The Oct motif cluster is required for both Nub-PD-dependent repression and Nub-PB-driven activation of the <i>CecA1-lacZ</i> reporter.

<p>(A) Schematic representation of the <i>CecA1-lacZ</i>-constructs carried by transgenic fly strains. The pA10 construct contains 760 bp of 5’ upstream region from the <i>CecA1</i> gene (horizontal line), and 62 bp of 5’ UTR (open box) fused to an SV40 leader (filled box), providing a translational start site in frame with the <i>E</i>. <i>coli lacZ</i> coding sequence (hatched box) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006936#ppat.1006936.ref015" target="_blank">15</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006936#ppat.1006936.ref047" target="_blank">47</a>]. 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 an additional κB site located 5′ in close proximity of the IRE [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006936#ppat.1006936.ref056" target="_blank">56</a>]. The cluster of Oct sequence motifs contains several consensus Oct sequences (●) and Oct-like () sequences [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006936#ppat.1006936.ref015" target="_blank">15</a>]. The <i>pA10</i>Δ<i>Oct</i> construct has an internal deletion of the whole Oct cluster (−336 to −150) but is otherwise identical to the <i>pA10</i> construct. (B-G) <i>CecA1</i>-driven β-gal staining in fat body and other tissues in female flies carrying either the <i>pA10</i> construct (B, D, F) or the <i>pA10ΔOct</i> construct (C, E, G), in combination with <i>c564-Gal4</i> driven overexpression of <i>nub-RB</i> (F, G) or in control flies without <i>c564-Gal4</i> (B-E), with the genotypes as indicated. Incubation with the β-gal substrate X-gal was either carried out overnight (O.N.) or for 2 h (D-G). Note the reporter gene expression in the thorax region and legs (F-G) as indicated by arrows. (H) Estimate of X-gal intensity, by conversion of blue pixels in images to 8-bit grayscale values ranging from 0 (white, no staining) to 255 (black) (<i>N</i> = 6). Group letters denote significantly different cohorts (<i>p</i><0.05). (I) Schematic model of <i>CecA1</i>-<i>lacZ</i> regulation by Nub-PD and Nub-PB via the Oct sequence cluster. Nub-PD (orange) binds to the Oct cluster and prevents <i>CecA1</i> expression in healthy flies, also in the presence of Relish (blue) and Nub-PB (green) (B and D). In constructs lacking the Oct cluster (<i>pA10ΔOct</i>), Nub-PD cannot bind and repress the <i>CecA1</i> promoter, which leads to moderate activation (++) of reporter gene expression (C and E). Overexpressed Nub-PB will compete with Nub-PD for binding to the Oct cluster and hyperactivates (++++) expression from the intact promoter (F), while the activation is not as prominent (+++) in constructs lacking the Oct cluster (G).