THE NF-BETA-A-BINDING ELEMENT, NOT AN OVERLAPPING NF-IL-6-BINDING ELEMENT, IS REQUIRED FOR MAXIMAL IL-1-BETA GENE-EXPRESSION

NF-beta A is a monocyte, neutrophil, and B cell-specific nuclear prote in that is involved in regulation of the IL-1 beta gene. These studies further define the functional role of NF-beta A in RAW264.7 monocytic cells by using transient transfection analysis. We showed that NF-bet a A was able to activate transcription from a heterologous promoter in a distance-independent and dose-dependent manner. NF-beta A also appe ared to function in a positionally independent manner within the IL-1 beta cap-site proximal (CSP) promoter. NF-beta A was required for maxi mal IL-1 beta gene expression directed by the upstream LPS-inducible e nhancer element. Deletion of the NF-beta A-binding sequence resulted i n an 80% reduction in basal reporter gene activity and an 86% reductio n in LPS-inducible reporter gene activity in constructs containing onl y the enhancer and CSP promoter. Other regulatory elements located bet ween the enhancer and the cap site were not able to substitute functio nally for the absence of NF-beta A. Recently, other investigators have reported that IL-1 beta CSP promoter function was decreased by introd ucing multiple mutations within both the NF-beta A-binding sequence, a nd a putative overlapping NF-IL-6-binding sequence. We have found that these mutations predominantly affect NF-beta A binding. Furthermore N F-beta A, and not NF-IL-6, was required for supporting basal and LPS-i nducible transcription from a minimal IL-1 beta CSP promoter (position s -58 to +11). This promoter region did not appear to direct monocyte- specific IL-1 beta gene expression because reporter constructs contain ing the IL-1 beta CSP promoter were also active in transiently transfe cted HeLa cells.