Fig. 6

Long-range interaction between E_156 and PTEN maintains melanoma-suppressive function. a Epigenetic and 3D genome profiles at the PTEN and its downstream enhancer locus (GRCh37/hg19 chr10: 89,400,000–90,140,000), including signals of A375 combined Hi-C, H3K27ac, H3K4me1, H3K4me3, ATAC-seq, and HiChIP loops. b Genomic profiles of recurrent DELs and CRISPRi enhancer screen results at the E_156 and PTEN locus on 297 melanoma samples. c 4C assay result of interaction between the PTEN promoter and E_156, light blue arrow refers to the 4C viewpoint (VP), and peak region highlighted by yellow arrow represents the chromosome region interacting with the VP. d Expression levels of PTEN in tumor (T, n = 461 samples) and normal tissues (N, n = 558 samples) were analyzed using the TCGA-SKCM data. e Disease-free survival analysis of PTEN expression were analyzed using the TCGA-SKCM data. Low expression of PTEN predicts shorter disease-free survival. f Western blotting results of MEF2A protein expression in the E_156 (E_155 or E_154 adjacent to E_156)-inhibited A375-KRAB cells. g Western blotting results of PI3K/AKT signaling pathway activation in the E_156 (E_155 or E_154 adjacent to E_156)-inhibited A375-KRAB cells with 1 µM vemurafenib or without treatment. P-AKT denotes Phosphorylated AKT, and T-AKT denotes total AKT. h, i Cell apoptosis (h) and Plate clone formation (i) assay results of the E_156 (E_155 or E_154 adjacent to E_156)-inhibited A375-KRAB cells with 1 µM vemurafenib or without treatment (n = 3 samples). j,k The representative three-dimension (3D) modeling graphs and statistical results in calculating tumor volumes for E_156-inhibited A375-KRAB and control cells at 2 or 3 weeks after subcutaneous injection in 5-week-old female nude mice (j), and then the tumors were weighed (k) after euthanasia of the mice (n = 7 mice). CI denotes CRISPR interference, and CA denotes CRISPR activation. Left graph is the representative result, and right graph is the statistical result. All the data are expressed as the means ± SD and analyzed by an unpaired two-tailed Student’s t test. Asterisks indicate significant differences between the indicated experimental groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001