Figure 6. CaZF promoter is a target of CAP2.

(A) The DNA binding ability of CAP2 to CRT in CaZF promoter was analyzed by gel mobility-shift assay (GMSA). Dimers of the sequences shown in the left panel were used as probes. CRT nucleotides are shown in bracket and the mutations (M1 and M2) created within the probe by replacing bases are shown in lower case. GMSA was performed with purified GST-CAP2 fusion protein expressed in bacteria using cold or ³²P-labeled probe. (B) ChIP assay indicating CAP2 interacts with CaZF promoter in vivo. Tobacco leaf explants were transformed with proCaZF:GUS and CaMV-35S:c-Myc-CAP2 constructs along with control plasmids. Antibiotic-selected shootlets were harvested and fixed with 1% formaldehyde. The DNA-protein complex was immunoprecipitated by anti-c-Myc antibodies. Quantitative PCR was performed using proCaZF specific primers flanking the CRT3 region. Lanes 1–3, Input (total DNA-protein complex); lanes 4–6, DNA-protein complex immunoprecipitated with anti-c-Myc antibody. Empty plasmids without proCaZF (pBI101) or c-Myc-CAP2 (pCAMBIA1302) were used as controls.