In Vivo Circadian Oscillation of dCREB2 and NF-kB Activity in the Drosophila Nervous System Anne K. Tanenhaus1., Jiabin Zhang1., Jerry C. P. Yin2* 1 Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI 53706, 2 Departments of Genetics and Neurology, University of Wisconsin-Madison, Madison, WI 53706 Abstract cAMP response element-binding protein (CREB) and nuclear factor kappa-B (NF-kB) are two ubiquitous transcription factors involved in a wide number of cellular processes, including the circadian system. Many previous studies on these factors use cellular assays that provide limited information on circadian activity or anatomical specificity. The ability to study transcription factors in defined tissue within intact animals will help to bridge the gap between cellular and in vivo data. We have used the GAL4-UAS and FLP-FRT systems to gain spatial control over reporter gene expression. Using a luciferase- based reporter, we show in vivo that Drosophila dCREB2- and NF-kB-mediated transcription oscillates in neuronal cells, glia, and in the mushroom body, a higher-order brain center in flies. This oscillation is under circadian control, cycling with a 24- hour rhythm, under both light-dark and dark-dark conditions. In light-light conditions, dCREB2 and NF-kB reporter flies exhibit a suppression of rhythmic activity. Furthermore, neuronal cycling of dCREB2 and NF-kB activity are modulated in period mutant flies, indicating these oscillations are controlled through the central clock. This study shows for the first time region-specific circadian oscillation of dCREB2/NF-kB activity in the Drosophila nervous system. Citation: Tanenhaus AK, Zhang J, Yin JCP (2012) In Vivo Circadian Oscillation of dCREB2 and NF-kB Activity in the Drosophila Nervous System. PLoS ONE 7(10): e45130. doi:10.1371/journal.pone.0045130 Editor: Paul H. Taghert, Washington University Medical School, United States of America Received April 24, 2012; Accepted August 14, 2012; Published October 15, 2012 Copyright: 2012 Tanenhaus et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by the United States National Institutes of Health (NIH) NIH NS063245, High Q Foundation (http://chdifoundation.org), AHAF (American Health Assistance Foundation) (www.ahaf.org), and the Neuroscience Training Program University of Wisconsin, Madison. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jcyin@wisc.edu . These authors contributed equally to this work. Introduction The biological importance of circadian rhythms is firmly established and widely accepted. The daily oscillation of internal function and behavioral output are present throughout evolution, and are conserved from invertebrates to humans. This circadian activity is self-sustaining, and can be synchronized to the local environment. Disruption of these rhythms has detrimental effects on health and cognitive function [1,2]. Cell-autonomous molecular oscillators govern circadian rhythms. In flies, the central pacemaker consists of a network of molecular interactions in a small population of cells. At the core of this machinery is a transcriptional feedback loop: the heterodimer CLOCK/CYCLE (CLK/CYC) activate the transcription of period (per) and timeless (tim). Once accumulation of cytoplasmic PER and TIM proteins is sufficient, they enter the nucleus and inhibit CLK/CYC, inhibiting transcriptional activity. The presence of these molecular oscillators in a subset of clock cells is sufficient to drive rhythmic responses throughout the organism [3]. Circadian changes in the expression of a large number of genes have been identified across many tissues in Drosophila [4–11]. While all of these studies identify transcripts with circadian expression patterns, there is often little overlap in the specific genes that are identified [12]. Identifying transcription factors that are under circadian regulation could provide a means of simplifying the interpretation of this data. However, whether most transcrip- tion factors have oscillations in their activities is unknown. In this study, we focus on two ubiquitous transcription factor families that have been linked to the circadian system: cAMP response element- binding protein (CREB) and nuclear factor kappa-B (NF-kB). The activity of the Drosophila CREB homolog (dCREB2) is under circadian regulation, oscillating in a 24-hour rhythm with nighttime and daytime peaks in activity [13]. Circadian control of CREB activity has important physiological implications. For example, CREB mediates the circadian modulation of gluconeo- genesis in mice [14]. There is also evidence that CREB acts upstream of the circadian system. In the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals, CREB plays a role in photic entrainment [15–19]. Suppression of CREB in the SCN inhibits expression of PER1 and PER2 [19]. Likewise, a mutation in dCREB2 shortens the 24-hour circadian rhythm in flies, and impairs the oscillation of per [13]. Recently, cAMP signaling has been demonstrated as an integral component of rhythmicity in the SCN [20–22]. There are similar indications that NF-kB interacts with the circadian system in mammals. NF-kB is present in the hamster SCN, and inhibition of NF-kB blocks light-induced phase shifts [23]. Circadian variation in NF-kB activity has also been detected in the rat pineal gland [24]. Finally, effects of the circadian clock on apoptosis are mediated by NF-kB activity in mouse cancer models [25]. In Drosophila, the effects of the immune response on sleep are under circadian control, and may require the NF-kB family member Relish [26]. PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e45130