Fig 2

Fig 2

(A) Above, schematic representation of the zebrafish ddb2-Luc promoter reporter construct. The transcription start site (TSS), ATG translation initiation codon and the luciferase reporter gene (luc+) are indicated. Below, schematic representation of the various zebrafish ddb2 promoter deletion, luciferase reporter constructs analysed. Below is shown the portion of the promoter that constitutes the 49bp LRR_ddb2 region within the LRR_ddb2-Luc reporter construct. (B,C) Representative real-time bioluminescence assays in PAC-2 (B) or EPA (C) cells transfected with the ddb2-Luc luciferase reporter. Bioluminescence, counts per second (CPS), is plotted against time (hrs). Black and white bars along the x-axes show dark and light periods, respectively. Each time-point represents the mean of n = 8 ± s.d. (D) Sequence alignment of the LRR promoter region in the zebrafish (zf) and P. andruzzii (pa) ddb2 genes. The E2F, D-box and ATF1/CREB enhancer elements are highlighted in red, green and blue respectively. Vertical bars denote identical sequences in the alignment. Blue horizontal arrows indicate the orientation of the two D-box enhancers. The locations of the aligned sequences in terms of nucleotides upstream from the ATG translation start codon (nt) are indicated on the right side of each sequence (in parentheses). Below: schematic representation of the set of LRR_ddb2 sub-deletion constructs where each of the enhancer elements in the LRR_ddb2-Luc reporter is deleted. The E2F, D-box and ATF1/CREB enhancer elements are represented by red, green and blue boxes respectively. (E,F) Above: Schematic representation of the ZF and PA sequence D-box_ddb2-Luc reporters. Below: Representative real-time bioluminescence assays from zebrafish PAC-2 (blue trace, left panel) and cavefish EPA cells (orange trace, right panel) transfected with ZF: and PA: D-box_ddb2-Luc respectively and exposed to light and dark periods. Bioluminescence (CPS) is plotted on the y-axes and time (hrs) on the x-axes. Each time-point represents the mean of n = 8 ± s.d. White and black bars below each panel represent the light and dark periods, respectively. (G,H) Above: Schematic representation of the ZF and PA E2F/D-box_ddb2-Luc reporter constructs. Below: Representative real-time bioluminescence assays from PAC-2 (left panel) and EPA cells (right panel) transfected with ZF: and PA: E2F/D-box_ddb2-Luc respectively and exposed to light and dark periods (as described for panel E-F). (I) Schematic representation of the E2F site, D-box and E-box elements as well as ATF1/CREB site present in the promoters of ddb2 genes in various fish species. These elements are conserved in a broad range of fish species (see S2 Table for the Ensembl accession numbers).