FIGURE 2.

Identification of a JHRR in Kr-h1 promoter. A and B, Kc cells were co-transfected with the pGL3 basic plasmids containing truncations of 2-kb (A) or 216-bp (B) promoter regions of Kr-h1α and the hsp70 basal promoter regulating expression of firefly luciferase (Fluc) and a reference reporter plasmid carrying Rellina luciferase (Rluc). At 48 h after transfection, the cells were treated with 1 μm methoprene (JHA) for 2 h, and then dual luciferase assays were performed. Arrow represents the transcriptional start site (+1), numbers indicate the distance from the transcriptional start site, and shaded box represent the hsp70 basal promoter. A, the luciferase reporter activity supported by the −2000 to 1 of Kr-h1α promoter region truncations are shown; B, the luciferase reporter activity supported by −440 to −224 of the Kr-h1α promoter region truncations are shown. C, schematic representation of the location of JHRR (−440 to −321), which contains three E-box-like motifs. The nucleotide sequences are shown below the gene structure. Bold plus underlined nucleotides are C box, bold nucleotides are B boxes, and the dash separates the JHRR into two regions: −440 to −387 and −386 to −321. D, elucidation of the individual contribution of each E-box-like motif in the JHRR using the dual luciferase assays. WT, wild type JHRR; 1B*, mutation to the first B box of JHRR; 2B*, mutations to both B boxes of JHRR; C*, mutation to C box of JHRR; 2B*+C*, mutations to the two B boxes and C box of JHRR. E, Kc cells were co-transfected with pGL3 basic plasmids containing the WT JHRR, two copies of the two B boxes, or four copies of the C box (2B or C), the hsp70 basal promoter regulating expression of firefly luciferase (Fluc) and a reference reporter plasmid carrying Rellina luciferase (Rluc). After 48 h of transfection, the cells were treated with 1 μm JHA for 2 or 12 h, and dual luciferase assays were performed.