drosophila circadian locomotor rhythms are generated by subsets of clock - containing neurons in the brain . under light - dark ( ld ) cycles , the small ventral lateral neurons ( s - lnvs ; m oscillators ) and a small subset of lateral and dorsal neurons named e oscillators drive morning and evening activity peaks . in constant darkness ( dd ) , the s - lnvs control the synchrony of clock neurons and drive behavioral rhythms , thus serving as the master pacemakers [ 35 ] .
nuclear receptors are ligand - dependent transcription factors that regulate diverse biological processes . a  number of nuclear receptors are known to play key roles in the molecular clock and its output pathways in mammals . with the exception of unfulfilled ( unf ; dhr51 ) , which is required for the pacemaker function of the s - lnvs , whether nuclear receptors provide important regulatory points in drosophila circadian rhythms remains unclear . to test the requirements of the nuclear receptors in drosophila circadian rhythms , we sought to knock down each of the 18 nuclear receptor genes in the s - lnvs .
we chose to use recently generated uas - micrornas ( mirnas ) targeting fly nuclear receptors because each uas line polycistronically expresses two independent mirnas , which permit the efficient silencing of the target with a minimum off - target effect [ 8 , 9 ] .
we expressed uas - mirnas with the lnv - specific gal4 driver , pdf - gal4 or gal1118 , and analyzed the locomotor activities of the flies in ld and dd .
consistent with our previous results , the knockdown of unf rendered flies arrhythmic in dd .
additionally , the knockdown of e75 ( also known as ecdysone - induced protein 75 , eip75b ) in the lnvs led to a similarly high proportion of arrhythmia in dd ( figures s1a and s1b ) .
the knockdown of seven - up ( svp ) or estrogen - related receptor ( err ) had a moderate effect on free - running rhythms ( table 1 ) .
the e75 gene produces three isoforms of e75 , named e75a , e75b , and e75c , all of which share a large part of the c - terminal region , including the ligand - binding domain , but differ in their n terminus structures . despite these differences ,
all the isoforms are functionally redundant to some extent in ecdysone - induced developmental processes .
e75 isoform redundancy is at least partly explained by the fact that all three isoforms , including e75b ( which lacks dna - binding domain ) , can heterodimerize with the dhr3 nuclear receptor and repress its transcriptional activity [ 12 , 13 ] .
our previous rna analysis from isolated lnvs showed that all e75 isoforms are expressed in both larval and adult lnvs [ 14 , 15 ] .
the uas - mirnas against e75 targets two sequences in the common c - terminal domain , thereby enabling the knockdown of all the isoforms .
expression of uas - e75 mirnas under the control of ubiquitously expressed tubulin - gal4 driver caused near 100% embryonic lethality , which recapitulates the lethal phenotype of the e75 null mutants lacking all isoforms .
by contrast , expression of uas - rnai targeting e75 from the vienna drosophila resource center ( vdrc ) collection with tubulin - gal4 had no effect on viability ( figure  s2a ) .
lnv - targeted expression of e75 mirnas rendered flies arrhythmic , whereas expression of either of the two vdrc uas - rnai lines had no effect on the locomotor rhythms ( figure  s2b ) .
these results indicate that uas - mirnas efficiently silences the e75 gene and confirm that behavioral arrhythmia is specifically caused by the reduction of the e75 expression in the lnvs .
e75 is a homolog of mammalian rev - erb  and rev - erb  , which are important transcriptional regulators contributing to the molecular clockwork and the clock output [ 1618 ] .
intrigued by the high proportion of arrhythmia in the knockdown and the relevance to the circadian rhythms in mammals , we further investigated how e75 contributes to the behavioral rhythm generation in the lnvs .
first , we overexpressed e75a , the isoform containing both dna - binding and ligand - binding domains , in the lnvs .
these flies showed no differences in locomotor behavior compared with the control ( figures s1a and s1b ) .
thus , loss of function , but not overexpression , of e75 in the lnvs impairs the free - running locomotor rhythms . because the knockdown of dhr3 had no effect on behavioral rhythms ( table 1 )
, the e75/dhr3 heterodimer is not a likely candidate that is involved in the behavioral control in the s - lnvs .
we next examined the effect of e75 loss of function in developing and adult lnvs separately via stage - specific knockdown using a combination of uas - e75 mirnas , pdf - gal4 , and a temperature - sensitive gal80 expressed under the control of the tubulin promoter ( tub - gal80 ) . to analyze the role of e75 in the developing lnvs
, we raised these flies until eclosion at 29c ( the restrictive temperature of gal80 ) , enabling the pdf > e75 mirnas expression .
the eclosed flies were transferred to 18c ( the permissive temperature of gal80 ) to stop the expression of e75 mirnas and subsequently tested for locomotor activity under this condition . strikingly , 100% of these flies were arrhythmic in dd .
this is consistent with the results of the e75 constitutive knockdown performed at 25c . by staining for pdf and per
, we found that all the pdf - positive s - lnvs were undetectable , whereas the l - lnvs and the pdf - negative fifth s - lnv were normal in number and morphology ( figure  1c ) .
although this does not exclude the possibility that the s - lnvs are still present , it nevertheless implies that at least multiple genetic programs are severely impaired in the s - lnvs .
these results indicate that e75 is critically required for the development of the s - lnvs , which are the master pacemaker neurons for free - running rhythms ; therefore , e75 gene silencing during development renders adult flies arrhythmic in dd .
to determine whether e75 in adult lnvs contributes to the generation of locomotor rhythms , we next knocked down e75 only during adulthood by raising the flies carrying uas - e75 mirnas , pdf - gal4 , and tub - gal80 at 18c until eclosion and incubating the adult flies at 29c . the adult - specific lnv - targeted e75 knockdown lengthened the free - running period by over 3  hr ( figures 1d and 1e ) . by staining brains at six time points on the third day in dd ( dd3 ) with anti - pdf / per double staining
, we found that both s - lnvs and l - lnvs were present and morphologically normal in the adult - specific e75 knockdown flies ( figure  1f ) , but per levels and oscillations were substantially reduced in the s - lnvs in dd ( figures 2a and 2d ) .
these results indicate that e75 is indispensable for robust free - running molecular rhythms in adult s - lnvs .
in contrast , per rhythms in the dorsal lateral neurons ( lnds ) were unaffected by this manipulation ( figures 2b and 2d ) . in the dorsal neurons 1 ( dn1s ) , the phase of per rhythms was approximately 12  hr delayed in the knockdown flies compared with the control , indicating that e75 knockdown in adult lnvs non - cell - autonomously slowed down the clocks in the dn1s by approximately 4  hr per day ( figures 2c and 2d ) .
the pace of the dn1 clocks matches the behavioral pace in the e75 knockdown flies , suggesting that the dn1s compensate for the dampened rhythms in m oscillators and help drive the behavioral rhythms in these flies .
these results are consistent with the previous findings that modulation of the properties of the s - lnvs alters the phase and amplitude of the dn1s [ 7 , 20 , 21 ] , and the dn1s can play a major role in behavioral rhythm generation when s - lnvs are dysfunctional [ 2124 ] . taken together with the results of the e75 developmental knockdown , we conclude that e75 is essential for the development of the s - lnvs and for the molecular clockwork in adult s - lnvs . similar to e75 , the nuclear receptor unf ; dhr51 is expressed in the lnvs throughout development and adulthood and is required for the proper functioning of the s - lnvs as the circadian master pacemakers [ 7 , 15 ] .
unf knockdown in developing lnvs does not affect the gross structure of the lnvs but leads to the complete disruption of free - running clocks in adult s - lnvs .
unf downregulation in adult lnvs dampens their molecular oscillation in dd , resulting in long - period free - running rhythms .
thus , the loss of function of unf in adult lnvs partially phenocopies that of e75 .
this further suggests that unf and e75 might function in the same pathway controlling the molecular clocks in the s - lnvs .
we next addressed this possibility by testing the genetic interaction between unf and e75 in adult lnvs .
interestingly , unf and e75 double knockdown in adult lnvs rendered flies arrhythmic in dd .
overexpression of e75 did not alter the lengthened free - running period in the unf knockdown flies ( figures s3a and s3b ) . because behavioral rhythms were normal when double knockdown flies were kept at 18c throughout developmental and adult stages , behavioral arrhythmia in the adult - specific knockdown flies was not caused by leaky mirna expression during development ( figure  s3c ) .
these results indicate that unf and e75 genetically interact in the pathway controlling the molecular clockwork in the s - lnvs , but e75 is unlikely to be downstream of unf .
to understand the mechanisms by which e75 controls molecular clockwork in the s - lnvs and how unf takes part in this process , we sought to identify the e75 downstream target genes . because the adult - specific e75 knockdown in the lnvs not only diminishes the molecular oscillation but also dramatically reduces per levels ( figure  2a and 2d )
, we speculated that e75 controls the transcription of the gene(s ) directly involved in the molecular oscillation .
because the dna - binding domains of e75 and mammalian rev - erb  and rev - erb  are highly conserved , we used the well - characterized rev - erb  and rev - erb  binding sequences to find potential e75 binding sites in drosophila core clock genes .
rev - erb / is known to bind to the ror element ( rore ) ( [ a / t]a[a / t]nt[a / g]ggtca ) as a monomer .
interestingly , a consensus rore was found in the first intron of the per gene .
moreover , the rore is located near the clk / cyc binding peaks identified in   ( figure  3c ) .
these bioinformatic analyses predict that e75 could modulate the clk / cyc - mediated transcription of per . to test  this possibility , we performed transactivation assays in schneider 2 ( s2 ) cells , which permit the analysis of transcriptional regulation in a non - cycling context .
we transiently transfected s2 cells with varying combinations of clk , e75a , and unf expression constructs and analyzed the endogenous per and tim mrna levels by qpcr ( figure  3a ) . in agreement with a previous study , because cyc is expressed in s2 cells , the expression of clk alone significantly upregulated the per and tim rna levels .
unf or e75 alone or together but without clk had no effect on the per and tim rna levels .
when either unf or e75 was co - expressed with clk , we observed a small but non - significant increase of per mrna levels compared with clk transfection alone .
strikingly , the addition of unf and e75 together to clk dose dependently increased per mrna levels by at least 4-fold .
neither unf nor e75 affected the tim rna levels when co - expressed with clk . to examine the extent to which unf and e75 transcriptionally control per expression , we generated luciferase reporters to monitor transcription from wild - type per promoter ( per - luc ) and per promoter lacking rore ( delta rore - luc [ drore - luc ] ) in s2 cell co - transcription assay .
expression of unf significantly increased clk - dependent transcription from both promoters , whereas e75 caused only a slight and non - significant increase .
unf / e75/clk co - expression yielded higher transcriptional activity than unf / clk transfection ; specifically , the increase of the drore - luc activity was statistically significant . intriguingly , drore - luc activity was significantly higher than per - luc in both unf / clk and unf / e75/clk co - transfections ( figure  3b ) .
this suggests that transcriptional repression through rore is involved in the overall transcriptional control of per , but other domains within the reporter construct mediate net transcriptional activation .
compared to the strong upregulation of per mrna level by unf / e75/clk co - transfection ( figure  3a ) , the difference between unf / clk and unf / e75/clk transfection on per - luc activation was less evident .
this probably reflects that regulatory sequences outside the region cloned in the luciferase reporters are also involved in the regulation of per transcription .
together with the data indicating that unf / e75 co - transfection synergistically activates transcription of drore - luc , these results suggest that unf upregulates clk / cyc - mediated per transcription and e75 enhances this process , at least via proximal and intronic promoters , except rore . to test whether unf and e75 bind to per regulatory sequences and thereby regulate transcription , we performed chromatin immunoprecipitation ( chip ) in s2 cells transfected with epitope - tagged clk , unf , and e75 and analyzed the binding by qpcr . as expected , clk was bound to the per gene regulatory sequences around circadian regulatory sequence ( crs ) , rore , and a downstream e - box ( e8 ) , reminiscent of prior chip studies [ 26 , 28 ] ( figure  s3d ) .
strikingly , although e75 binding was unaffected by the co - expression of unf , unf binding was approximately 3- to 4-fold reduced when co - expressed with e75 ( figure  3c ) .
these data suggest that both unf and e75 can independently bind to per regulatory sequences , but the presence of e75 increases the turnover of unf binding .
this high turnover of unf binding is concomitant with higher transcriptional activity ( figure  3d ) .
this model is in concert with the notion that transcriptional activation from highly regulated promoters often requires the turnover of the nuclear receptors and their co - regulators . taken together with the behavioral results ( figure  s3a ) , these in  vitro data suggest that coordinated action of e75 and unf on per transcription is essential for the molecular clock machinery in the s - lnvs .
here , we identified the nuclear receptors e75 and unf as components of the molecular clocks in the s - lnvs .
e75 is the closest homolog of mammalian rev - erb  and rev - erb  , which play important roles in the molecular clock feedback loops .
in contrast with rev - erb / , which represses transcription , our results demonstrated that e75 is neither a potent repressor nor a strong activator but potentiates the activation of per transcription by unf . despite these mechanistic divergences ,
the notion that rev - erb homologs are integral to the molecular oscillators in both drosophila and mammals highlights the significance of transcriptional regulations via nuclear receptors in metazoan circadian clocks .
rev - erb  and rev - erb  are rhythmically transcribed by the clock / bmal1 transcriptional activators , and rev - erbs periodically repress the transcription of bmal1 , thereby forming a feedback loop to ensure robust molecular oscillations of the mammalian clock .
a previous study demonstrated that e75 is a cycling target of clk / cyc in the fly head . because e75 has three isoforms , we were unable to determine whether any of the isoforms were rhythmically expressed in the lnvs from the rna profiles of the isolated lnvs .
nonetheless , our results indicate that e75 together with unf ( which is not a clk / cyc target ) reinforces the main loop of the core fly clock composed of clk / cyc and per / tim through a feedforward mechanism , showcasing the mechanistic parallels between fly and mammalian clocks .
e75 has been demonstrated to covalently bind to heme , and its binding appears to stabilize the e75 and facilitates the binding of nitric oxide ( no ) and carbon monoxide ( co ) .
the no / co binding to e75 modulates the transcriptional activity of its known heterodimeric partner dhr3 [ 13 , 30 ] . to test whether similar mechanisms are involved in the action of e75 in the s - lnvs
, we attempted to disrupt cellular heme metabolism by knocking down the enzymes in the heme biosynthesis pathway , coproporphyrinogen oxidase ( coprox ) and protoporphyrinogen oxidase ( ppox ) , and the key enzyme in the heme degradation pathway , heme oxygenase ( ho ) .
these experiments were inconclusive , as we did not observe any effect on the behavioral rhythms by any knockdown with pdf - gal4 , and knockdown with tim - gal4 was lethal ( data not shown ) .
s2 cell experiments showed that unf is a transcriptional activator of per , and concurrent expression of e75 and unf increases the turnover of unf binding to per regulatory sequences .
the finding that e75 acts through unf on transcription is consistent with our in  vivo data : ( 1 ) depletion of both unf and e75 in adult lnvs abolishes the behavioral rhythms ( figure  s3a ) ; ( 2 ) e75 overexpression has no effect on the behavioral rhythms ( figure  s1a ) ; and ( 3 ) e75 overexpression does not rescue unf knockdown ( figure  s3a ) .
although unf mrna levels do not oscillate , unf protein levels cycle in the s - lnvs , peaking at zeitgeber time ( zt)2 and lowest at zt14 .
indeed , per is most actively transcribed around zt13   when unf levels are minimum in the s - lnvs .
nonetheless , downregulation and arrhythmia of per levels in the s - lnvs is most probably not the sole cause of the altered locomotor rhythms in the unf knockdown , e75 knockdown , and unf / e75 double knockdown .
a recent study showed the implication of e75 in the repression of clk transcription , although our results are not in concordance with this observation probably due to the differences in the reagents used for e75 knockdown and the timing of knockdown . deciphering whether e75 and unf heterodimerize or bind to adjacent sequences , how they cooperate with clk / cyc , and whether any ligand is involved in their transcriptional regulation will yield new insights into the diverse mode of nuclear receptor crosstalk and their critical roles in circadian biology .
 summarycircadian clocks in large part rely on transcriptional feedback loops . at the core of the clock machinery , the transcriptional activators clock / bmal1 ( in mammals ) and 
 clock / cycle ( clk / cyc ) ( in drosophila ) drive the expression of the period ( per ) family genes . 
 the per - containing complexes inhibit the activity of clock / bmal1 or clk / cyc , thereby forming a negative feedback loop [ 1 ] . 
 in mammals , the ror and rev - erb family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the  generation of robust circadian molecular oscillation [ 2 ] . despite the overall similarities between mammalian and drosophila clocks , whether comparable mechanisms via nuclear receptors are required for the drosophila clock 
 remains unknown . 
 we show here that the nuclear receptor e75 , the fly homolog of rev - erb  and rev - erb  , and the nr2e3 subfamily nuclear receptor unf are components of the molecular clocks in the drosophila pacemaker neurons . in  vivo assays in conjunction with the in  vitro experiments 
 demonstrate that e75 and unf bind to  per regulatory sequences and act together to enhance the clk / cyc - mediated transcription of the per gene , thereby completing the core transcriptional feedback loop necessary for the free - running clockwork . 
 our results identify a missing link in the drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks . 
Results
Discussion
Experimental Procedures