Enhancers and transcription factors controlling the inducibility of the tumor necrosis factor-α promoter in primary macrophages

In macrophages, the TNF-α promoter is specifically induced by bacterial endotoxin, and provides a good model for gene regulation during bacterial infections. We have analyzed the protein-binding characteristics and enhancer activity of four κB-like enhancers and of a MHC class II-like Y box found in the mouse TNF-α promoter. In addition to members of the NF-κB/rel transcription factor family, at least two of the κB sites also bound a nuclear protein identified as NF-GMa, a factor that binds to promoter sequences from many cytokines. When inserted upstream of an enhancer-less promoter, two of the κB sites were active as LPS-inducible enhancers in primary macrophages, whereas the other two were not. Mutations in nucleotides known to contact nuclear factors severely reduced affinity of the κB sites for NF-κB. Introduction of the same mutations into a construct containing 1059 bp of the TNF-α promoter coupled to a CAT reporter gene resulted in a stepwise reduction in inducibility by LPS; mutation of all four sites (11 bp of 1059) reduced inducibility by 90%, providing compelling evidence for the role of transcription factors belonging to the NF-κB/rel family in the activation of the TNF-α promoter. The TNF-α Y box bound an abundant nuclear factor, but had no detectable activity in our assays, either as an enhancer or as a mutation-sensitive controlling element.