Fig. 5.

Ectopically expressed ATF3 binds to the promoters of the pro-apoptotic and pro-inflammatory target genes. a INS-1 cells were infected with adenovirus expressing ATF3 (Ad-ATF3) or βGal (Ad-βGal) for 24 hours prior to ChIP analysis using primer pairs (schematics in Supplemental Fig. 2) for the potential binding sites at the indicated distance from the transcriptional start site (TSS). For each primer pair, the ChIP signals were determined by qPCR and normalized to input chromatin. The normalized ChIP signals from the Ad-ATF3-infected cells were divided by that from the Ad-βGal-infected cells to obtain the “fold enrichment.” Fold enrichment from immunoprecipitation using either IgG (solid bars) or ATF3 antibodies (open bars) is shown. In four independent experiments, the pattern of binding (relative ATF3 recruitment to different sites on a given promoter) was largely the same, although the absolute fold enrichment varied to some extent from one experiment to another. A representative result is shown. b ChIP signals were analyzed by agarose gel electrophoresis at the end of the linear phase of PCR reactions. The most proximal site for each promoter relative to the corresponding TSS was examined, except those indicated as distal (D). TNFα(D): −4800bp; IL-6(D): −2400 bp. Shown is a representative of four experiments.