Log-rank (Mantel-Cox) test. J, K ChIP analysis of PHB2 (J), H3K4me3 (K) and MLL2 (K) at the CANT2 core promoter. Rabbit normal IgG served as the negative control. ChIP enrichment was presented as the percentage of bound/input signal. All the experiments were performed in triplicate and data are presented as the mean ± SD using a Dunnett’s multiple comparisons test; *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001. L, M PCR of ChIP analysis of H3K4me3 (L) and MLL2 (M) at the CANT2 core promoter in melanoma and normal cells. Representative blots from three independent experiments. N Co-IP assay was performed to show the interaction between PHB2 and MLL2 in normal and melanoma cells. IgG was used as a negative control. Representative blots from three independent experiments.