Fig. 4.

ATF3 and ATF4 form a complex to activate the NOXA promoter. (A) Schematic representation of the reporter constructs. A predicted p53 binding site was labeled. (B-C) ATF3 and ATF4 synergistically activate the NOXA promoter. (B) Luciferase activity in the extract of HeLa cells transfected with N1-Luc together with the indicated plasmids was determined. Shown is the mean of 2 independent experiments. A fraction of the extracts was analyzed by immunoblotting to verify the expression of ATF3 and ATF4. (C) As in (B) except that ATF3 was cotransfected with the indicated ATF4 variants. (D) ATF3 and ATF4 regulate NOXA expression in a p53-independent manner. (E) Interaction of ATF4 with ATF3. Extracts from 293T cells expressing His-ATF3 together with the indicated Flag-ATF4 variants were subjected to immunoprecipitation and immunoblotting analyses. (F) ATF3 and ATF4 form a complex on the NOXA promoter. A DNA fragment corresponding to the NOXA regulatory sequence was radiolabeled and incubated with purified ATF3 and ATF4 either singly or in combination. The reaction was analyzed by native gel electrophoresis and autoradiography. (Right) An example of the purified ATF3 and ATF4. (G) ATF3 and ATF4 associate with the endogenous NOXA promoter. JEKO-1 cells were treated with EerI (10 μM, 8 h). Chromatin immunoprecipitated with the indicated antibodies was analyzed by PCR using primers corresponding to the different regions of the NOXA promoter as shown in Fig. 6E.