DNase I footprinting assay

DNase I footprinting assays were performed with two different methods: the radioactive labeled probe and DNA sequencing methods. For the radioactive labeled probe method [41, 42], the promoter-proximal DNA region with a single 32P-labeled end was PCR amplified with either sense or antisense end-labeled primers. The PCR products were purified using QiaQuick columns (Qiagen). Increasing amounts of His-ToxR were incubated with the purified and labeled DNA fragments (2–5 pmol) for 30 min at room temperature. The final reaction volume was 10 μl and contained binding buffer used in EMSA. Before DNA digestion, 10 μl of Ca2+/Mg2+ solution (5 mM CaCl2 and 10 mM MgCl2) was added, followed by incubation for 1 min at room temperature. The optimized RQ1 RNase-Free DNase I (Promega) was then added to the reaction mixture, and the mixture was incubated at room temperature for 40–90 s. The reaction was quenched by adding 9 μl of stop solution (200 mM NaCl, 30 mM EDTA, and 1% SDS), followed by incubation for 1 min at room temperature. The partially digested DNA samples were then extracted with phenol/chloroform, precipitated with ethanol, and analyzed in 6% polyacrylamide/8 M urea gels. Protected regions were identified by comparison with sequencing ladders. The templates for these sequencing ladders were the same as the DNA fragments for DNase I footprinting. Radioactive species were detected by autoradiography.

For the DNA sequencing method [26, 28], a DNA fragment of promoter DNA region of each indicated genes was PCR amplified using the primers target-FP-F (M13F) and target-FP-R (M13R) with ExTaq DNA ploymerase. After being purified, the amplicon was used as the template for labeling the probes with different primer pairs: M13F-FAM and target gene-FP-R (M13R) for preparation of 6-carboxyfluorescein (FAM)-labeled coding strand, and target gene-FP-F (M13F) and M13R-HEX for preparation of 5’-Hexachlorofluorescein phosphoramidite (HEX)-labeled noncoding strand. The PCR products were purified by using the Qiaquick columns (Qiagen) and quantified with a NanoDrop 2000 (Thermo). Approximately 350 ng of the purified, FAM/HEX-labeled DNA fragments were incubated with increasing amounts of His-CalR in a final 10 μl reaction volume containing the binding buffer used in EMSA. The DNA digestion procedure was the same as the radioactive labeled probe method. The digested DNA samples were extracted with a Beaver Beads ™PCR Purification Kit (Beaver) according to the manufacturer’s instructions, and the sample pellets were dissolved in 15μl modified water (HiDi: water: 600 LIZ=90: 60: 1). For sequencing, the BigDye® Terminator v3.1 Cycle Sequencing Kits (ABI) was used. The volume of each sequencing reaction was increased to 20 μl that contains 10 ng of target promoter region as template, 3.2 pmol of sense or antisense primer as the sequencing primer, and 8 μl of BigDye reaction mix (BigDye: 5× buffer = 1: 3). After pre-denaturation at 96°C for 1 min, PCR amplification was conducted at 25 cycles of denaturation at 96°C for 10 s, annealing at 50°C for 5 s and extension at 60°C for 4 min. The sequencing samples were precipitated with the same method as above, and then dissolved in 10 μl HiDi and 1μl 600 LIZ. The digested DNA fragments were analyzed by using ABI 3500XL DNA Genetic analyzer with GeneMarker software 2.2. The sequencing products were examined with Sequence Scanner software v1.0.