Regulatory studies of murine methylenetetrahydrofolate reductase reveal two major promoters and NF-κB sensitivity

Two promoters of the murine methylenetetrahydrofolate reductase gene (Mthfr), a key enzyme in folate metabolism, were characterized in Neuro-2a, NIH/3T3 and RAW 264.7 cells. Sequences of 189 bp and 273 bp were sufficient to achieve maximal activity of the upstream and downstream promoter, respectively. However, subtle differences in minimal promoter lengths and in promoter activities were observed between the cell lines. Both promoters demonstrated comparable activity in NIH/3T3 and RAW 264.7 cells, while in Neuro-2a cells, the upstream promoter was 15-fold more active than the downstream promoter. Alignment and data mining tools identified a candidate nuclear factor kappa B (NF-κB) binding site at the 3′end of the downstream promoter that is conserved throughout several species. NF-κB activation experiments in cultured cells were associated with increased Mthfr mRNA. Co-transfection of NF-κB and promoter constructs demonstrated Mthfr up-regulation by at least 2-fold through its downstream promoter in Neuro-2a cells; this increase was significantly reduced when the putative binding site was mutated. EMSA analysis demonstrated direct binding of NF-κB to this non-mutated site. This study, a first step into the elucidation of Mthfr regulation, demonstrates that two TATA-less, GC-rich promoters differentially drive transcription of Mthfr in a cell-specific manner, and provides a novel link of Mthfr to possible roles in the immune response and cell survival.