ZNF148 and E4F1 bind to WT and −57A > C TERT promoter sequence in vitro. (A) Schematic showing the coverage of the TERT promoter sequence by the DNA probes used in this study. (B) Workflow for a quantitative SILAC-based protein–DNA interaction screen. Biotinylated concatenated DNA oligonucleotides from A were immobilized on magnetic streptavidin beads, and bound proteins were detected by MS or western blot. −57WT and −57A > C probes are shown as an example of WT and mutant sequences. Specific protein binders display a differential SILAC ratio, with background binders showing a 1:1 ratio. (C) Two-dimensional interaction plot for WT versus −124C > T using SILAC-labeled U87MG nuclear protein extracts. Mutant-specific binders are labeled in red, and WT-specific binders are labeled in green. (D) Two-dimensional interaction plot for WT versus −146C > T. Mutant- and WT-specific binders are labeled similar to those in C. (E) Two-dimensional interaction plot for WT versus −57A > C. Mutant- and WT-specific binders are labeled similar to those in C. (F) Sequence specific pull-down of endogenous ZNF148 and E4F1 with HeLa nuclear extracts using the nine probes shown in A, top. Sequence-specific pull-down of endogenous ZNF148 with HeLa nuclear extracts with probes for rs36115365 major SNP-G and minor SNP-C, rs509813 major and minor SNP, and CDKN1A promoter (containing ZNF148 binding site) probes (bottom). Please note the occurrence of a double band for endogenous ZNF148, which may be owing to isoforms and/or post-translational modifications.
