Figure 4.

ATF4 binds to a 33-bp binding element within the TRB3 promoter. (A) EMSA performed with 10 μg of dialyzed nuclear extracts from HT22 cells transfected with ATF4WT, mutant ATF4 (ATF4ΔRK), or GFP, respectively. Extracts were incubated with a radioactively labeled WT oligonucleotide containing the TRB3 promoter binding site or with a mutant oligonucleotide. Binding of ATF4WT to the TRB3WT promoter binding site was confirmed by supershift analysis (arrow) performed with an antibody (Ab) directed against ATF4. (B) Overexpressed ATF4WT protein occupies its putative binding site within the TRB3 promoter in HT22 cells, as shown by chromatin immunoprecipitation assay. HT22 cells were transfected with ATF4WT, mutant ATF4 (ATF4ΔRK), or GFP. An anti-myc antibody was used to precipitate the proteins in nuclear extracts of cross-linked HT22 cells. Coprecipitated DNA fragments were detected using PCR with a set of primers specific for the ATF4 binding site in the TRB3 promoter, yielding a 190-bp product. A representative example of three experiments is shown. (C) HT22 cells were transfected with the expression plasmids for ATF4WT, mutant ATF4 (ATF4ΔRK), or GFP. The cells were cotransfected with either a luciferase reporter vector containing the 33-bp ATF4WT binding site (33 bp WT), a reporter vector containing a mutant form of this binding site (33 bp MUT), or empty vector (pGL3 basic). In parallel, the transfection mix contained a plasmid expressing Renilla to allow normalization for transfection efficiency. The value for empty pGL3 cotransfected with GFP was arbitrarily defined as 1. Shown are ratios of luciferase and Renilla activities (mean ± SD for three independent experiments; each data point was performed in duplicate).