Regulation of the Human TNF Promoter by the Transcription Factor Ets

Tumor necrosis factor (TNF) affects the growth, differentiation, and function of a multitude of cell types and is viewed as a potent mediator of inflammation and cellular immune responses. In order to delineate functional domains that control TNF gene transcription, we have analyzed a 5′ flanking region of the human TNF promoter spanning base pairs −115 to −98. This region contains a PEA3/Ets-1 binding motif 5′ GAGGA 3′ in direct juxtaposition to an AP-1/ATF-like palindromic sequence motif 5′ TGAGCTCA 3′. Specific binding of Ets and Jun to their respective elements is demonstrated by competition analysis as well as by supershift assays. As shown by promoter deletion analysis, these two binding sites were essential for both basal promoter activity and responsiveness to the phorbol ester phorbol 12-myristate 13-acetate. Co-transfection of c- ets or c- jun expression plasmids along with TNF promoter-CAT reporter constructs revealed the participation of both transcription factors in the regulation of TNF gene transcription. Correspondingly, site-specific mutation of either Ets or Jun sites led to a complete loss of responsiveness to the respective transcription factor. These data suggest an essential role of Ets for the activation of TNF gene transcription.