Trihelix DNA-binding Protein with Specificities for Two Distinct Cis-elements: Both Important for Light Down-regulated and Dark-inducible Gene Expression in Higher Plants

The DE1 sequence is a cis-regulatory element necessary and sufficient for light down-regulated and dark-inducible expression of the pea GTPase pra2 gene. This sequence does not show any sequence similarity to the previously reported ones involved in light-regulated gene expression. A one-hybrid screen isolated a cDNA encoding a DNA-binding protein, named DF1, with specificity for the DE1 sequence 5'-TACAGT. DF1 has domains similar to the trihelix DNA-binding domain found in the GT-1 and GT-2 proteins, which are plant transcription factors. The DE1-binding domain of DF1 is most similar to the carboxyl-terminal trihelix domain of the rice GT-2 protein with specificity for the GT2 sequence 5'-GGTAATT, which is also necessary for dark-inducible expression of the rice phyA gene. An electrophoretic mobility shift assay showed that this DNA-binding domain specifically binds to two types of DNA sequences, DE1 and GT2. Additionally, using DF1/GT-1 chimeras, we show that the second and third helices of the trihelix DNA-binding domain of DF1 are responsible for this dual DNA binding specificity. Our results show that DF1 has specificity for the two distinct cis-regulatory elements, both important for light down-regulated and dark-inducible gene expression in higher plants.