Abstract
The 1311 bp TNF-α promoter region fused to a luciferase reporter vector was used in a transient transfection system to study the regulation of TNF-α promoter activity by E1A 13S in the U937 macrophage cell line and the MLA 144 T cell line. Co-transfections of the TNF-α promoter with an E1A expression vector resulted in a strong trans-activation of the promoter in both cell lines. Sequential truncation of the promoter mapped the E1A responsive region to sequences contained between −120 bp and the transcription start site. Truncation to −95 bp caused a dramatic 87% reduction of E1A activation in MLA 144 cells and further truncation to −36 bp caused a complete loss of E1A activation. In U937 cells, each truncation lowered E1A responsiveness but activity was never completely abolished. Site-directed mutagenesis of putative cis-acting sequences in the TNF-α promoter identified the AP-1 site as important for E1A trans-activation in the U937 cell line; the AP-2 and CRE sites also appeared to contribute to a lesser degree. In contrast, only the CRE mutation caused a reduction in E1A induced activity in the MLA 144 cell line. Co-transfection of the E1A expression vector with expression vectors for the cellular transcription factors AP-1, AP-2 and CREB indicated that none of these transcription factors showed any co-operativity with E1A. Thus, cis-acting sequences which contribute to E1A trans-activation of the TNF-α promoter have been delineated.
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