A distal promoter region of CecA1 is required for Nub-PD-dependent repression. (A) Schematic representation of the CecA1-lacZ and CecA1-luc constructs, either carried by transgenic Drosophila or used for cell transfections. The pA10 construct contains 760 or 751 bp of 5′ upstream region from the CecA1 gene (horizontal line), and 62 or 71 bp of 5′ UTR (open box) fused to a SV40 leader (filled box), providing a translational start site in frame with the Escherichia coli lacZ or luc coding sequence (hatched box) [7,41]. Numbers refer to positions relative to the transcription start site (+1). Location of regulatory sequence motifs, as indicated by symbols and letters, is in scale. A previously characterized infection-induced response element (IRE) contains a κB-like site (κ), GATA site (G) and R1 site (R), and one additional κB site is located just 5′ of the IRE. A cluster of Oct sequence motifs (rectangle) contains several consensus Oct sequences (black circles) and Oct-like (gray circles) sequences (see Additional file 11 for sequences and exact locations). The pA12 construct contains 111 bp of 5′ sequence including the IRE, but not the Oct cluster. The pA10∆Oct-luc construct has an internal deletion of the whole Oct cluster (−336 to −150) but is otherwise identical to the pA10-luc construct. (B-G)CecA1-driven β-gal staining in the fat body of abdomens from flies carrying either the pA10 construct (pA10 CecA1-lacZ / TM3) (B,-C) or the pA12 construct (pA12 CecA1-lacZ) (D-G). Two independent transgenic lines, pA12-4 (D,E) and pA12-5 (F,G) were used. (H) Transfection of mbn-2 cells with CecA1-luc constructs confirms that the Oct cluster region is involved in repression of the CecA1 promoter. The graph shows the mean values of relative luciferase activity and standard deviation (n = 6). Statistical significance was calculated using paired t-test, P = 0.0012.
Dantoft et al. BMC Biology 2013 11:99 doi:10.1186/1741-7007-11-99