We next investigated how BRAF V600E and TERT promoter mutations synergistically activated TERT. It was recently demonstrated that the ETS transcription factor GABPA selectively bound and activated the mutant TERT promoter, but not the wild-type TERT promoter, in human cancer cells31,32. We hypothesized that the BRAF V600E/MAPK pathway might regulate the GABP transcriptional machinery of TERT. To confirm the role of GABP in the regulation of mutant TERT in our cell systems, we demonstrated that, similar to GABPA, GABPB also bound to TERT promoter in cells harboring TERT promoter mutations, but not in cells harboring the wild-type TERT (Fig. 4a). Co-immunoprecipitation (Co-IP) assay revealed that GABPA and GABPB formed a complex in the cell (Fig. 4b), consistent with the notion that GABPA complexes with GABPB to form tetramers, creating a fully functional GABP complex that binds DNA and activates gene transcription33–35. We next investigated whether BRAF V600E/MAPK pathway regulated the binding of GABP to TERT promoter. Chromatin immunoprecipitation (ChIP) assay showed that BRAF knockdown decreased the binding of GABP to the mutant TERT promoter when anti-GABPA antibody was used in the assay (Fig. 4c). Interestingly, BRAF knockdown decreased the expression of GABPB, but not GABPA (Fig. 4d), suggesting that the BRAF V600E/MAPK pathway selectively upregulated the GABPB gene, resulting in increased production of GABPB, which in turn drove the formation of the GABPA-GABPB transcriptional complex. To further support this concept, luciferase reporter assay showed that BRAF knockdown decreased the promoter activity of GABPB, but not that of GABPA (Fig. 4e). These results, taken together, demonstrate a mechanism in which by upregulating the expression of GABPB, the BRAF V600E/MAPK pathway promotes the formation of the GABPA-GABPB complex and consequent activation of the mutant TERT promoter, upregulating TERT expression.Fig. 4BRAF V600E promoted GABP binding to mutant TERT promoter by upregulating GABPB. a Chromatin-immunoprecipitation (ChIP) assay for GABPA and GABPB occupancy at TERT promoter in K1 and A375 cells with TERT promoter mutation and in WRO and HTORI3 cells without promoter mutation. IgG was used as negative control. b Co-immunoprecipitation (Co-IP) analysis of the interaction of GABPA with GABPB in K1 cells. c ChIP assay for GABPA occupancy at TERT promoter in K1 and A375 cells with or without stable BRAF knockdown. d Western blotting analyses of GABPA, GABPB, and beta-actin in K1 and A375 cells with/without stable BRAF knockdown. e Luciferase reporter assays for GABPA and GABPB promoters in A375 cells with or without stable BRAF knockdown. Scramble shRNA was used as control. *P < 0.05, ***P < 0.001, by two-tailed Student’s t test. n.s. not significant. All the values represent the average ± standard deviation (SD) of triplicate samples and similar results were obtained in at least two independent experiments