Bsx directly binds to exorh promoter via PIRE sites. a A position-specific score matrix of Bsx recognition sequences obtained by SAAB. See Supplementary Fig. 7 for raw sequence data. b EMSA with BSXRE probe in the presence of GST-Bsx protein. BSXRE probe harbors the 18-bp highest matching sequence determined by SAAB (BSXRE; indicated by capital letters), which is flanked by a pair of short arm sequences (indicated by lowercase letters). Mutated sequences in BSXRE-mut probe were highlighted with dots. c A schematic structure of zebrafish exorh promoter. Gray rectangles represent DNA elements matching the consensus sequence of PIRE (TAATC/T), whereas white ones represent other potential sites for Bsx binding. See (d) for the nucleotide sequences. Arrows indicate the direction of each element. Nucleotide positions are given relative to the translation initiation site. d Competitive EMSA against BSXRE probe. Each competitor oligonucleotide has an 18-bp sequence of interest, which is flanked with the arm sequences shown in (b). Matching scores are calculated as the sum of relative appearance frequencies of nucleotides using the score matrix in (a) at the positions 4 through 15. P2 oligonucleotide contains the 12-bp sequence of PIPE, in which the nucleotide at the 5′-most position overlaps the left arm sequence (indicated by a lowercase letter). e EMSA with exorh promoter sequence (wt147bp) or its mutated ones. Multiple shifted bands possibly represent the variety of higher order structures of protein-probe complex. Full images of the electrophoreses (b, d, e) are shown in Supplementary Fig. 8
|
