Functional analysis of linker-scan mutants spanning the-376-308,-244, and-238 polymorphic sites of the TNF-α promoter

Tumor necrosis factor alpha (TNT-alpha) promoter polymorphisms have been linked to a large number of diseases but studies examining. the possible direct functional effects of these polymorphisms have been contradictory. Previous studies compared TNF-alpha promoter constructs containing single nucleotide changes. We have now made a series of mutant constructs in which regions of the TNF-alpha promoter containing suspected functional single nucleotide polymorphisms, including - 376, - 308, - 244 and - 238, were replaced by a 10 bp linker scan sequence. These constructs were transiently transfected into the T cell line Jurkat, the B cell line Raji, and the monocytic cell line U937, and tested for basal and induced transcriptional activity. Mutant constructs covering both the - 308 and - 376 polymorphisms showed no significant differences in either basal or induced transcriptional activity. Constructs covering the - 244/ - 238 region showed a small increase in basal activity in the U937 cell line. These results indicate (i) that the - 308 and - 376 regions are of no functional relevance for TNF-alpha promoter transcription, and (ii) that the - 244/ - 238 region does not influence transcription in some cell lines but may have some role in transcription in others. (C) 2001 Academic Press.