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Originally published In Press as doi:10.1074/jbc.M702319200 on July 16, 2007

J. Biol. Chem., Vol. 282, Issue 43, 31349-31357, October 26, 2007
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Circadian Transcription Depends on Limiting Amounts of the Transcription Co-activator nejire/CBP*Formula

Hsiu-Cheng Hung{ddagger}1, Christian Maurer{ddagger}1, Steve A. Kay§, and Frank Weber{ddagger}2

From the {ddagger}Biochemie-Zentrum Heidelberg, Ruprecht-Karls Universität Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany and §The Scripps Research Institute, La Jolla, California 92037

Received for publication, March 16, 2007 , and in revised form, July 12, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The circadian clock orchestrates physiological and behavioral activities, including metabolism, neuronal activity, and cell proliferation in synchrony with the environmental cycle of day and night. Here we show that the Drosophila ortholog of the CBP/p300 family of transcription co-activators, nejire (nej), is an intrinsic component of the circadian clock that performs regulatory functions for circadian controlled transcription. Screening of overexpression mutants revealed that gain of nej function was associated with a loss of behavioral and molecular rhythms. Overexpression of NEJ suppresses the long period phenotype of a mutation in the clock gene period (per). NEJ physically interacts through two binding sites with CLOCK and the CLOCK·CYCLE (CLK·CYC) complex. Induction of CLK·CYC-dependent transcripts upon induction of nej expression from a heat-shock promoter showed that NEJ is limiting. Reduced CLK·CYC-mediated transcription in a nej hypomorphic mutant indicates an essential function of NEJ/CBP for CLK·CYC activity and a regulation of circadian transcription by availability of the co-activator. Competition for recruitment of NEJ/CBP provides a potential mechanism for cross-talk between circadian transcription and other CBP-dependent physiological processes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The circadian clock controls genome wide transcription of many key regulatory components in a diverse selection of vital pathways (1–3) that ultimately allow a coordination of physiological and behavioral activities and their synchronization with the environmental cycles of day and night. The analogous and homologous clock mechanisms in Drosophila and mammals are based on two interconnected feedback loops (4, 5). In Drosophila, the heterodimeric complex of transcription factors CLOCK (CLK)3 and CYCLE (CYC) (BMAL1 in mammals) activates expression of its own inhibitors PERIOD (PER) and TIMELESS (TIM) forming the first feedback loop. This loop is interconnected with CLK·CYC-mediated expression of the transcription repressor vrille (vri) and the activator par-domain protein 1 (pdp1). VRI and PDP1 control the rhythmic transcription of Clk and contribute to the robustness of molecular oscillations (6, 7).

Oscillations in cyclic nucleotide, calcium, and MAPK signaling (8–10) likely contribute to a circadian control of physiological processes such as cell proliferation (11) and the sleep/wake cycle, which is important for memory formation (12). However, these pathways also feedback on the molecular oscillator at least in part through control of CLK·CYC activity (13). Cross-talk between circadian and cell signaling may increase the robustness of circadian oscillations and allow a coordination of circadian transcription with physiological requirements. Previous studies showed that recruitment of the CREB-binding protein (CBP) from a limiting cellular pool mediates cross-talk between the transcription factors E2F, JAK/STAT, AP1, and nuclear hormone receptors (14–16) that control e.g. entry into the cell cycle and the immune response. Here we show that CLK·CYC-mediated transcription is also dependent on CBP, and importantly circadian transcription responds to changes in limiting levels of the co-activator. These findings suggest a novel mechanism for cross-talk between CLK·CYC and other CBP-dependent transcription factors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Fly Stocks—w[*];;P{w[+mC] = hs-nej[+]}1 (hs-nej) and y[1] nej[Q7] v[1] f[1]/Dp(1;Y)FF1, y[+]/C(1)DX, y[1] w[1] f[1] (nejQ7) were obtained from Bloomington Drosophila stock center and EP(X)950, EP(X)1410, EP(X)1149, and EP(X)1179 were obtained from Szeged Drosophila stock center. nejQ7 flies were crossed with Canton S flies to obtain +/Dp(1;Y)FF1, y[+] flies (Dp(1;Y)FF1). hs-nej and Dp(1;Y)FF1 males were used as nej overexpressing flies together with ClkJrk for arrhythmic and wild-type Canton S and w1118 for rhythmic controls. The BGluc reporter gene (17) that expressed luciferase from a per promoter was crossed into these backgrounds to generate BGluc and BGluc;; ClkJrk as well as BGluc/Dp(1;Y)FF1 and BGluc;; hs-nej flies. EP lines were crossed with tim-GAL4 flies to generate EP(X)/Y; tim-GAL4/+ as shown in figures.

Locomotor Activity Assays—Flies were entrained during eclosion for 4 days in cycles of 12-h light and 12-h darkness (LD) at 20 or 22 °C. 1–4-day old flies were analyzed in locomotor activity assays using the DAM system IV (TriKinetics, Waltham, MA) for 3 days in LD cycles, with or without application of a 37 °C heat shock during the last hour in darkness and subsequently for 7 days in constant darkness at 20 or 22 °C. Data from the first 5 days in constant darkness were analyzed using Clocklab (Actimetrics, Wilmette, IL) and IandA software (17) to identify rhythmic flies and determine period estimates.

Real-time Bioluminescence Measurements—Bioluminescence from luciferase reporter gene expression in live flies was determined for 3 days in constant darkness as described previously (17) after entrainment of flies for 4 days in 12 h LD cycles. Rhythmic flies were identified by analysis of the first 3 days in constant darkness with IandA software (17).

Quantitative Real-time PCR (RT-PCR)—1–7-day old flies were harvested after incubation for at least 48 h in constant light at either constant 20 °C or after application of a 30-min heat shock at 37 °C. Heads were isolated and total RNA was purified by homogenization in peqGOLD TriFast (PEQLAB, Erlangen, Germany) following manufacturer's instructions. 1 µg of total RNA was reverse transcribed with random hexamer primers using the Quantitect Reverse Transcription kit (Qiagen, Hilden, Germany) following manufacturer's instructions. cDNA products were amplified in an ABI PRISM 7000 (Applied Biosystems, Foster City, CA). Forward (fwd) and reverse (rev) primers and probes for TaqMan quantitative RT-PCR were designed with the ABI PRISM Primer Express software (Applied Biosystems) as follows: n-synaptobrevin (fwd, GGC GGC GTG TAA GCA ATC; rev, CCC GCT GAA GGA GCA CAC TA; probe, 6-FAM-CGC TGC CAG GAC GAA AGT TTC TCG A-TAMRA); nej (fwd, GGT GCA AGT TCC ACG TCA TC; rev, AGT CGA TAC CGA GCT GAG TGG T; probe, 6-FAM-TCC TCG GCG GGC TCG GGT-TAMRA); pdp1 (fwd, CTT GGT CTT GGC CAC ATA ACC; rev, GGT TCG CGG ATC AAA GTC A; probe, 6-FAM-CGG CCG AGT CAA CAT TTT CGT TCG-TAMRA); per (fwd, CCA ATG GCA CCA ACA TGC T; rev, TGT GGC GTA TGG CGA ACT T; probe, 6-FAM-AGC AGC TAC AAG GTT CCC GAC GAG ATT C-TAMRA); tim (fwd, CTG GCT GCA GTT GGT CAT G; rev, TGG CTG CAC TGA TGG ACT TG; probe, 6-FAM-TCC CAG CGA TTG CAT TGG CTC CT-TAMRA); vri (fwd, CGT CCG GCT ATC CAA TAT ATC G; rev, GGA CAA CGG ATG CAA GTT AGA AG; probe, 6-FAM-TCG ATG AAC GGC AGC TCC AAC GA-TAMRA). n-synaptobrevin mRNA levels were determined as a constitutively expressed internal control. mRNA levels for individual clock genes were normalized toward n-synaptobrevin transcript levels and quantified by the 2-{Delta}{Delta}Ct method as described (18, 19) and according to the manufacturer's instructions (Applied Biosystems).

Western Blotting—CLK protein levels were determined by Western blot analysis as detailed in the legend of Fig. 4 using an antibody that was raised in rabbit against a peptide composed of the C-terminal 15 amino acids of CLK.

Immunohistochemistry—Third instar larvae were entrained for 3 days in cycles of 12 h LD, and the brains were dissected during the first day of constant darkness at times indicated in the figures. Circadian time (CT) 0 marks time of subjective "lights on" and CT12 marks time of subjective "lights off". The experiments were repeated twice, and for each time point at least 8 brains were dissected. Brains were fixed in 4% formaldehyde in PEM buffer (100 mM Pipes, pH 6.9, 1 mM EGTA, 2 mM MgSO4) for 2 h, blocked with 10% normal goat serum (NGS) in PT buffer (phosphate-buffered saline plus 0.3% Triton X-100) for 2 h at room temperature, and subsequently incubated for 48 h at 4 °C in 50 µl of primary antiserum solution containing 1:200 diluted rabbit anti-PER (Alpha Diagnostics, San Antonio, TX) and 1:1000 diluted guinea pig anti-PAP (20) in PT buffer with 10% NGS. Brains were rinsed with PT buffer and PT plus 5% NGS for 20 min each and then incubated at room temperature for 2 h in secondary antiserum solution containing 1:200 diluted TRITC-conjugated donkey anti-guinea pig (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and 1:200 diluted fluorescein isothiocyanate-conjugated goat anti-rabbit (Calbiochem) in PT buffer with 10% NGS. After incubation, brains were rinsed with PT buffer and PT plus 5% NGS for 20 min each and mounted in the mounting medium (50 mM Tris-Cl, pH 8, 90% glycerol, 2.5% DABCO (Sigma)).

Confocal Microscopy and Quantification of Staining—Optical sections of larval lateral neurons (LNs) were imaged on a Carl Zeiss LSM 510 META confocal microscope. For each CT, LNs from at least 16 brain hemispheres per fly strain were scanned. For each LN sample, PAP staining was used to identify and select an optical section, which was then scanned for PER immunoreactivity. Three images were taken per LN with single laser at 488 nm, 543 nm, and with both lasers together. Images taken with single 488-nm laser were imported to NIH ImageJ 1.34s, and the localization of neuronal PER staining was determined by the double-laser images. Total pixel intensity of PER staining was measured, and the mean ± S.E. of all brains at a particular CT is reported in the graphs.

Expression Constructs and Co-transfection Assays—Clk, E1A(12S), and a E1A(12S) construct that carries a deletion of the first 72 amino acids (E1A({Delta}N)) were cloned into XhoI and PmeI sites of pAc5.1/V5-HisA vector (Invitrogen) for expression in Drosophila S2 cells. pHT control vector, reporter plasmids pRLcopia for Renilla luciferase control and pGL3-(4-per-E-box)hs::luc+, pGL3-per::luc+, and pGL3-tim::luc+ for expression of firefly luciferase from a minimal heat-shock promoter with four per-E-box elements, a per promoter, and a tim promoter, respectively, are described previously (13, 21). Co-transfection assays were performed as described previously (13) with either 200 ng of pHT control, pAc-E1A(12S), or pAc-E1A({Delta}N) vectors. Averages of at least three independent experiments are shown as percent of control. Control is CLK·CYC-activated LUC activity set to 100% in the presence of pHT control vector. Expression levels of E1A constructs were determined by Western blot analysis using an anti-E1A(12S) antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

For dsRNA production, approximately 500-bp long DNA fragments of the coding region of target genes Clk, cyc, nej, and EGFP (enhanced green fluorescent protein) control were generated by PCR using primers that contained a 5' T7 RNA polymerase-binding site followed by gene specific sequences. Primers were as follows: Clk (fwd, TAA TAC GAC TCA CTA TAG GGG TGG TCT GCA CCC ATA AGG; rev, TAA TAC GAC TCA CTA TAG GGC GTG CGT CAT CAT GGA CT); cyc (fwd, TAA TAC GAC TCA CTA TAG GGT GGG TTG TGA CCG AGG AC; rev, TAA TAC GAC TCA CTA TAG GGA GTT GCG AAC GTT GGG C); nej (fwd, TAA TAC GAC TCA CTA TAG GGC AAT CTG ACG GGT CTG GTA GTG GAT; rev, TAA TAC GAC TCA CTA TAG GGT GGG TTG CTG CTG TTG TTG CTG ATG); EGFP (fwd, TAA TAC GAC TCA CTA TAG GGC ACA TGA AGC AGC ACG ACT T; rev, TAA TAC GAC TCA CTA TAG GGA CTG GGT GCT CAG GTA GTG G). Purified DNA fragments were in vitro transcribed using the T7 RiboMAX Express Large Scale RNA Production System (Promega, Madison, WI). The RNA products were isolated by using the SV Total RNA Isolation System (Promega) and annealed to dsRNA by incubation at 65 °C for 30 min and slow cooling to room temperature.


Figure 1
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  		FIGURE 1.
Overexpression of nej increases behavioral arrhythmicity. A, average locomotor activity over all flies of indicated genotypes for the last day in LD cycles and 5 days in constant darkness. B–D, overexpression of nej by heat-shock induction or the GAL4/UAS system. B, nej transcript levels, determined by quantitative RT-PCR, in w1118 control and hs-nej flies either after incubation at constant 20 °C (black bars) or after application of a 30-min heat shock at 37 °C (gray bars). Average nej mRNA levels ± S.E. from at least eight independent experiments per genotype are shown with nej mRNA levels in w1118 flies set to 100. C, representation of different EP-element insertions in the 5'-untranslated region of nej that either allow overexpression of nej from the UAS promoter (EP(X)1149 and EP(X)1179) or that contain an inverted insertion of the UAS promoter not allowing overexpression of nej (EP(X)950 and EP(X)1410). D, nej transcript levels, determined by quantitative RT-PCR, in EP lines with a tim-GAL4 driver. Average nej mRNA levels ± S.E. from at least four independent experiments per genotype are shown with nej mRNA levels in w1118 flies set to 100.

 
To test effects of dsRNAs on reporter gene expression in co-transfection assays, 250 µl of Drosophila S2 cells (2 x 106 cells/ml) in serum-free Schneider's insect medium (Sigma) were incubated with 7.5 µg of dsRNA for 45 min prior to addition of 250 µl of Schneider's medium containing 20% fetal bovine serum. Cells were incubated for 24 h, and co-transfection assays were performed essentially as described previously (13) with the difference that another 7.5 µg of dsRNA was added 45 min prior to the addition of 20% fetal bovine serum. Averages of at least three independent experiments are shown as percent of control. Control is CLK·CYC-activated LUC activity in the absence of dsRNA set to 100%.

Co-immunoprecipitation Experiments—FLAG-tagged CLK or CLKJrk as well as CYC and domains of NEJ were expressed from a pAc5.1 vector, which carried a SP6 promoter insertion in the KpnI site, in reticulocyte lysate using the SP6 quick-coupled transcription/translation system (Promega) as instructed by the manufacturer. NEJ domains include amino acids 1–897 (NEJ1), 898–1667 (NEJ2), 1668–2653 (NEJ3) and 2677–3190 (NEJ4). FLAG-tagged CLK or CLKJrk was expressed with unlabeled methionine to reduce background, whereas CYC and NEJ fragments were labeled with [35S]methionine. The immunoprecipitation was performed as described previously (22). In brief, 10 µl of NEJ-expressing lysate was pre-incubated with 10 µl of anti-FLAG-M2-conjugated-agarose (Sigma) in 150 µl of IB solution (5 mM Tris-Cl, 10 mM HEPES, pH 7.5, 10% glycerol, 50 mM KCl, 0.05% Triton X-100, 1 mM EDTA, 1 mM dithiothreitol, 1x complete protease inhibitor (Roche Diagnostics)) to eliminate nonspecific interaction. The agarose was then removed from the lysate. The NEJ lysate was added to different interaction reactions either with or without 10 µl of CLK or CLKJrk lysate in the absence or presence of 10 µl CYC lysate (as indicated in Fig. 3), and the final volume of the interaction mixture was adjusted to 500 µl with IB solution. The protein mixture was incubated at 25 °C with gentle rotation for 30 min. Subsequently, 10 µl of anti-FLAG-M2-conjugated-agarose, previously pre-blocked in IB solution with 1% milk, was added and incubated for 2 h at 25 °C. The beads were then collected and washed three times with 500 µl of IB solution for 10 min each. Proteins were eluted with 30 µl of 2x sodium dodecyl-sulfate (SDS) sample buffer, and radiolabeled proteins were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
We identified a circadian function of nejire (nej), which is the CBP ortholog in Drosophila, by screening of duplication mutants for phenotypes in circadian behavioral rhythms. A duplication of the X-chromosomal nej locus on the Y chromosome in Dp(1;Y)FF1 caused a large increase in the number of arrhythmic flies (Fig. 1A; Table 1). Specific overexpression of nej from a heat-shock promoter (hs-nej) resulted in a similar phenotype. Interestingly even a moderate increase in nej levels by about 45%, as observed in hs-nej flies in the absence of heat shock (Fig. 1B), was associated with a loss of behavioral rhythms in 71% of the flies (Table 1, Fig. 1A, and supplemental Fig. S1). The remaining rhythmic flies displayed a wild-type period length. This phenotype was reminiscent to the effects observed for the overexpression of Clk, which also increased the number of arrhythmic flies without affecting period length (23). A similar increase in arrhythmicity of nej overexpressing flies was observed after entrainment in temperature cycles under subsequent free-running conditions (Table 1). This finding indicated that nej did not affect a specific entrainment pathway but the circadian clock itself. To further test specificity of the behavioral phenotype, we overexpressed nej from a UAS enhancer after transcription of a GAL4 driver from a tim promoter (tim-GAL4). Several EP lines carry an insertion of the UAS enhancer in the 5'-untranslated region of nej (24). EP(X)1179 overexpressed nej by the tim-GAL4 driver, whereas two lines, EP(X)950 and EP(X)1410, carried the UAS enhancer in the inverse orientation, serving as a control that lacked nej overexpression (Fig. 1, C and D). Overexpression of nej by tim-GAL4 in EP(X)1179 resulted in an increase of behavioral arrhythmicity, without significant effects on general activity levels (Table 1). In contrast, EP(X)950 and EP(X)1410 showed wild-type rhythms. These results demonstrate that nej gain of function causes an increase in the number of behaviorally arrhythmic flies.


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  		TABLE 1
Results of free-running locomotor activity measurements

 
To analyze whether nej has a role in the molecular oscillator itself, we investigated the circadian profile of clock gene expression by real-time monitoring of CLK·CYC-dependent expression of a luciferase reporter from a per promoter in individual BGluc flies (25). After entrainment of BGluc flies in cycles of 12-h light and 12-h darkness, CLK·CYC-dependent luciferase bioluminescence was monitored for 3 days in constant darkness. Luciferase expression oscillated in BGluc control flies, whereas the antimorphic ClkJrk mutation caused molecular arrhythmicity (Fig. 2A). A moderate overexpression of nej from the heat-shock promoter at 20 °C induced arrhythmicity in 40% of hs-nej flies (Fig. 2A). Similarly, duplication of the nej locus caused molecular arrhythmicity in 64% of the flies (Fig. 2A). These results show that overexpression of nej affects the ability to maintain free running molecular rhythms, consistent with the observed increase of behaviorally arrhythmic flies.

We also analyzed PER protein expression in PDF positive pacemaker lateral neurons of larval brains from EP lines that carried a tim-GAL4 driver (Fig. 2, B and C and data not shown). Although EP(X)950 and EP(X)1410 control flies displayed normal PER oscillations with trough levels at CT 10 and a peak at CT 24, the EP(X)1179 line showed a strongly reduced amplitude of PER protein oscillations. For both time points PER staining intensities in nej overexpressing EP(X)1179; tim-GAL4 flies were similar to trough PER levels in the EP(X)950; tim-GAL4 control line (supplemental Fig. S2). In summary the data show that gain of nej function is associated with a strong increase in molecular and behavioral arrhythmicity.


Figure 2
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  		FIGURE 2.
Overexpression of nej increases molecular arrhythmicity. A, average real-time luciferase bioluminescence rhythms over all rhythmic or arrhythmic BGluc flies of indicated genotypes for the last day in LD cycles and 3 days in darkness. Number of rhythmic (upper panels) or arrhythmic flies (lower panels) versus total number of flies is given in brackets together with percentage. B, PER immunohistochemistry in PDF expressing lateral neurons of larval brains harvested at CT 10 and 24 during the first day in constant darkness after entrainment in LD cycles (CT 0 is subjective time of lights on; CT 12 is subjective time of light off) from genotypes as indicated in the figure. The PER signal from double-stained brains is shown in green; and the signal from cytoplasmic PDF precursor PAP is in red; yellow indicates co-localization of PER and PAP. C, mean ± S.E. staining intensity of PER in immunohistochemistry images as shown in B for at least 14 brain hemispheres per genotype and time point.

 
In contrast to gain of function, a nej loss of function cannot be analyzed because of embryonic lethality. Two heterozygous nej loss of function mutants (nej3 and nejQ7) did not display a phenotype because of the remaining wild-type allele (data not shown). The lack of a period phenotype for nej partial loss of function is reminiscent to the effects of decreased CLK·CYC activity in partial Clk loss of function flies, which show a reduced amplitude but wild-type period length of circadian oscillations (26). However, the insertion of the UAS enhancer in the 5'-untranslated region of nej in EP(X)1149 flies has also been shown to cause a nej partial loss of function, when investigated in the absence of a GAL4 driver (24). We observed a severe long period phenotype in EP(X)1149 flies (Table 1). However, this phenotype did not segregate with the EP-element insertion. Sequencing revealed that this line carried an additional mutation in the per locus that substitutes serine 45 to tyrosine and has previously been coined perSLIH (27). The perSLIH mutation causes a temperature-sensitive phenotype with a long period oscillation of about 27.6 h at low temperature and a shorter period length at higher temperatures. Because nej can be overexpressed in EP(X)1149 flies by a tim-GAL4 driver (Fig. 1D), we analyzed the effects of nej overexpression on the perSLIH phenotype. In the presence of the tim-GAL4 driver, overexpression of nej in EP(X)1149 caused a strong increase in arrhythmic flies, similar to the observations in EP(X)1179 (Fig. 1A, Table 1). The remaining rhythmic flies showed, however, a wild-type period length at low temperatures demonstrating that nej overexpression suppressed the temperature-sensitive effect of the perSLIH mutation. The genetic interaction between per and nej supports an intrinsic clock function of nej as a co-activator for CLK·CYC-dependent transcription.

To directly test this hypothesis we first analyzed the effects of decreased nej activity on CLK·CYC function in cell culture. CLK·CYC-dependent luciferase expression from a four per-E-box containing reporter in cell culture (21) was strongly reduced by co-expression of the adenoviral E1A(12S) protein (Fig. 3A), a well characterized inhibitor of CBP/p300 (28). Specificity of NEJ/CBP inhibition was verified by co-expression of E1A({Delta}N), a truncated construct that lacked the N-terminal CBP/p300 binding domain, which showed no inhibition of CLK·CYC activity. The same results were obtained using a reporter construct that expressed luciferase from a genomic fragment of the tim or per promoter (Fig. 3B and data not shown). Both E1A(12S) and E1A({Delta}N) were expressed to similar levels (Fig. 3C). To confirm the specificity of reduced CLK·CYC function by inhibition of nej, we analyzed the effects of a knock-down of nej expression by small interfering RNAs (siRNA) in the co-transfection assay (Fig. 3D). siRNA targeting either Clk or cyc expression strongly reduced CLK·CYC activity as expected. In contrast, control siRNA targeting the EGFP gene had no effect on CLK·CYC function. When siRNA targeting nej transcription was included in the reporter assay, CLK·CYC activity was significantly reduced. The decrease in CLK·CYC-dependent transcription was almost proportional to the reduction in nej mRNA levels (compare Fig. 3D and supplemental Fig. S3). An almost complete inhibition of CLK·CYC activity (less than 10%) was observed after reduction of nej transcription by siRNA and additional inhibition of residual NEJ by E1A(12S) co-expression (supplemental Fig. S3). These results strongly suggest that nej plays an important role for CLK·CYC-dependent transcription.


Figure 3
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  		FIGURE 3.
NEJ co-activates CLK·CYC-dependent transcription and physically interacts with CLK and CLK·CYC complexes. CLK·CYC-dependent luciferase reporter gene expression in Drosophila Schneider 2 cells expressing luciferase either from a four per-E-box containing promoter (A and D) or a genomic fragment of the tim promoter (B). A and B, CLK·CYC-dependent luciferase reporter gene expression in the absence (control) or presence of co-expressed E1A(12S) or E1A({Delta}N). C, Western blot analysis of E1A(12S) and E1A({Delta}N) expression in S2 cells after transfection of 2 µg for each expression construct or for untransfected control. Results shown are from the same blot and exposure. D, CLK·CYC-dependent luciferase reporter gene expression in the absence (control) or presence of siRNA targeting either EGFP, Clk, cyc, or nej. Values are mean ± S.E. LUC activities expressed as a percentage of the control value (set to 100%) in the absence of nej inhibitors or siRNA. E, representation of NEJ fragments that were cloned and expressed in reticulocyte lysate for interaction studies (cysteine/histidine rich zinc finger domains, C/H1, C/H2, and C/H3; CREB binding domain, CBD; histone acetyl transferase domain, HAT). F–I, autoradiograph images from interaction studies with [35S]methionine-labeled proteins. F, input (15%) controls for [35S]methionine-labeled FLAG-CLK (lane 1), FLAG-CLKJrk (lane 2), and CYC (lane 3) as well as precipitation of FLAG-CLK (lane 4) or FLAG-CLKJrk (lane 5) with anti-FLAG antibody. G, co-immunoprecipitation of 35S-CYC (lane 1, input) with anti-FLAG beads alone (lane 2) or with non-radiolabeled FLAG-CLK (lane 3) or FLAG-CLKJrk (lane 4). H, co-immunoprecipitation of 35S-labeled domains of NEJ with non-labeled FLAG-tagged CLK with or without 35S-CYC by anti-FLAG antibody (lanes 1, 5, 9, 13 input controls for NEJ fragments; lanes 2, 6, 10, 14 control pull down in the absence of CLK; lanes 3, 7, 11, 15 pull down in the presence of CLK; lanes 4, 8, 12, 16 pull down in the presence of CLK and 35S-CYC). I, co-immunoprecipitation of NEJ 1 and NEJ 3 fragments with non-radiolabeled FLAG-tagged CLK or CLKJrk in the presence or absence of 35S-CYC as in H (lanes 1 and 7 input controls for NEJ fragments; lanes 2 and 8 pull down in the absence of CLK or CLKJrk; lanes 3 and 9 pull down with CLK; lanes 4 and 10 pull down with CLK and 35S-CYC; lanes 5 and 11 pull down with CLKJrk; lanes 6 and 12 pull down with CLKJrk and 35S-CYC).

 
A direct interaction between NEJ and the CLK·CYC complex would be expected, if NEJ acts as a transcription co-activator for CLK·CYC-dependent transcription. Yeast two-hybrid assays indicated a binding between CLK and the N terminus of NEJ (data not shown). To map the interaction between Drosophila NEJ and either CLK or CLK·CYC, we performed co-immunoprecipitation experiments of reticulocyte lysate-expressed domains of NEJ (Fig. 3E) with FLAG-tagged CLK in the presence or absence of CYC (Fig. 3F). The intensity of the CYC signal served as a control for the efficiency of co-immunoprecipitation with CLK because binding of CYC was specific (Fig. 3G). We identified two domains of NEJ that bound to CLK as well as to CLK·CYC complexes (Fig. 3H), the N-terminal fragment from amino acid 1 to 898 (NEJ 1) that also interacted with nuclear hormone receptors such as the retinoic acid (RAR, RXR), estrogen, progesterone, thyroid hormone, and glucocorticoid receptors (29), as well as the middle domain of NEJ from amino acids 1668 to 2653 (NEJ 3) that contained the histone acetyl transferase activity and the zinc finger domains C/H2 and C/H3. Binding of the N-terminal NEJ 1 fragment to CLK was independent of CYC, whereas the interaction of the middle domain NEJ 3 to CLK appeared to be enhanced by the presence of CYC (Fig. 3H). The interaction was independent of the glutamine-rich domain of CLK because both fragments of NEJ bound to the C-terminally truncated CLKJrk protein that lacked this region (Fig. 3I). The antimorphic nature of the CLKJrk mutation may be because of effects on the histone acetyl transferase activity of CLK that has previously been identified in mammals (30). Our results show that NEJ physically interacts through two binding sites, the N terminus, and a middle domain, with an N-terminal part of CLK, which is consistent with a role of NEJ as a co-activator for CLK·CYC-dependent transcription in Drosophila. The interaction of the mammalian CLK·BMAL1 complex with CBP and p300 family members suggests that this function is conserved between the invertebrate and vertebrate clock (31–33), although mutant phenotypes await to be investigated in a mammalian model.

We next addressed the question of whether recruitment of NEJ to the CLK·CYC complex is a constituent of the basal transcription machinery or NEJ is a regulatory component of the circadian clock. NEJ as well as the mammalian ortholog of NEJ, CBP, affects transcription through different modes of action: 1) through histone acetylation; 2) by mediating interactions between transcription factors and the basal transcription machinery (co-activation); 3) by mediating cross-talk between transcription factors (29). Previous studies in mammals showed that the cellular concentration of CBP is limiting, and CBP-dependent transcription factors such as AP1, nuclear hormone receptors, the JAK/STAT pathway, and E2F compete for recruitment of CBP, thereby establishing a CBP-mediated cross-talk between these signaling pathways (14–16). The molecular arrhythmicity induced by overexpression of NEJ in flies suggests that NEJ is a limiting factor for CLK·CYC activity as well. To further analyze a co-activator function of NEJ for CLK·CYC-mediated transcription in vivo and to test whether trans-activation by CLK·CYC responds to changes in nej levels, we assayed constitutive CLK·CYC-dependent transcription in wild-type and nej mutant flies under constant light conditions, when the Drosophila circadian clock did not oscillate. Under such conditions at least the per/tim feedback loop is not functional because of degradation of PER and TIM proteins in light. Therefore feedback mechanisms that may compensate for an increase or decrease of CLK·CYC activity are at least partially non-functional, which allows to monitor effects on CLK·CYC activity in flies more directly. tim and pdp1 transcript levels were decreased in the nej partial loss of function mutant EP(X)1149 compared with EP(X)950 control, consistent with a co-activator function of nej for CLK·CYC-mediated transcription (Fig. 4, A, C, and E). hs-nej flies showed an induction of tim and pdp1 mRNA levels upon heat shock, whereas wild-type flies displayed no significant changes or rather decreased transcript levels (Fig. 4, B, D, and F). Similar results were obtained for per and vri expression (Supplemental Fig. S4). CLK levels were not significantly different in these fly strains and conditions as determined by Western blotting (Fig. 4, G and H). These results show that overexpression of nej increases CLK·CYC-dependent transcription by ~50% suggesting that under wild-type conditions about one-third of CLK·CYC complexes lack NEJ to reach maximal activity. The decrease in CLK·CYC-dependent transcripts in EP(X)1149 flies is proportional to the decrease in nej levels (Fig. 4, A, C, and E). These experiments further demonstrate a co-activator function of NEJ for CLK·CYC activity, and they show that trans-activation by CLK·CYC responds to changes in limiting cellular levels of NEJ.


Figure 4
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  		FIGURE 4.
CLK·CYC-dependent transcription is sensitive to cellular levels of NEJ in flies. A–F, results from quantitative RT-PCR for transcript levels of nej (A and B), tim (C and D), and pdp1 (E and F) from fly heads of indicated genotypes. Flies were previously incubated in constant light for at least 48 h and harvested either at 20 °C (black bars) or after application of a 30-min heat shock at 37 °C (gray bars). Average mRNA levels ± S.E. from at least five independent experiments per genotype are shown with mRNA levels in EP(X)950 (A, C, and E) or in the absence of heat shock (B, D, and F) set to 100. G and H, Western blot analysis of CLK levels for genotypes and conditions as in A–F showed no effect of NEJ levels on CLK expression. Representative Western blot results are shown with indicated CLK and an asterisk marking an unspecific band detected by the anti-CLK antibody, which served as a loading control (EP(X)950 (lane 1), EP(X)1149 (lane 2), w1118 at 20 °C (lane 3), w1118 after heat shock (lane 4), hs-nej at 20 °C (lane 5), hs-nej after heat shock (lane 6)). Mean CLK levels ± S.E. from two or three experiments are shown below with CLK levels in EP(X)950 (G) or in the absence of heat shock (H) set to 100.

 
Consistent with our results, a parallel study by Lim et al. (34) found a role for nej in the Drosophila circadian clock. From the finding of low expression levels of CLK·CYC-dependent genes after constitutive overexpression of nej it was concluded that NEJ acted as a repressor on CLK·CYC function. We show, however, that a reduction in nej levels results in a proportional decrease in CLK·CYC activity in flies as well as in cell culture, and induction of nej expression in flies induces CLK·CYC-dependent transcripts, which demonstrates that NEJ acts as a co-activator for CLK·CYC function. Reduced levels of CLK·CYC-dependent genes observed after constitutive overexpression of nej (see also PER levels in Fig. 2 and supplemental Fig. S2) are likely because of circadian feedback regulation that compensates for hyper-activation of CLK·CYC by NEJ.

The involvement of NEJ in the functional organization of the Drosophila circadian clock adds additional modes to the regulation of circadian transcription. The finding that moderately reduced or increased levels of NEJ decrease or enhance CLK·CYC-dependent transcription, respectively, demonstrates that CLK·CYC activity is sensitive to limiting levels of NEJ, and availability of NEJ regulates circadian transcription (Figs. 1 and 4). Studies on transcriptional activation by AP1, nuclear hormone receptors, the JAK/STAT pathway, and E2F have shown that multiple transcription factors compete for recruitment of the mammalian ortholog CBP from a limiting pool, thereby establishing negative cross-talk between these pathways (14–16). Our results suggest that CLK·CYC joints this competition for recruitment of NEJ (or CBP in mammals) allowing a cross-talk between circadian transcription and NEJ/CBP-dependent transcriptional regulation of metabolism, development, cell proliferation, memory formation, and other physiological processes. In addition it has been shown that a number of signaling pathways such as cyclic nucleotides, calcium, and Ras/MAPK regulate NEJ-dependent transcription through both, direct phosphorylation of NEJ as well as phosphorylation-dependent recruitment of NEJ to target transcription factors (35). The regulation of CLK·CYC-dependent transcription by these signaling pathways, is in addition to direct phosphorylation of CLK (13), likely mediated through the control of NEJ co-activator function. NEJ thereby provides an interface for cross-talk between circadian transcription and vital physiological processes, which likely assists the circadian orchestration of life.


   FOOTNOTES
 
* This work was supported by the National Institutes of Health Grant MH51573 (to S. A. K.) and by an Emmy-Noether Fellowship (WE2608/1-2) of the Deutsche Forschungsgemeinschaft (to F. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Biochemie-Zentrum Heidelberg, Ruprecht-Karls Universität Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. Tel.: 49-6221-548573; Fax: 49-6221-544769; E-mail: Frank.Weber{at}bzh.uni-heidelberg.de.

3 The abbreviations used are: CLK, CLOCK; CBP, CREB-binding protein; CYC, CYCLE; PER, PERIOD; TIM, TIMELESS; MAPK, mitogen-activated protein kinase; STAT, signal transducers and activators of transcription; LD, light and darkness; RT, real-time; fwd, forward; rev, reverse; Pipes, 1,4-piperazinediethanesulfonic acid; NGS, normal goat serum; TRITC, tetramethyl-rhodamine isothiocyanate; LN, lateral neurons; LUC, luciferase; dsRNA, double-stranded RNA; EGFP, enhanced green fluorescent protein; nej, nejire; siRNA, small interfering RNAs; CT, circadian time; UAS, upstream activating sequence; PDF, pigment dispersing factor; PAP, PDF-associated peptide. Back


   ACKNOWLEDGMENTS
 
We thank Hilmar Bading for E1A(12S); Sarah Smolik for nej; Paul Taghert for PAP antibody; Tatjana Wüst for technical assistance; Justin Blau for discussing data prior to publication; and Paul Hardin, Urs Albrecht, Charlotte Förster, Michael Young, and Michael Brunner for helpful discussions and comments on the manuscript.



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 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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P. Taylor and P. E. Hardin
Rhythmic E-Box Binding by CLK-CYC Controls Daily Cycles in per and tim Transcription and Chromatin Modifications
Mol. Cell. Biol., July 15, 2008; 28(14): 4642 - 4652.
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