Figure 3. bZIP transcription factors associate with the cat-1 promoter during amino acid starvation.
(A and B) C6 glioma cells were starved of amino acids for the indicated times and ChIP was performed with antibodies to RNA polymerase II (Pol II), Sp1, ATF4, ATF3 and C/EBPβ as described in the Materials and methods section. In (A), PCR was used to amplify bases −37 to +156, relative to the cat-1 transcription start site. Samples that used input DNA as a template are also shown. In (B), samples precipitated with ATF4 antibody were amplified with the indicated primers. Representative gels are shown. (C) Time course of endogenous cat-1 mRNA accumulation during amino acid starvation. Cells were starved of amino acids for the indicated times and the 3.4 and 7.9 kb forms of the cat-1 mRNA were determined by Northern blot analysis. These two forms arise by the use of alternate polyadenylation sites. 18S ribosomal RNA was measured by ethidium bromide staining as a loading control. (D) ATF3 is required for a decrease in cat-1 transcription late in starvation. Wild-type and ATF3−/− MEF cells were starved for amino acids and the level of cat-1 mRNA was analysed by reverse transcription and real-time PCR as described in the Materials and methods section. The means±S.E.M. from three experiments is shown. **Significantly different from wild type (P<.001). (E) Wild-type and ATF3−/− MEF cells were starved of amino acids for the indicated times and protein levels in nuclear extracts were determined by Western blotting as described in the Materials and methods section.