Retinoic acid induction of the tissue transglutaminase promoter is mediated by a novel response element

We recently cloned and sequenced two kilobases of the upstream flanking region of the mouse tissue transglutaminase gene. Transfection experiments showed that this region of the transglutaminase flanking sequence was sufficient to mediate a 4-fold induction in reporter gene expression by retinoic acid. The goal of these studies was to identify retinoid receptor binding sites within this proximal 2 kilobase sequence and then to determine if these binding sites had ligand-dependent enhancer activity. To accomplish this we first employed a competitive band-shift assay using PCR-synthesized genomic DNA fragments as probes. This assay identified a receptor binding site located 1.7 Kb upstream from the transcription start site that contained three consensus hexanucleotide 'half sites' separated by 7 and 5 bp, respectively. This tripartite response element was shown to mediate retinoic acid activation of a heterologous promoter in transient transfection assays in RAW264.7 cells. Our results suggest that this novel retinoid response element is responsible for the retinoic acid induction of mouse tissue transglutaminase gene expression observed in numerous cells.