NF-kappaB/rel proteins, tumor suppressor p53, and oncogene c-Myc are critical transcription factors involved in coordinating cellular decision-making events in response to external stimuli. Consensus sequences for binding these three transcription factors are found in the promoter region of IEX-1 (Immediate Early response gene X-1) gene that can either suppress or induce apoptosis in a cell- and stimulus-dependent manner. Utilizing an electrophoretic mobility shift assay (EMSA) and a promoter/reporter assay, we show that the NF-kappaB/rel consensus sequence in the IEX-1 promoter is specifically bound and activated by multiple NF-kappaB/rel complexes in descending order p65-c-rel-->p65-50-->p50-50. Interestingly, NF-kappaB/rel-mediated activation of IEX-1 expression was synergized by p53, but strongly inhibited by c-Myc in a dose-dependent fashion. Moreover, the ability of c-Myc to inhibit IEX-1 expression requires the presence of functional p53, which may partially contribute to the varying effects of p53 on IEX-1 expression in different cells. In support of coordinated regulation of IEX-1 expression by these three transcription factors in vivo, binding of endogenous p53, c-Myc and NF-kappaB/rel proteins, including p50, p65 and c-rel, to the IEX-1 promoter was demonstrated in living cells by chromatin immunoprecipitation using specific antibodies. The study reveals a novel integrative regulation of specific gene expression by NF-kappaB/rel, p53 and c-Myc transcription factors.