Oscillating dCREB2 and NF-kB Activity Figure 4. The NF-kB reporter activity oscillates under circadian control. (A) NF-kB reporter activity in multiple tissues oscillates in constant darkness. Reporter activity is plotted as a function of time for different genotypes. Triply transgenic flies, where the reporter is activated in all neurons (using the elavc155-GAL4 driver, black squares), in a pan-glial pattern (using the repo-GAL4 driver, open circles), or in the mushroom body (using the ok107-GAL4 driver, open triangles), exhibit oscillatory activity when flies are maintained in light:dark (first 24 h period) or dark:dark (subsequent time) conditions. These plots are compared to those from doubly transgenic flies (solid line; UAS-FLP/+; NFkB-F-luc/+) that do not contain the tissue- specific driver. (B) The activity of the NF-kB reporter in different tissues is dampened under constant light. Reporter activity is shown as a function of time, with the shift to constant light occurring during the second 24 h period. The reporter is activated in triply transgenic flies in neurons (denoted elavc155-GAL4, elavc155-GAL4; UAS-FLP/+; NFkB-F-luc/+), glia (denoted repo-GAL4, UAS-FLP; NFkB-F-luc/repo-GAL4) or the mushroom body (denoted ok107-GAL4; UAS-FLP; NFkB-F-luc/+; ok107-GAL4/+) when compared to doubly transgenic flies without a GAL4 driver (solid line, UAS-FLP/+; NFkB-F- luc/+). (C–F) Neuronal reporter activity is plotted over time as flies are shifted from light:dark to constant darkness. For all of these panels, the same transgenes (UAS-FLP/+; NFkB-F-luc/elav-GAL4) exist in all flies, but the flies are examined in a wild type (C), per0 (D), perS (E) or perL (F) genetic background. doi:10.1371/journal.pone.0045130.g004 transcription. Because NFkB-F-luc reporter activity responds to known modulators of NF-kB activity (Fig. 2C–E), the oscillations we observe seem to reflect genuine changes in NF-kB transcrip- tional activity across the daytime and nighttime. However, we cannot discriminate which of the forms of NF-kB controls expression off of our reporter since Dorsal, Relish and Dif/Relish can bind to the consensus binding site used in our reporter fly [51]. Low-amplitude circadian fluctuations in luciferase reporter activity, even in predicted non-circadian reporters, are common [28], though it is unclear if these rhythms are due to changes in transcription, or to non-transcriptional circadian changes that might affect luciferase activity (such as substrate feeding or locomotor activity). We can not definitively exclude the possibility that the oscillations in dCREB2 and NF-kB reporter activity are due to, or include, other non-transcriptional changes that are PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e45130