The adenoviral transcription factor, E1A 13S, trans-activates the human tumor necrosis factor-alpha promoter.
The 1311 bp TNF-alpha promoter region fused to a luciferase reporter vector was used in a transient transfection system to study the regulation of TNF-alpha promoter activity by E1A 13S in the U937 macrophage cell line and the MLA 144 T cell line. Co-transfections of the TNF-alpha 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-alpha 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-alpha promoter have been delineated.
Rhoades
K L
KL
Department of Microbiology and Immunology, UCLA School of Medicine 90024, USA.
Golub
S H
SH
Economou
J S
JS
D017500:Adenovirus E1A Proteins / Q000235:genetics / Q000378:metabolism*; D000260:Adenoviruses, Human / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics; D004247:DNA; D004268:DNA-Binding Proteins / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D018808:Transcription Factor AP-1 / Q000235:genetics; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism
D017500:Adenovirus E1A Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D018808:Transcription Factor AP-1; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D004247:DNA