Figure 5

ZNF143 binds to the zebrafish pax2a promoter in vitro. (A) Map of zebrafish pax2a proximal promoter showing three potential SPH sites. The sequences of the potential SPH sites are compared to the zebrafish U6-1 element [29] and the consensus SPH sequence from [37]. Capital letters depict nucleotides matching the consensus. To facilitate binding assays two separate radiolabeled zebrafish pax2a probes were prepared by PCR as diagrammed. (B) Electrophoretic mobility shift assay. Approximately 3 fmol of radiolabeled probes depicted in (A) were incubated with 3 μL of human ZNF143-(88-638) expressed by in vitro transcription/translation (lanes 3-9; 12-18; 21-24) or with unprogrammed extract (lanes 2, 11, 20), and electrophoresed. In addition, in samples shown in lanes 4-6, 13-15, and 22-24, an unlabeled double-stranded oligonucleotide with the sequence including the human U6-1 SPH element was added with the labeled probe in amounts noted, or similarly, an unlabeled oligonucleotide containing the unrelated, OCT sequence was added to samples in lanes 7-9 and 16-18. (C) DNase I footprinting assay. Approximately 3 fmol of singly end-labeled zebrafish pax2a probe from -154 to +16 were incubated with amounts of purified human ZNF143 DBD protein as indicated. The wt pax2a promoter was used for samples in lanes 1-5, whereas SPH2 or SPH3 mutant promoters were used for samples in lanes 6-9, or lanes 10-13, respectively.