Screening of Novel Factors for TNF-α Expression in LPS-stimulated Macrophages

Objectives: Tumor necrosis factor alpha (TNF-α) is a major cytokine implicated in the pathogenesis of periodontitis. The nuclear factors associated with human TNF-α gene regulation is still under investigation. We previously identified a novel region located from -550 to -487 in human TNF-α promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-κB) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to this promoter region.
Methods: Yeast one-hybrid system was used to identify proteins that bind to the TNF-α promoter region. Three tandem copies of the TNF-α promoter region (66-bp) were cloned into the pHIS2 vector carrying the auxotrophic marker HIS3. Total RNA was extracted from THP-1 cells after differentiation with phorbol 12-myristate 13-acetate (PMA) and stimulation with LPS, and purified mRNA was used to synthesize cDNA fragments. The cDNA was connected to pGADT7-Rec2 vector by in vivo homologous recombination. The resulting library and the bait vector were then transformed into the yeast strain Y187. The transformants were selected on SD/-His/-Leu/-Trp medium containing 80mM 3-amino-1,2,4-triazole. Positive clones were isolated by insert-screening PCR. The PCR fragments were sequenced and confirmed by BLAST search.
Results: We isolated 104 positive clones, and 100 clones were identified as 95-100% homologous to human genes. We found that 41 of the 100 clones encoded protein sequences; 56% of these 41 clones were MYL6 (myosin, light chain 6, alkali, smooth muscle and non-muscle), 15% were SFRS3 (splicing factor, arginine/serin-rich 3), and three clones of the remaining 29% encoded DNA-binding proteins.
Conclusions: We identified several candidates for novel TNF-α transcription factors in LPS-stimulated human macrophages.