To decipher how BRAF V600E regulated GABPB expression, we took the next step to test our hypothesis that certain target protein molecules of the BRAF/MAPK pathway might function as transcription factors to activate GABPB. Our bioinformatics analyses of the regulatory 5′-untranslated region (5′-UTR) of GABPB revealed that two classical target molecules downstream of the MAPK pathway, FOS and MYC, were predicted to bind to 5′-UTR of GABPB (Fig. 5a). To test this, we constructed a luciferase reporter containing the wild-type 5′-UTR of GABPB and two mutant reporters containing disrupted FOS-binding motif and MYC-binding motif, respectively. If a transcription factor normally binds to the regulatory region and activates a target gene, disruption of the binding site would be expected to lead to decreased activities of the target gene. We found that disruption of the predicted FOS-binding site, but not the MYC-binding site, in the 5′-UTR region of GABPB significantly decreased the reporter activities of the GABPB gene (Fig. 5b), suggesting that FOS, but not MYC, regulated GABPB. We next performed ChIP assay to directly test whether FOS or MYC bound to GABPB in the cell and found that FOS, but not MYC, bound to the 5′-UTR of GABPB (Fig. 5c). We then used specific shRNA to knock down FOS or MYC and found that knockdown of FOS, but not MYC, could decrease the expression of GABPB (Fig. 5d). These results demonstrated that FOS could bind to the 5′-UTR of GABPB and activate its expression. We next examined the role of FOS in TERT expression. Luciferase reporting assay showed that FOS knockdown specifically inhibited the activities of the mutant TERT promoter but not the wild-type TERT promoter (Fig. 5e). Correspondingly, FOS knockdown suppressed TERT expression in cells harboring the TERT promoter mutation, but not in cells harboring the wild-type TERT (Fig. 5f). Taken together, these results demonstrated that FOS was a transcription factor of the GABPB gene, which could directly bind and activate the promoter of GABPB, leading to the upregulation of TERT expression in a TERT promoter mutation-dependent manner.Fig. 5FOS bound to 5′-UTR of GABPB and upregulated mutant TERT expression. a Diagrammatic illustration of the putative FOS- and MYC-binding sites in the 5′-UTR of GABPB identified by bioinformatics analyses. The predicted palindromic FOS consensus binding site (5′-TGACTCA-3′) and the canonical MYC binding site (5′-CATGTG-3′) located at +222 to +228 and +238 to +243, downstream of the transcriptional start site, respectively. b GABPB 5′-UTR region-luciferase-reporter assays for the wild-type and artificially-induced mutated putative FOS- and MYC-binding sites. c ChIP assay for FOS and MYC occupancy at the 5′-UTR of GABPB in K1 and A375 cells. Pol II and IgG were used for positive and negative controls, respectively. d Western blotting analyses of GABPB, MYC, FOS, and beta-actin in K1 cells with stable FOS or MYC knockdown. e TERT promoter-luciferase reporter assays in K1 cells with stable FOS knockdown. f Western blotting analyses for TERT, FOS, and beta-actin in K1, A375 and WRO cells with or without stable FOS knockdown. *P < 0.05, **P < 0.01, by two-tailed Student’s t test. n.s. not significant. All values represent the average ± standard deviation (SD) of triplicate samples, and similar results were obtained in three independent experiments