Abstract
Tumor necrosis factor alpha (TNF-alpha) is an important mediator of inflammation, apoptosis, and the development of secondary lymphoid structures. Multiple polymorphic microsatellites have been identified in and around the gene, and there are also multiple single-base pair biallelic polymorphisms in the introns and promoter. The TNF-alpha -308 promoter polymorphism is a G-to-A transition which has been statistically associated with various autoimmune disorders. Some studies have found that it may directly mediate the increased transcription of TNF-alpha in some circumstances. This study characterizes proteins interacting at the polymorphic promoter site. Affinity purification of binding proteins and confirmatory chromatin immunoprecipitation assays were used to identify the proteins. Electrophoretic mobility shift analyses and surface plasmon resonance were used to define binding characteristics. Proteins interacting at this site include GCF2/LRRFIP1 and Ets-1. GCF2/LRRFIP1 appears to act as a repressor and occupies the -308 site in cells that do not make TNF-alpha. Cells competent to produce TNF-alpha have Ets-1 bound to the -308 promoter site. Active transcription is accompanied by NF-kappaB and c-Jun binding to the proximal promoter. Thus, dynamic changes on the TNF-alpha promoter, particularly at the -308 site, accompany the transition from repressed to active transcription. GCF2/LRRFIP1 is the first TNF-alpha repressor identified.