The CREB-binding protein affects the circadian regulation of behaviour

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1 The CREB-binding protein affects the circadian regulation of behaviour Christian Maurer, Tobias Winter, Siwei Chen, Hsiu-Cheng Hung and Frank Weber Biochemistry Center Heidelberg, University of Heidelberg, Germany Correspondence F. Weber, Biochemistry Center Heidelberg, University of Heidelberg, Im Neuenheimer Feld 328, Heidelberg, Germany Fax: Tel: (Received 26 May 2016, revised 7 July 2016, accepted 8 July 2016, available online 28 August 2016) doi: / Edited by Ivan Sadowski Rhythmic changes in light and temperature conditions form the primary environmental cues that synchronize the molecular circadian clock of most species with the external cycles of day and night. Previous studies established a role for the CREB-binding protein (CBP) in molecular clock function by coactivation of circadian transcription. Here, we report that moderately increased levels of CBP strongly dampen circadian behavioural rhythms without affecting molecular oscillations of circadian transcription. Interestingly, light dark cycles as well as high temperature facilitated a circadian control of behavioural activity. Based on these observations we propose that in addition to its coactivator function for circadian transcription, CBP is involved in the regulation of circadian behaviour down-stream of the circadian clock. Keywords: behaviour; circadian rhythms; CLOCK/CYCLE; CREBbinding protein; Drosophila Most species synchronize their physiological and behavioural activities with the environmental cycles of day and night through regulation by a molecular circadian clock. The circadian clock is built by a set of transcription factors that control each other s rhythmic activity by interlocked transcriptional/post-translational feedbackloops [1 3]. Light and temperature are the primary environmental cues that facilitate a synchronization of the molecular clock with the external cycles of day and night. Drosophila and mammalian clock genes share high homology and analogous function [1,4,5]. The core oscillating activity is formed by the complex of transcription factors CLOCK (CLK) and CYCLE (CYC) that controls genome-wide transcription of many key regulatory factors in a rhythmic fashion. The circadian clock thereby facilitates a temporal synchronization of most homeostatic and behavioural activities. Activation of CLK/CYC-dependent transcription requires recruitment of the transcription coactivator CREB-binding protein (CBP) [6,7], as well as specific phosphorylation of the CLK protein [8 12]. In Drosophila, two CLK/CYC-activated genes, period (per) and timeless (tim), negatively feedback on their own transcription through PERmediated inhibition of CLK/CYC [9,10,13]. Two other CLK/CYC-activated genes, PAR-domain protein 1e (Pdp1e) and vrille (vri), engage in a second, interconnected feedback loop that rhythmically controls Clk expression due to VRI-mediated repression and PDP1emediated activation of Clk transcription [14]. Light and temperature are crucial environmental effectors of the circadian clock [15 17]. Synchronization of the molecular clock with environmental light dark cycles is achieved through the circadian photoreceptor cryptochrome (cry) [18], which facilitates a light-induced degradation of TIM [19,20]. Different light-sensitive structures, such as the compound eye, Hofbauer Buchner eyelet and ocelli, also contribute to circadian light reception in Drosophila [17,21]. Entrainment of the circadian clock by cycles of high and low temperature is Abbreviations CBP, CREB-binding protein; CLK, CLOCK; CREB, camp-response element-binding protein; CYC, CYCLE; PAP, PDF-associated peptide; PDFR, PDF receptor. FEBS Letters 590 (2016) ª 2016 Federation of European Biochemical Societies 3213

2 CBP affects circadian behaviour C. Maurer et al. independent of light entrainment [22,23] and mediated by specific subgroups of circadian neurons [16,22,24,25]. Here, we investigated the circadian regulation of locomotor activity in flies that moderately overexpress the coactivator CBP. Previous studies established that CBP is important for circadian transcription [6,7,26] thereby affecting molecular clock function as well as downstream behavioural rhythms. Our findings suggest an additional role of CBP in the circadian regulation of behaviour down-stream of the molecular clock. Materials and methods Fly stocks and locomotor activity assays w 1118 ; hs-nej flies (referred in here as hs-nej flies) carry a P- element insertion in the 5 0 -UTR of the nejire (nej) locus that allows the overexpression of nej by a heat shock-inducible promoter [27]. nej encodes the Drosophila homologue of the mammalian cbp gene. We previously observed a lowlevel expression of nej from the hs-nej transgene at constant temperature in the absence of heat shock [6]. hs-nej flies were obtained from the Bloomington Drosophila stock centre and compared to w 1118 control and Canton S wild-type flies. Flies were entrained in cycles of 12 h light and 12 h darkness (LD) with about 4000 lux light intensity during the light phase before transfer into constant darkness (DD). Locomotor activity assays at either 20, 25 or 30 C, as indicated in the figures, were performed as described previously [6,12] and analysed using CLOCKLAB (Actimetrics, Wilmette, IL, USA) software. Quantitative Real-time PCR (RT-PCR) 1 7-day-old flies were harvested after 7 days of entrainment in cycles of 12 h light and 12 h darkness (LD) during the last day in LD and a subsequent day in constant darkness (DD). Light intensity during light phase was about 4000 lux. Lights were on from Zeitgeber Time 0 (ZT0) to ZT12 and off from ZT12 to ZT24. Circadian Time 0 (CT0) to CT12 marks subjective daytime during DD. Flies were harvested at time-points indicated in the figure. Total RNA was purified from head extracts, reverse transcribed with random hexamer primers and cdna products were quantified by TaqMan quantitative RT-PCR as described previously [6]. Forward (fwd) and reverse (rev) primers as well as probes for clock genes per, tim, vri, Pdp1e, cbp and the constitutive control gene n-synaptobrevin (n-syb) were as described previously [6]. mrna levels for individual clock genes were normalized towards n-syb transcript levels and quantified by the 2 DDCt method as described [28,29] and according to manufacturers instructions (Applied Biosystems, Foster City, CA, USA). Statistical analysis Data from quantitative RT-PCR was statistically evaluated by nonparametric one-way ANOVA using the software GRAPHPAD PRISM 5 (GraphPad Software, La Jolla, CA, USA) to determine whether differences in the expression profiles of clock genes per, tim, vri and pdp1 in hs-nej and control flies at 20 C and at 30 C were significant. Immunohistochemistry Third instar larvae were entrained for 3 days in cycles of 12 h light and 12 h darkness (LD) and the brains were dissected during the first day in constant darkness at times indicated in the figures. Circadian Time 0 (CT0) marks time of subjective lights on and CT12 marks time of subjective lights off. The experiments were repeated twice and for each time-point a total of at least 8 brains was dissected. Data for CT18 (Fig. 4) are from one experiment. Immunohistochemical double-staining of brains with an antibody against PER and the PDF-precursor PDF-associated peptide (PAP) was performed as described previously [6,12]. Results Moderately increased levels of cbp cause a loss of free-running behavioural rhythms CREB-binding protein has previously been shown to act as a coactivator of CLK/CYC-dependent transcription in mammals and in Drosophila [6,7]. Overexpression as well as loss of CBP function affect molecular oscillations of the circadian clock and thereby clockcontrolled locomotor activity rhythms [6]. We recently observed that a moderate ~ 50% increase in cbp levels (Fig. 1A) caused a loss of locomotor activity rhythms in the majority of hs-nej flies when investigated under constant dark conditions at 20 C (Fig. 1B) [6]. Consistent with these findings an average activity profile over the whole population of hs-nej flies revealed strongly dampened behavioural rhythms under constant darkness due to the small number of rhythmic flies as compared to control flies (Figs 1B and 2B). Interestingly, locomotor activity of hs-nej flies was rhythmic during entrainment in light dark cycles (Fig. 2A). Light dark cycles are known to drive locomotor activity rhythms in the absence of a functional molecular clock, for example, in per 0 mutant flies. Light-driven behavioural rhythms in arrhythmic animals are due to a mere light response that lacks the anticipation of cyclic changes in light conditions, which is characteristic for circadian-controlled locomotor activity. Similar to control flies, the activity profile 3214 FEBS Letters 590 (2016) ª 2016 Federation of European Biochemical Societies

3 C. Maurer et al. CBP affects circadian behaviour Fig. 1. A temperature-sensitive behavioural phenotype in CBP overexpressing flies. (A) Average cbp transcript levels SEM determined by quantitative RT-PCR for w 1118 ; hs-nej and w 1118 control flies at either constant 20 C or 30 C are shown. cbp levels in w 1118 flies at 20 C were set to 100 as control. (B) A summary of locomotor activity analysis at 20, 25 and 30 C during the first 5 days in constant darkness is shown for genotypes as indicated. of hs-nej flies revealed an anticipatory increase in behavioural activity prior to the changes in light conditions (Fig. 2A). Morning and evening anticipation indicated a circadian control of locomotor activity in hs-nej flies during exposure to light dark cycles, which, however, is lost in most flies under free-running conditions (Fig. 2B). Effects of CBP on behavioural rhythms independent of molecular oscillations Consistent with a CBP-dependent coactivation of CLK/CYC-mediated transcription, [6,7,30] a strong 20- fold overexpression of cbp compared to wild-type caused behavioural as well as molecular arrhythmicity [6]. We therefore investigated whether the behavioural phenotype in hs-nej flies, which showed only 50% elevated levels of cbp compared to wild-type (Fig. 1A), correlated with effects on molecular clock oscillations. Transcript levels of clock genes were measured in fly head extracts over the course of a day in light dark cycles and in subsequent constant darkness at 20 C (Fig. 3). Although a slight phase advance is apparent at 20 C for per, tim and vri accumulation in DD, this difference was not significant by ANOVA analysis. Differences in Pdp1 accumulation are difficult to interpret, due to a lower amplitude oscillation as compared to other clock genes. Thus, no significant differences were observed in the expression profiles of per, tim, vri and Pdp1 between hs-nej and w 1118 control flies, revealing largely intact molecular oscillations of CLK/CYC-controlled transcription in the majority of hs-nej flies in constant darkness (Fig. 3). In contrast, free-running locomotor activity rhythms were lost or strongly dampened in most hs-nej flies under these conditions (Fig. 1B and 2B). hs-nej flies did not show a gradual loss of behavioural rhythms under free running conditions, but locomotor activity rhythms were lost or strongly dampened already during the first day in constant darkness. Immunohistochemistry of PER protein levels in lateral neurons of the larval brain revealed largely intact molecular oscillations in circadian neurons of hs-nej flies similar to the control (Fig. 4). The results indicate that moderate overexpression of CBP dampens circadian behavioural rhythms although molecular clock oscillations remain largely intact. These observations suggest that CBP is not only important for circadian clock function, as shown previously, but in addition also appears to affect the translation of molecular oscillations into rhythmic behavioural output. Temperature-sensitive circadian control of locomotor activity We next analysed the effects of temperature on behavioural activity rhythms in hs-nej flies. In contrast to the large number of behaviourally arrhythmic hs-nej flies under free-running conditions at 20 C (Figs 1B and 2B), robust behavioural rhythms were observed under constant conditions at high temperature of 30 C (Figs 1B and 5). At 30 C, the average activity profiles (Fig. 5) as well as the percentage of rhythmic hs-nej flies were very similar to controls (Fig. 1B). An intermediate phenotype was observed at 25 C. Since FEBS Letters 590 (2016) ª 2016 Federation of European Biochemical Societies 3215

4 CBP affects circadian behaviour C. Maurer et al. Fig. 2. Moderate overexpression of CBP affects the circadian control of locomotor activity under free-running conditions. (A) The upper panels show double-plotted average actograms over all flies of indicated genotypes (19 flies for w 1118, 31 flies for hs-nej, and 32 flies for Canton S) during 3 days in cycles of 12 h light and 12 h darkness (LD), as indicated by open and closed bars in the figure. The lower panels show the results from Chi-square periodogram analysis of average activity profiles as above, indicating robust rhythms for all genotypes in LD. The anticipatory increase in locomotor activity prior to the change in light conditions in the morning and in the evening (arrows in the actograms) indicates a circadian control of behavioural activity. (B) The upper panels show double-plotted average actograms over all flies of indicated genotypes for 5 days in constant darkness (DD) after entrainment in LD (as in A). The lower panels show the results from Chisquare periodogram analysis of average activity profiles as above, indicating robust free-running rhythms for Canton S and w 1118 control flies, but strongly dampened rhythms for hs-nej flies (see also Fig. 1B). hs-nej flies were not heat-shocked, but incubated at constant temperature, cbp-expression was not induced. However, hs-nej flies showed 40 50% higher constitutive levels of CBP expression at constant 20 C and at constant 30 C as compared to control flies (Fig. 1A). Consistent with the intact behavioural rhythms, clock gene transcription revealed no significant differences between hs-nej and w 1118 control flies at high temperature (30 C) (Fig. 3). These findings suggest a temperature-sensitive behavioural phenotype in hs-nej flies with intact free-running behavioural rhythms at high temperature of 30 C that are lost in most animals at a low temperature of 20 C. Discussion The coactivator CBP is an established interaction partner of CLK/CYC complexes in Drosophila and mammals, facilitating a coactivation of circadian transcription that is accompanied by rhythmic changes in chromatin structure [6,7,26]. CBP thereby plays a part in the assembly and regulation of the molecular circadian clock, controlling cyclic changes in physiological and behavioural activities. Consistent with this role of CBP in molecular clock function, overexpression and down-regulation of the transcriptional coactivator alters period-length and rhythmicity of molecular as well as behavioural oscillations [6,26]. Here, we investigated the effects of moderately, ~ 50% increased levels of CBP on molecular clock function and behavioural rhythms. A moderate constitutive increase in expression levels was achieved by a leaky expression of CBP from a heat-shock promoter transgene (hs-nej) at constant temperatures of 20 and 30 C. In the absence of heat shock, CBP expression was not induced, but constitutively elevated by about 3216 FEBS Letters 590 (2016) ª 2016 Federation of European Biochemical Societies

5 C. Maurer et al. CBP affects circadian behaviour Fig. 3. Intact molecular oscillations of circadian transcription in hsnej flies. Transcript levels of clock genes per, tim, vri and Pdp1 as indicated in the figure were determined by quantitative RT-PCR from fly heads of hs-nej (open squares) and w 1118 (black squares) control flies that were harvested over the last cycle of 12 h light and 12 h darkness and a consecutive day in constant darkness. Flies were incubated either at constant 20 C (left panels) or constant 30 C (right panels). Each time-point represents average transcript levels SEM from at least three independent experiments that were normalized with respect to average transcript levels. Statistical analysis by ANOVA did not show significant differences in phase or peak values of transcript accumulation between hs-nej and w 1118 flies except for pdp1 at 30 C in LD (for details see Results) %. At 20 C, the moderate overexpression of CBP caused a loss or dampening of behavioural rhythms in the majority of flies under free-running conditions of constant darkness (Figs 1B and 2B). At first sight this result appeared consistent with earlier observations that overexpression of high levels of CBP [6,26] cause a loss of behavioural as well as molecular oscillations. In contrast, weak overexpression of CBP allowed, however, overall molecular oscillations of clock gene expression to remain rhythmic and similar to wild-type flies (Figs 3 and 4). Thus, moderately increased levels of CBP dampen free-running behavioural rhythms over the whole population of hs-nej flies, although molecular clock oscillations remain largely intact. These observations suggest that CBP is able to affect circadian-controlled behaviour independent of its function in the molecular clock. Interestingly, effects of moderately elevated levels of CBP on behavioural rhythms appear to depend on environmental conditions. At 20 C, free-running behavioural rhythms were largely lost or dampened in hs-nej flies, while light dark cycles were able to drive behavioural oscillations that revealed characteristics of circadian regulation such as the anticipatory increase in behavioural activity prior to the changes in light conditions (Fig. 2A). The circadian control of behavioural activity was therefore enhanced in light dark cycles. Light dark cycles increase the amplitude of molecular oscillations, which may enforce a downstream circadian control of behaviour. Similar phenotypes have been observed in flies that carry mutations, for example, in PKA [31], neurofibromatosis 1 (Nf1) [32], disco [33] and pdf [34]. Some of these mutants act in the same signalling pathways. The neuropeptide, PDF, is secreted by small and large ventral-lateral neurons (LN v ) that control free-running behavioural rhythms and light-induced arousal respectively [35]. Secretion of the neuropeptide is important for synchronization of circadian neurons and for the control of behavioural activity [34,36]. The PDF receptor (PDFR) is a widely expressed G protein-coupled seven trans-membrane receptor that stimulates adenylate cyclase activity [37 39] and downstream PKA. Nf1 is a regulator of the Ras/MAPK signalling pathway. Both cyclic nucleotide/pka as well as MAPK are well established regulators of the camp-response elementbinding protein (CREB) and its coactivator CBP, which facilitate immediate light-induced transcription in the circadian system, providing a potential lightresponsive signalling pathway that controls circadian behaviour down-stream of the circadian clock. Similar to light dark cycles also elevated temperature facilitated robust free-running locomotor activity rhythms (Fig. 5). While free-running behavioural rhythms of hs-nej flies were largely lost at low temperature (Figs 1B and 2B), they remained intact at high temperature (Figs 1B and 5), giving rise to a temperature-sensitive phenotype in hs-nej flies. Together these results support an effect of light and temperature on CBP-dependent regulation of circadian behaviour. In conclusion, our findings indicate that CBP affects the circadian regulation of behaviour not only as a molecular clock component that coactivates CLK/ CYC-dependent transcription [6,7,26], but in addition by affecting a down-stream signalling pathway that translates molecular clock oscillations into rhythmic behavioural output. Unfortunately, a similar moderate increase in CBP levels could not be achieved by an independent strategy such as by use of the GAL4/ UAS system. The effects of moderately increased levels FEBS Letters 590 (2016) ª 2016 Federation of European Biochemical Societies 3217

6 CBP affects circadian behaviour C. Maurer et al. Fig. 4. Intact molecular oscillations of PER expression in circadian neurons of hs-nej larvae. (A) PER immunohistochemistry in PDF expressing lateral neurons of larval brains for genotypes as indicated. Brains were harvested over a time-course in constant darkness at indicated time-points after prior entrainment of larvae for 3 days in LD at 20 C (Circadian Time 0 (CT0) refers to subjective lights on and CT12 is subjective lights off ). The PER signal from double-stained brains is shown in green and the signal from cytoplasmic PDF-precursor PAP in red, yellow indicates colocalization of PER and PAP. (B) Mean SEM staining intensities for PER from images as in (A) for at least 16 brain hemispheres per genotype and time-point (eight brain hemispheres for CT18). Fig. 5. Robust free-running locomotor activity rhythms of hs-nej flies at 30 C. (A) The upper panels show double plotted average actograms over all flies of indicated genotypes (13 flies for w 1118,28 flies for hs-nej, and 21 flies for Canton S) during 3 days in cycles of 12 h light and 12 h darkness (LD), as indicated by open and closed bars in the figure at 30 C. The lower panels show the results from Chisquare periodogram analysis of average activity profiles as above, indicating robust rhythms for all genotypes in LD. (B) The upper panels show double plotted average actograms over all flies of indicated genotypes for 5 days in constant darkness (DD) after entrainment in LD at 30 C (as in A). The lower panels show the results from Chi-square periodogram analysis of average activity profiles as above, indicating robust rhythms for all genotypes. of CBP on behavioural rhythms depend on environmental light and temperature conditions, which may be due to enhanced molecular oscillations in light dark cycles and at high temperature that enforce a control of behaviour by the molecular clock. Alternatively, downstream signalling pathways that translate 3218 FEBS Letters 590 (2016) ª 2016 Federation of European Biochemical Societies

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