O-GlcNAcylation of PERIOD regulates its interaction with CLOCK and timing of circadian transcriptional repression

Circadian clocks coordinate time-of-day-specific metabolic and physiological processes to maximize organismal performance and fitness. In addition to light and temperature, which are regarded as strong zeitgebers for circadian clock entrainment, metabolic input has now emerged as an important signal for clock entrainment and modulation. Circadian clock proteins have been identified to be substrates of O-GlcNAcylation, a nutrient sensitive post-translational modification (PTM), and the interplay between clock protein O-GlcNAcylation and other PTMs is now recognized as an important mechanism by which metabolic input regulates circadian physiology. To better understand the role of O-GlcNAcylation in modulating clock protein function within the molecular oscillator, we used mass spectrometry proteomics to identify O-GlcNAcylation sites of PERIOD (PER), a repressor of the circadian transcriptome and a critical biochemical timer of the Drosophila clock. In vivo functional characterization of PER O-GlcNAcylation sites indicates that O-GlcNAcylation at PER(S942) reduces interactions between PER and CLOCK (CLK), the key transcriptional activator of clock-controlled genes. Since we observe a correlation between clock-controlled daytime feeding activity and higher level of PER O-GlcNAcylation, we propose that PER(S942) O-GlcNAcylation during the day functions to prevent premature initiation of circadian repression phase. This is consistent with the period-shortening behavioral phenotype of per(S942A) flies. Taken together, our results support that clock-controlled feeding activity provides metabolic signals to reinforce light entrainment to regulate circadian physiology at the post-translational level. The interplay between O-GlcNAcylation and other PTMs to regulate circadian physiology is expected to be complex and extensive, and reach far beyond the molecular oscillator.


Introduction
Circadian clocks are endogenous protein machines that integrate external time cues and internal metabolic states to impose temporal organization on physiology, metabolism, and behavior (reviewed in [1][2]). They allow organisms from all kingdoms of life, which experience perpetual 24-hour day-night cycles, to anticipate daily environmental changes and execute biological tasks, from molecular to behavioral levels, at the optimal time of day. Over the years, great progress in elucidating the molecular mechanisms driving circadian rhythms has been made by studying the core circadian oscillator. The molecular oscillator consists of two interlocked transcriptional translational feedback loops (TTFLs) that produce daily oscillations in clock mRNAs and proteins to drive rhythms in diverse cellular processes. During the day and into the early parts of the night, two basic helix-loop-helix (bHLH)-PAS transcription factors, CLOCK (CLK) and CYCLE (CYC; homolog of BMAL1 in mammals), activate transcription of their own repressors, period (per) and timeless (tim) and other clock-controlled output genes [3]. After a time-delay and TIM-assisted entry into the nucleus [4], PER interacts with and inhibits CLK-CYC activity [5]. This repression is relieved upon proteasomal-dependent degradation of PER during late night into early morning, thus initiating another round of CLK-CYC-mediated transcription [6][7]. Among CLK-activated genes are two bZIP transcription factors, vrille (vri) and par domain protein 1ε (Pdp1ε) [8][9]. Due to differential kinetics of VRI and PDP1ε protein accumulation, VRI accumulates first and inhibits Clk expression. As VRI level decreases, PDP1ε accumulates and activates Clk transcription, and the cycle of per/ tim expression starts again. More recent evidence however suggested that the main role of VRILLE could be to control clock output by driving rhythms in expression of the neuropeptide PDF (Pigment Dispersing Factor) and neuronal arborization [10]. CLOCKWORK ORANGE (CWO) is another direct CLK target that feedbacks and represses CLK activity by competing with CLK-CYC complexes for E-box binding at circadian promoters [11]. The TTFLs are synchronized to the 24-hour day-night cycle through light-dependent degradation of TIM [12][13][14], which interacts with the photoreceptor CRYPTOCHROME 1 (CRY1) [15].
To extend the duration of the TTFL to last a 24-hr circadian cycle, post-translational regulation of core clock proteins overlays on the TTFL and has been recognized to be critical in maintaining the functionality of the circadian oscillator (reviewed in [1,16]). In Drosophila, the phase-specific phosphorylation state of PER is closely linked to its time-of-day specific function and the speed of the oscillator [17][18][19][20][21]. De novo synthesized hypophosphorylated PER goes through a multi-site phosphorylation program that progressively increases its phospho-occupancy until it gets hyperphosphorylated. In particular, phosphorylation at a N-terminal phosphodegron targets PER for degradation in a proteasome-dependent manner [17]. In a study by Robles et al. [22], 25% of the 20,000 phosphosites identified in mouse liver proteins were found to oscillate over the circadian cycle. This suggests that widespread and dynamic oscillations in phosphorylation occur beyond core circadian transcription factors to transition cellular proteins between functional states to regulate circadian physiology.
More recently, O-GlcNAcylation has emerged as another PTM that can regulate the temporal function and activity of circadian transcription factors [23][24][25]. In contrast to protein phosphorylation, which is mediated by a wide selection of kinases and phosphatases, protein O-GlcNAcylation is regulated by a single pair of enzymes with opposing functions [26]. O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) facilitate the O-GlcNAcylation and de-O-GlcNAcylation of cellular proteins respectively. In Drosophila, PER and CLK have been identified as substrates of O-GlcNAcylation, and there is evidence from overexpression of ogt to support that O-GlcNAcylation of these clock transcription factors regulates their nuclear translocation, stability, and transcriptional activity [23][24]. In mammalian clocks, BMAL1 and CLOCK have been shown to be rhythmically O-GlcNAcylated over the circadian cycle, and O-GlcNAcylation functions to counteract ubiquitination to stabilize these proteins [25]. In a separate study, PER2 was shown to be modified by O-GlcNAcylation at the S662-S674 region, which is important for regulating clock speed via CK1 phosphorylation [24]. A S662G mutation in humans is known to cause the familial advanced sleep phase syndrome (FASPS) [27]. Interestingly, S662 can also be O-GlcNAcylated, suggesting in vivo interplay between phosphorylation and O-GlcNAcylation in this domain. Increasingly, the interplay between phosphorylation and O-GlcNAcylation is shown to be prevalent in the regulation of diverse cellular processes (reviewed in [1]). As O-GlcNAcylation is a nutrient-sensitive PTM that relies on the availability of UDP-GlcNAc, an end product of the hexamine biosynthetic pathway (HBP), it is expected that levels of cellular protein O-GlcNAcylation may be highly dependent on daily feeding-fasting cycles.
Many unknowns regarding PTM regulation of clock proteins remain, including the mechanisms by which phase-specific phosphorylation and O-GlcNAcylation collaborate to regulate their time-of-day functions. A significant barrier to understanding these mechanisms is the identification of O-GlcNAcylated residues. In fact, although both Drosophila PER and CLK are known to be O-GlcNAcylated, specific O-GlcNAcylated residues have not been identified [23][24]. The effects of O-GlcNAcylation on these clock proteins have only been investigated by global overexpression and knockdown of ogt and oga, which impact all O-GlcNAcylated residues simultaneously. Lessons learned from previous studies on clock kinases [17][18][19][20][21] highlight the likelihood that valuable mechanistic insights may be overlooked by global manipulation in PTM enzyme expression. Other important questions concern the temporal requirement of clock protein O-GlcNAcylation and the relationship between protein O-GlcNAcylation status and feeding-fasting cycles.
In this study, we sort to understand how metabolic input influences the O-GlcNAcylation status of PER and regulates its function. We used Mass Spectrometry (MS) proteomics to identify PER O-GlcNAcylation and phosphorylation sites from adult flies, and characterized the function of site-specific O-GlcNAcylation events in vivo. We focused on PER as its phasespecific function has been shown to be highly dependent on its progressive phosphorylation program over the circadian cycle. Investigating PER O-GlcNAcylation can therefore set the stage for understanding the interactions between phosphorylation and O-GlcNAcylation in regulating its circadian function. We observe that loss of O-GlcNAcylation at multiple residues affect PER repressor function. In particular, loss of O-GlcNAcylation at PER(S942), which is located in the PER-CLK interaction or CLK binding domain (CBD), leads to stronger PER-CLK interaction and premature entry into the circadian repression phase. Conversely, overexpression of OGT in clock neurons weakens PER-CLK interaction, contributing to its period-lengthening phenotype in locomotor activity rhythms. Finally, we report that daily rhythms of PER O-GlcNAcylation in adult head tissues correlate with feeding-fasting cycles. This correlation is expected to be even stronger in peripheral tissues, which are more sensitive to metabolic signals. Specifically, PER O-GlcNAcylation exhibits circadian rhythmicity and is higher during the day when flies are actively feeding. Our results suggest that metabolic input collaborates with other entrainment signals to regulate time-of-day PER function in circadian transcription.

Mass spectrometry analysis identifies PER O-GlcNAcylation and phosphorylation sites in fly tissues
Although O-GlcNAcylation has been shown to influence PER function, specific residues that are modified by O-GlcNAcylation have not been identified [23][24]. This represents a critical barrier to understand the function of site-specific O-GlcNAcylation events and the interplay between O-GlcNAcylation and phosphorylation to regulate the phase-specific functions of PER. We therefore sought to identify PER O-GlcNAcylation sites and obtain temporal data on their occupancy. We purified FLAG-tagged PER from heads of wper 0 ; p{3XFLAG-per(WT)} flies at specific time-points over the circadian cycle using FLAG affinity purification and performed quantitative mass spectrometry (MS) to obtain a circadian profile of PER phosphorylation and O-GlcNAcylation using fly tissues as starting materials. Although we attempted to perform this study using both fly head and body tissues, we were only able to identify PER O-GlcNAcylation sites in head tissues since we were unable to pull down sufficient PER proteins in bodies for comprehensive PTM identification. Nevertheless, we postulate that PER residues that are O-GlcNAcylated in head tissues will also be O-GlcNAcylated in peripheral tissues due to the ubiquitous expression of ogt [28]. To enable quantitation of PTM sites, flies were fed with 15 N-labeled (heavy) yeast or 14 N (light) yeast for two generations to ensure complete labeling in flies (herein termed 15 N and 14 N flies) [29]. To ensure that 15 N-fed and 14 Nfed flies show similar behavioral rhythms, we examined their locomotor activity rhythms using Drosophila activity monitoring [30]. Both types of flies displayed strong behavioral rhythms with periods close to 24-hr in constant darkness (S1A and S1B Fig). Furthermore, we measured PER daily abundance in 15 N and 14 N flies to confirm that temporal expression of PER was not altered as a result of the diets and observed no difference (S1C Fig). To profile PER PTMs in vivo, 15 N and 14 N flies were collected at six time-points over the circadian cycle (ZT1, 3,12,16,20,24), and protein extracts from heads of 15  Despite the large amount of fly head tissues we used for our protein extraction and FLAG--PER purification, our 15 N/ 14 N quantitative MS did not yield satisfactory temporal resolution of PER PTM cycling. We therefore consolidated our MS data from multiple time-points with the goal of identifying PER O-GlcNAcylation sites qualitatively (Fig 1 and Table 1). We observed that multiple residues located in the CBD and within the CLK:CYC inhibition domain (CCID) [31,32] are O-GlcNAcylated. These include PER(S942) as well as a potential sites at S951, T952, or T954. Our MS analysis yielded O-GlcNAc-modified peptides in which only PER(S942) is modified, providing unambiguous identification of PER(S942) as an O-GlcNAcylated residue (Fig 1 and Table 1). In addition, O-GlcNAc-modified peptides spanning S942 to T954 were also identified, but we were not able to narrow down the single modified residue within these peptides. It is possible that only S942 is O-GlcNAcylated within this region. Alternatively, either S951, T952, or T954 may represent a second O-GlcNAcylated residue within this region. Taken together, we hypothesize that O-GlcNAcylation in the CBD may be important in modulating PER-CLK interaction and PER repressor activity. In addition to the O-GlcNAcylated residues in the CBD, a number of other potential sites were identified in other parts of the PER protein, but most of them are not located in characterized functional domains.
https://doi.org/10.1371/journal.pgen.1007953.g001 points over the circadian cycle (ZT3, 16,20,24). S1 Table summarizes the label-free data and compares it to the phosphorylation sites identified in the 15 N/ 14 N-labeled MS analysis. PER O-GlcNAcylation site identification was not performed as part of the label-free MS analysis. We observed a high level of congruence between the PER phosphorylation sites identified in our two fly head tissue data sets, as well as those generated using Drosophila S2 cells as starting materials [17][18][19]21]. In summary, our MS analysis sets the stage for future studies to understand the functional interplay between PER O-GlcNAcylation and phosphorylation.

O-GlcNAcylation of PER regulates its repressor activity
Subsequent to the identification of PER O-GlcNAcylation sites, we proceeded to analyze the function of site-specific PER O-GlcNAcylation by mutating one or a cluster of S/T residues to O-GlcNAcylation regulates PER-dependent repression of CLK non-O-GlcNAcylatable alanine on the per gene and evaluated PER repressor function on CLK activity using the per-luciferase (per-luc) reporter assay in Drosophila S2 cells [3,37]. To prioritize, we analyzed PER O-GlcNAcylation residues with a Mascot score >50 (Table 1). We compared per-luc activity in S2 cells expressing per wild type or mutant variants (Fig 2A). In order to take into account the varying expression level of the PER variants ( O-GlcNAcylation regulates PER-dependent repression of CLK and S3B Fig), we normalized CLK-activated per-luc activity observed for PER(WT) and PER mutant variants to their respective protein expression levels to more accurately assess the impact of blocking PER O-GlcNAcylation at specific residues on repressor activity ( Fig 2C). We observed that all but one PER O-GlcNAcylation site mutant (PER(S174A)) exhibited significant increase in repressor activity. Interestingly, PER(S942A) and PER(T951A-S954A) are the two O-GlcNAc site mutants that exhibited the strongest repressor activity, further strengthening the rationale for testing the hypothesis that O-GlcNAcylation in the CBD may be important in modulating PER-CLK interaction and PER repressor activity. For this reason, this current study will focus on investigating the function of PER O-GlcNAcylation in the CBD, while analysis of other PER O-GlcNAcylation residues will be pursued in the future.
To confirm this finding in whole animals, we generated transgenic flies expressing p{per (WT)-HA10HIS} (herein referred to as per(WT) and mutant variants) in the per 0 genetic background [38] so that only transgenic per (WT or mutant) was expressed. First, we performed quantitative RT-PCR to measure clock gene expression (per and tim) in heads of per(S942A) flies to examine the function of PER(S942) O-GlcNAcylation in central clock oscillators. All flies used for molecular analysis are homozygous for the per transgene. As predicted from the elevated PER repressor activity observed in S2 cells, we found that cycling of per and tim mRNAs was significantly dampened in per(S942A) flies as compared to per(WT) flies in both LD cycles and constant darkness (DD) (Fig 2D and 2E). Furthermore, per and tim mRNAs exhibited earlier initiation of repression phase in DD ( Fig 2E). Since the per(S942A) mutant exhibited lowered levels of clock gene mRNAs, PER protein level is expected to decrease. As expected, peak PER abundance was significantly reduced in the heads of per(S942A) flies than in per(WT) flies in both LD and DD conditions (Fig 2F and 2G). To ensure that lower level of PER protein is a result of increased PER(S942A) repression on CLK-dependent transcription of per rather than reduced PER stability, we monitored the rate of PER degradation by cycloheximide (CHX) chase assay in S2 cells. The PER(S942A) mutant degraded at a similar rate as PER(WT) in the presence of OGT, demonstrating that the S942A mutation has no significant effect on PER stability (S3C and S3D Fig).
Since peripheral tissues are known to be more sensitive to metabolic fluxes [39,40] and O-GlcNAcylation is a nutrient-sensitive PTM, we also analyzed the effect of blocking PER(S942) O-GlcNAcylation in oscillators of peripheral tissues, specifically the fat body. The fat body, which is analogous to mammalian liver and adipose tissue, plays an essential role in regulating energy metabolism in insects [39,40]. We assayed per and tim mRNA levels in the fat body of per(WT) and per(S942A) flies on DD1 after LD entrainment to evaluate PER-dependent repression of CLK activity. Consistent with what we observed in fly heads, per(S942A) flies displayed significantly dampening of per and tim mRNA cycling as compared to per(WT) flies in the fat body ( Fig 2H  and 2I). This suggests that PER(S942) O-GlcNAcylation normally weakens the activity of PER to repress CLK-dependent transcription in both head and fat body oscillators.
As in PER(S942A) mutant, PER(S951A/T952A/T954A) mutant also exhibited a significant difference in repressor activity as compared to PER(WT) in per-luc reporter assay ( Fig 2C). To evaluate the effects of these residues, which are also located in the CBD, we generated It is important to stress that PER(S942) has never been identified as a phosphorylation site in all previous comprehensive mapping studies [17][18][19]21], so it is highly unlikely that the effects for S942A mutation is due to disruption of PER(S942) phosphorylation.

Site-specific inhibition of PER O-GlcNAcylation modulates locomotor activity and feeding rhythms in flies
Given that several of the non-O-GlcNAcylatable per mutants exhibited elevation in PER repressor activity in S2 cells and in flies, we proceeded to investigate if this molecular phenotype can translate to alterations in output behavioral rhythms. We first evaluated daily locomotor activity rhythms of per(WT) and mutant flies. Activity rhythm is a robust readout that reflects the function and speed of the central oscillators located in fly heads [30]. Flies were entrained for 3 days in LD cycles followed by 7 days in DD. As expected, heterozygous per (WT) flies manifested robust rhythms with~24-hr periods, indicating full rescue of arrhythmic per 0 mutation ( Fig 3A). Homozygous per(WT) flies displayed somewhat shorter behavioral rhythms due to the extra copy of per. Interestingly, as compared to per(WT), per(S942A) and the triple per(S951A/T952A/T954A) mutants homozygous for their respective transgenes exhibited shorter periods by 1.4 and 0.7 hrs. The earlier initiation of repression previously observed in per(S942A) mutant likely contributes to its short-period phenotype. In comparison, per(S951A/T952A/T954A) mutants displayed a smaller though significant change in repression activity, which likely accounts for the smaller change in period length.
In addition to monitoring locomotor activity rhythms of per(S942A) flies, we also examined the effects of blocking PER(S942) O-GlcNAcylation in peripheral oscillators by measuring feeding rhythms. Feeding assays were only performed to compare per(WT) and per(S942A) flies as per(S951A/T952A/T954A) mutants displayed minor changes in activity rhythms and clock gene repression. Feeding activity rhythms are governed by oscillators of metabolic tissues (i.e. in the fat body) [40], which are more sensitive to nutrient flux than central oscillators in the brain. Since we demonstrated earlier that clock gene cycling was dampened in the fat body of per(S942A) flies as a result of PER being a stronger repressor, we expected that feeding rhythms will also be disrupted. We entrained per(WT) and per(S942A) flies in 12h:12h LD cycles and assayed their feeding activity rhythms for two consecutive days either in LD or DD conditions using the CAFE assay [41]. The amount of food consumed over a 2-hr period was determined at 4-hr intervals over the circadian cycle. As control, per 0 flies were measured in parallel, and as expected they exhibited arrhythmic feeding activity in LD and DD (Fig 3B and  3C) as determined by JTK-cycle (P = 1) [42].
per(WT) flies displayed robust rhythms of feeding behavior (JTK-cycle (P < 0.01)); they feed during daytime and fast at night in both LD and DD conditions (Fig 3B and 3C). Robust feeding rhythms were also observed in per(S942A) flies in LD (JTK-cycle (P < 0.01)), but the peak was phase advanced. This is consistent with the shorter period length in locomotor activity observed in the per(S942A) mutant. The consequence of blocking O-GlcNAcylation at PER (S942) on feeding behavior is even more severe in DD in the absence of light cues as rhythmic feeding was abolished in DD (JTK-cycle (P = 1)). Together, our data demonstrates that O-GlcNAcylation at PER(S942) modulates the function of central and peripheral oscillators by regulating PER activity.

Timing of PER nuclear entry is not regulated by PER(S942) O-GlcNAcylation
Premature PER nuclear entry during the night could explain why per(S942A) mutant exhibits stronger and/or premature initiation of repression on CLK activity. A previous study showed that PER O-GlcNAcylation is involved in regulating nuclear entry [23]. To examine this possibility, we monitored timing of PER nuclear entry in adult clock neurons in LD cycles. In the fly brain, rhythmic expression and nuclear localization of PER in lateral clock neurons (LN v s) are necessary for proper clock function and maintenance of rhythmic locomotor activity (reviewed in [2]). The lateral neurons (small and large; l-LN v s and s-LN v s) express pigment dispersing factor (PDF), which has been commonly used to label the cytoplasm of the LN v s [43]. Despite having shorter behavioral rhythms, we found that per(S942A) mutants did not exhibit a significant difference in accumulation of nuclear PER as compared to per(WT) flies at time-points when nuclear entry is most prominent, ZT18 to ZT22 [4] (S5A and S5B Fig).
Although a previous report demonstrates that global knockdown or overexpression of OGT in clock neurons affects the timing of PER nuclear entry [23], it is not surprising that site-specific non-O-GlcNAcylatable per mutants, in this case per(S942A), may not show the same phenotype. As such, our results suggest that the stronger and premature initiation of CLK repression observed in heads and fat bodies of short-period per(S942A) flies cannot be explained by altered timing of PER nuclear localization in circadian oscillators.

PER(S942) O-GlcNAcylation is necessary for proper timing and level of repression by mediating PER-CLK interaction
Given that PER does not enter the nucleus prematurely in the LN v s of per(S942A) mutant flies, we speculate that the PER(S942) may have a higher affinity to CLK that results in stronger and/or premature initiation of repression. We therefore examined PER-CLK interactions by performing co-immunoprecipitations (coIP) using head extracts from per(WT) and per (S942A) flies. These experiments were performed in head tissues as IP reactions of clock proteins are more efficient in head tissues and produce more robust results. Indeed, the per (S942A) flies showed significantly higher PER-CLK interaction at ZT16 than per(WT) flies (Figs 4A, 4B and S6A). To validate our results, we performed coIPs using S2 cells coexpressing per(WT)-cmyc or per(S942A)-cmyc with clk-V5, in the presence or absence of pMT-ogt-FLAG. Consistent with the fly data, we observed that PER(S942A) proteins exhibited significantly stronger binding to CLK as compared to PER(WT) (Fig 4C and 4D). Interestingly, when PER(S942A) mutant was coexpressed with CLK in the presence of OGT, the level of PER-CLK binding was lower than the levels when PER(S942A) mutant was coexpressed with CLK in the absence of OGT. This suggests that O-GlcNAcylation of CLK or perhaps at other PER residues may negatively impact PER-CLK interaction. Nevertheless, our results suggest that stronger and/or premature initiation of repression phase in per(S942A) flies is due to higher affinity between CLK and PER(S942A).
In comparison to per(S942A) mutant flies, per(S951A/T952A/T954A) flies only displayed minor changes in activity rhythms and clock gene expression in flies. Based on these observations, the PER(S951A/T952A/T954A) mutant protein is predicted to exhibit minor changes on PER-CLK interactions, despite that these three residues are also localized within the CBD. As expected, no significant changes in PER-CLK interactions were observed between per(WT) and per(S951A/T952A/T954A) flies at the indicated time-points (S7A and S7B Fig). Thus, our results suggest that O-GlcNAcylation events at PER(S951/T952/T954) only have minor modulatory effects on PER-CLK interactions. Alternatively, it is possible that only PER(S942) is O-GlcNAcylated within the CBD (Fig 1).
Since part of PER repressor function is to remove CLK from clock gene promoters, we would expect that CLK may be removed prematurely when PER(S942) O-GlcNAcylation is blocked. We therefore measured CLK occupancy at the E-box elements of per and tim promoters by performing chromatin immunoprecipitation (CLK-ChIP) using extracts from adult fly heads. Consistent with our hypothesis, we found that flies expressing per(S942A) showed significantly reduced CLK occupancy on per and tim promoters at ZT18 and ZT20 as compared to per(WT) flies (Fig 4E). To rule out the possibility that decreased CLK occupancy observed at ZT18 and ZT20 in per(S942A) flies was due to decreased CLK levels, we examined CLK levels after the ChIP reactions and found that CLK was not limiting in flies expressing per(WT) or per(S942A) (S6B Fig).

OGT overexpression delays the timing of PER-CLK interaction
To further support our hypothesis that dynamic O-GlcNAcylation at PER(S942) is critical for regulating the strength and/or timing of PER-dependent clock gene repression, we overexpressed 3XFLAG-ogt in tim-expressing clock neurons using a tim(UAS)-gal4 driver (referred to as TUG) [44] and examined PER-CLK interactions in head extracts of these flies. First, we verified that ogt overexpressors (TUG>FLAG-ogt) exhibited a significant~2-hr period-lengthening of behavioral rhythms, as previously observed [23,24] (Fig 5A). All 4 independent ogt overexpressor fly lines we generated showed similar period-lengthening phenotypes as compared to parental controls. To confirm that the overexpressed OGT enzyme is functional, we optimized O-GlcNAc chemoenzymatic labeling to measure levels of O-GlcNAcylated proteins in fly head extracts.
Currently, detecting protein O-GlcNAcylation remains to be a challenge when using traditional protein analytical techniques because the addition of a sugar group does not influence the migration of a polypeptide in gel electrophoresis (reviewed in [45]). Additionally, commercially available O-GlcNAc-specific antibodies or lectin yield non-specific signals in our hands. Thus, we chose to use a more sensitive and specific chemoenzymatic labeling method to examine in vivo protein O-GlcNAcylation status in this study [46][47][48].
We first tested the specificity of this approach by detecting O-GlcNAcylated PER and OGT in Drosophila S2 cell culture. S2 cells were transiently transfected with V5 tagged per with or without pMT-ogt-FLAG. Immunoprecipitated PER and OGT were labeled with resolvable mass tags prior to SDS-PAGE and western blotting. Results showed successful labeling of O-GlcNAcylated PER with 20 kD PEG mass tag in the presence of OGT, leading to clear mobility shift of O-GlcNAcylated PER isoforms (S8A Fig). As OGT is known to be auto-O-GlcNAcylated, addition of 10kD PEG mass tag in the reaction resulted in O-GlcNAcylated OGT isoforms that appeared as slower migrating bands as detected by Western blots (S8B Fig). The use of PEG mass tag labeling provides two advantages. First, both modified and nonmodified isoforms can be detected, providing information on stoichiometry. Second, epitope- tagged antibodies or target-specific antibodies can be used for detection of all isoforms. Alternatively, O-GlcNAcylated proteins can also be labeled with biotin to facilitate detection with α-streptavidin antibodies [46][47][48]. Our success in detecting and visualizing in vivo O-GlcNAcylation status of proteins in S2 cells by chemoenzymatic labeling allowed us to proceed and confirmed that PER O-GlcNAcylation was elevated in head extracts of ogt overexpressor flies, an indication that overexpression of ogt led to increase in enzyme activity (Fig 5B and 5C).
Since blocking PER(S942) O-GlcNAcylation promotes PER-CLK interaction, we expect that PER-CLK interaction should be reduced in ogt overexpressors assuming PER(S942) is hyper-O-GlcNAcylated. As anticipated, we observed that control TUG flies exhibited significantly higher PER-CLK interaction at ZT18 as compared to ogt overexpressor flies in coIP assays using head extracts of flies (Fig 5D and 5E). For ogt overexpressor flies, the reduction in PER-CLK interaction observed in fly heads may partially account for the physiological effect of OGT overexpression, i.e. period-lengthening of clock-controlled locomotor activity rhythms (Fig 5A).

Daily rhythms in PER O-GlcNAcylation correlate with feeding rhythms and suggest that PER(S942) O-GlcNAcylation prevents premature initiation of circadian repression
We speculate that PER(S942) O-GlcNAcylation occurs either during the daytime or early night to prevent de novo PER from prematurely binding to CLK or at late night to facilitate CLK dissociation from PER after PER-dependent repression. Both of these scenarios would result in stronger clock gene repression in per(S942A) flies. Since we observed that the differences in PER-CLK interactions between per(WT) and per(S942A) flies as well as between WT TUG control and ogt overexpressor flies occurred at around the start of the circadian repression phase (i.e. ZT16 and ZT18 respectively; Fig 4B and  We examined the temporal profile of PER O-GlcNAcylation in head extracts of per(WT) flies in LD condition by labeling PER with either 20kD PEG or a biotin tag [46][47][48]. In both cases, maximal PER O-GlcNAcylation occurred at around ZT4 to ZT8 with subsequent decline from ZT12 to ZT24 (Fig 6A and 6B). Our results were more in line with the temporal PER O-GlcNAcylation profile observed in Kaasik et al. [24]. Daily PER O-GlcNAcylation cycle was found to be significantly rhythmic (JTK cycle; P < 0.0005) (Fig 6C). Our results reveal that PER is more highly O-GlcNAcylated during the day. This suggests that PER(S942) is likely O-GlcNAcylated during the day and may persist into early night to prevent de novo PER from prematurely interacting with CLK to initiate the repression phase.
O-GlcNAcylation of cellular protein is sensitive to nutrient input [25,49]. Since we observed that PER O-GlcNAcylation is higher during the day and gradually decreases over the circadian cycle, we expect that this temporal pattern may correlate to daily feeding activity. We measured feeding rhythms in per(WT) flies fed ad libitum using the CAFE assay [41]. The flies used for these assays were entrained simultaneously with flies used for PER O-GlcNAc labeling to better assess correlation between feeding activity and PER O-GlcNAcylation. We found that mixed sexes of per(WT) flies displayed rhythmic feeding that peaked during early day in LD condition (S9A Fig). Similar results were obtained when male and female per(WT) flies were housed and tested separately ( S9B Fig). Furthermore, a separate experiment comparing feeding activity rhythms of per(WT) and per(S942A) flies also showed higher daytime feeding activity in per(WT) flies (Fig 3B). Taken together, our data suggest that nutrient flux via feeding activity provides time-of-day metabolic signals to the circadian oscillator via temporal O-GlcNAcylation of PER. Specifically, PER(S942) O-GlcNAcylation, which is expected to occur during the day and persist into early night, may prevent interaction of PER and CLK prematurely to regulate timing of circadian repression.

Discussion
Recent studies reveal that O-GlcNAcylation of circadian transcription factors, PER and CLK in Drosophila and BMAL1 and CLOCK in mice, plays an important role in modulating their function in the circadian oscillator [23][24][25]50]. However, to more fully understand the mechanisms by which site-specific O-GlcNAcylation events regulate circadian physiology and to set the stage for investigating the interplay between phosphorylation and O-GlcNAcylation, it is necessary to identify O-GlcNAcylated residues in core clock proteins and other cellular proteins and characterize their site-specific functions. Furthermore, the relationship between feeding-induced nutrient influx and the temporal regulation of clock protein O-GlcNAcylation warrants investigation in whole animals. Since feeding activity is controlled by the circadian clock, we hypothesize that food intake will increase HBP influx, leading to increase in clock protein O-GlcNAcylation during the feeding period or soon after. This could serve as a mechanism by which metabolic input reinforces circadian entrainment by other zeitgebers and regulates oscillator function in a time-of-day specific manner. We therefore set out to (i) identify PER O-GlcNAcylation sites and characterize their site-specific functions in regulating the circadian oscillator; and (ii) determine if there is a correlation between time of feeding and O-GlcNAcylation levels of PER. Although overexpression or knockdown of OGT and OGA has provided insights into the global effects of protein O-GlcNAcylation on circadian clock regulation, we expect that site-specific characterization of O-GlcNAcylation events will alleviate confounding effects resulting from having multiple O-GlcNAcylated residues within a single protein or the involvement of multiple O-GlcNAcylated proteins in clock regulation, leading to new mechanistic insights.
By utilizing MS-based proteomics, we observed that in addition to being heavily phosphorylated, PER is O-GlcNAcylated at at least 6 residues, some of them in the CBD. To understand the role of these O-GlcNAcylation events in regulating PER function and circadian physiology, we analyzed these O-GlcNAcylation sites by replacing serine or threonine to non-GlcNAcylatable alanine either singly or in clusters. Several of these non-O-GlcNAcylatable per mutants exhibit changes in PER repressor function, which consequently result in period-changing phenotypes in their corresponding transgenic fly lines. In particular, we observed that O-GlcNAcylation at PER(S942), which is located in the CBD, reduces PER-CLK interaction (Fig 7A and  7B). Using O-GlcNAc chemoenzymatic labeling, we show that PER O-GlcNAcylation primarily occurs during daytime and correlates with the time period when animals are feeding. We therefore postulate that PER(S942), as in the case for most PER O-GlcNAcylation sites, is O-GlcNAcylated during the day. This ensures that PER does not interfere with CLK activity in the circadian activation phase and its repression of CLK activity does not initiate prematurely when de novo PER starts to translocate into the nucleus. This suggests that OGA may need to actively remove O-GlcNAc from PER residues prior to circadian repression phase. Indeed, OGA level has been shown to oscillate over the circadian day, peaking prior to initiation of circadian repression phase [24]. Moreover, O-GlcNAcylation has been shown to regulate PER nuclear entry [23], suggesting that OGA-dependent removal of O-GlcNAc at unknown PER residue(s) is likely required to facilitate PER nuclear translocation independent of OGA activity on PER(S942). Finally in the evening, since flies are fasting, the level of O-GlcNAcylation at PER(S942) will remain low allowing PER to bind strongly to CLK to repress its activity.
Our findings that PER O-GlcNAcylation at S942 reduces PER repression of CLK activity is not congruent to the observation in [24], where they observed that the repressor activity of PER is enhanced when coexpressed with OGT in S2 cell per-luc reporter assay. However, this apparent incongruence could be explained by the combined activities of other O-GlcNAcylation events on PER, CLK, or other cellular proteins that impact per-luc reporter gene expression. Nevertheless, it is important to point out that none of our non-O-GlcNAcylatable per mutants showed a decrease in repressor activity (Fig 2C).
It is interesting to note that unlike some phosphorylation sites previously identified on PER proteins, residues we identified to be O-GlcNAcylated in Drosophila PER are not conserved in mouse PER proteins. This is perhaps not surprising since nutrient-dependent O-GlcNAcylation on cellular proteins likely depends on when organisms are actively feeding, i.e. whether they are diurnal or nocturnal. For instance, mice are nocturnal, which may restrict protein O-GlcNAcylation of cellular proteins to nighttime. This nighttime peak in O-GlcNAcylation levels corresponds to when mouse PER2 proteins are abundant and active as repressors, suggesting that O-GlcNAcylation may act to promote PER2-dependent repression on circadian transcription. On the other hand, given that flies are diurnal and feed during the day (Fig 3B), nutrient flux promoting O-GlcNAcylation of Drosophila PER inhibits PER function as a transcriptional repressor (Fig 7). Overall, the differences in timing of feeding activity and nutrient flux between diurnal and nocturnal animals will present interesting opportunities for comparative analysis with regard to site-specific and global effects of O-GlcNAcylation on cellular protein function.
In addition to regulating PER-CLK interaction in the Drosophila circadian clock, O-GlcNAcylation is expected to affect oscillator function via other mechanisms [23][24][25].
Besides regulating clock proteins directly, it is likely that O-GlcNAcylation can modify the activity of clock kinases, just as GSK3β can regulate the activity of OGT [24]. CK2 and GSK3β are two clock kinases that have been shown to be substrates of OGT [51][52], and currently it is unclear how OGT might modulate their activities in clock regulation. Furthermore, O-GlcNAcylation can also impact the activities of chromatin modifiers and transcription machineries, including RNAPII [53]. The interplay between O-GlcNAcylation and other PTMs to regulate circadian physiology is expected to be complex and extensive, and reach far beyond the molecular oscillator and circadian transcription.
In summary, our results support that clock-controlled feeding activity provides metabolic input to reinforce entrainment signals by light-dark cycles to regulate circadian physiology via clock protein O-GlcNAcylation. We expect that circadian rhythms in peripheral systems, where oscillators are more sensitive to metabolic input, to be particularly sensitive to modulation via O-GlcNAcylation of clock proteins. Finally, our results imply that disruptions in daily feeding rhythms, e.g. irregular meal times and late night eating common in modern societies, will likely affect rhythms in protein O-GlcNAcylation and interplay with other PTMs, thereby disrupting circadian rhythms in physiology. Future experiments to manipulate feeding schedules by time-restricted feeding (TRF) [54] can further solidify the causal relationship between feeding-induced nutrient influx and O-GlcNAcylation of cellular proteins. They can also provide mechanistic insights into the benefits of TRF.

Transgenic Drosophila construct design and fly transformation
To profile PER PTMs, we generated transgenic flies that expressed a 13.2 kb genomic clone of per in w 1118 per0 (wper 0 ) background. A previously characterized vector that contains a 13.2kb per genomic fragment tagged with HA and 10X histidine at the carboxyl terminal (pCaSpeRper(13.2WT)-HA-10HIS) [55] was used as the template for inserting 3XFLAG at the amino terminal before the starting Methionine to facilitate FLAG Affinity Purification. Transformants were generated by P-element transformation (BestGene Inc., Chino Hills, CA), and the 3XFLAG-per(13.2WT)-HA-10HIS transgene was tested for functionality by determining whether it can rescue wper 0 flies in behavioral and molecular assays.
To generate transgenic flies carrying wild-type (WT) or O-GlcNAcylation site mutants of per, we opted to use PhiC31 site-directed recombination [56]. The genomic per(13.2WT)-HA-10HIS was excised from pCaSpeR-per(13.2WT)-HA-10HIS using the restriction sites XhoI and BamHI and subcloned into pattB vector (kind gift from Amita Seghal) to yield pattB-per(13.2WT)-HA-10HIS. The pattB vector was modified so that the sites KpnI and XbaI were removed, the BgIII site was replaced by a BamHI site, and the BamHI site was replaced by a BgIII site. For generating flies expressing non-O-GlcNAcylatable mutants on per, a 3.4 kb XbaI-BamHI or 1kb Bam-HI-KpnI genomic fragment was excised from the pattB-per(13.2WT)-HA-10HIS plasmid and subcloned into a pGem7 vector [20]. The resulting pGem7-per(XbaI-BamHI) or pGem7-per(Bam-HI-KpnI) plasmid served as a parent template for site-directed PCR mutagenesis (Agilent Technologies, Santa Clara, CA) depending on the location of the O-GlcNAc site (S2 Table for mutagenic primer sequences). After mutagenesis and confirmation by Sanger sequencing (UC Davis Sequencing), the mutant variants of 3.4 kb or 1 kb per subfragments were used to replace the WT fragments in pattB-per(13.2WT)-HA-10HIS. Plasmids were injected into w 1118 fly embryos carrying attP sites on chromosome 3 (attP2) (BestGene, Inc.). Transformants were crossed with wper 0 flies to remove endogenous copies of per prior to behavioral and molecular analyses.

CAFE assay
CAFE assay was performed as described [41] with modifications. Mixed-sex population of five male and five female wper 0 , wper 0 ; per(13.2WT), and wper 0 ; per(S942A) flies or separately housed male or female (10 per group) flies were fed Bloomington Drosophila Stock Center standard fly food during entrainment in 12 h light/12 h dark day-night cycles and food consumption were measured starting on the third day of LD or the first day of DD. Prior to the day of measurement, grouped flies were transferred to a vial containing 2% agar as the medium with 5% sucrose solution maintained in calibrated glass micropipettes (VWR). After 24 hours of training, old micropipettes were replaced by fresh experimental micropipettes filled with 5% sucrose solution approximately 2 hours before each of the indicated time-points. After 2 hours, the amount of liquid consumed from the experimental micropipette was recorded, and the evaporation effect was evaluated by measuring the change in liquid volume in a micropipette placed in a vial without flies. Food consumption for each group/vial was determined by subtracting the amount of liquid consumed from the experimental micropipette with the amount of evaporated liquid. These values were normalized to the amounts of flies in the vial that survived until the end of the experiment. These experiments were performed in biological triplicates (one group/vial represents one independent experiment). Error bars = SEM. Rhythmicity of feeding was determined by JTK Cycle [42].

Stable isotope labeling of Drosophila proteins and affinity purification of PER for 15 N/ 14 N-labeled proteomics
wper 0 ; p{3XFLAG-per(13.2WT)-HA10HIS} flies were fed with an 15 N diet (0.2g 15 N yeast, 1% Bacto agar, 15% unsulfured molasses, phosphoric and propionic acid mix, and tegosept). Saccharomyces cerevisiae were metabolically 15 N-labelled as described [29,60]. As control, flies were also fed with 14 N diet. The adult progenies of 15 N-or 14 N-fed parental flies were reared in 15 N-or 14 N diet and entrained for 3 days in 12hr light:12hr dark at 25˚C and collected every four hours over a period of 24 hours on the fourth day. Upon collection, flies were immediately frozen on dry ice until protein extraction. For each time-point, 4ml of fly heads were homogenized into fine powder by a chilled mortar and pestle and resuspended in Lysis Buffer (20mM HEPES pH 7.9, 5% glycerol, 350mM NaCl, 0.1% Triton X-100, 1mM DTT, 1mM MgCl 2 , 0.5mM EDTA, 25mM sodium fluoride, 1x protease inhibitor (Sigma, St. Louis, MO), 1x Phos-STOP (Roche, South San Francisco, CA). Homogenate was dounced for 15 strokes using a 50ml tissue grind tight pestle (Kimble-Chase, Vineland, NJ) and were filtered using a 70μm cell strainer. Samples were spun at 300 rcf for 1 minute and then incubated at 4˚C on a nutator for 30 minutes. Additional Lysis Buffer was added to dilute the sample from 350mM to 150mM NaCl before centrifuging at 15,000 rpm for 15 minutes at 4˚C. Supernatant was collected and incubated with 200μl α-FLAG resin (Sigma) overnight over a nutator at 4˚C. Next day, beads were washed twice for 15 mins with Lysis Buffer without EDTA, DTT, or Phos-STOP. Samples were eluted in 300ul R+A buffer (30% glycerol, 3% SDS, 6mM EDTA, 0.15M Tris-HCl pH 6.8) at 95˚C. Eluate was reduced with 15μl 1M DTT for 10 minutes at 65˚C and then alkylated with 35μl 1M IAA in room temperature for 20 minutes in the dark. Eluates were then flash frozen using liquid nitrogen immediately. We used the pooled standard approach to enable more accurate comparisons between different time points. 14 N eluate from six time-points were pooled together and split evenly to mix with each 15 N eluate at a 1:1 ratio on ice. For each time-point, 600μl of cold acetone was added to the 14 N/ 15 N eluate mixture and placed in -20˚C overnight. Precipitate were spun at 14,000 rpm for 10 minutes at 4˚C and the resulting pellet were washed briefly with 1ml cold acetone. Precipitated eluate was resuspended in 80μl R+A sample buffer containing 3μl of 4X SDS sample buffer. The 14 N/ 15 N eluate was resolved in 12% SDS-PAGE and the excised PER band was used for protease digestion and analysis by mass spectrometry.

Processing of stable isotope labeled proteins for LC-MS/MS and data analysis
For analysis of 14 N/ 15 N-labeled samples, proteins were digested in-gel with trypsin and elastase in separate reactions to result in overlapping peptides, such that individual modified sites can be determined. We have previously used this multi-protease approach [61] to maximize high sequence coverage when mapping PER phosphorylation sites [17]. For in-gel digestion the excised gel bands were destained with 30% ACN, shrunk with 100% ACN, and dried in a Vacuum Concentrator (Concentrator 5301, Eppendorf, Hamburg, Germany). Digests with trypsin and elastase were performed overnight at 37˚C in 0.05 M NH 4 HCO 3 (pH 8). About 0.1 μg of protease was used for one gel band. Peptides were extracted from the gel slices with 5% formic acid.
NanoLC-MS/MS analyses were performed on an LTQ-Orbitrap Velos Pro or an Orbitrap Fusion (Thermo Fisher Scientific, Waltham, MA) equipped with an EASY-Spray Ion Source and coupled to an EASY-nLC 1000 (Thermo Fisher Scientific). Peptides were loaded on a trapping column (2 cm x 75 μm ID. PepMap C18, 3 μm particles, 100 Å pore size) and separated on an EASY-Spray column (25 cm x 75 μm ID, PepMap C18, 2 μm particles, 100 Å pore size) with a 90-minute linear gradient from 3% to 30% acetonitrile and 0.1% formic acid. For the Oribtrap Velos MS scans were acquired in the Orbitrap analyzer with a resolution of 30,000 at m/z 400, MS/MS scans were acquired in the Orbitrap analyzer with a resolution of 7,500 at m/ z 400 using HCD fragmentation with 30% normalized collision energy. A TOP5 data-dependent MS/MS method was used; dynamic exclusion was applied with a repeat count of 1 and an exclusion duration of 30 seconds; singly charged precursors were excluded from selection. Minimum signal threshold for precursor selection was set to 50,000. Predictive AGC was used with AGC target a value of 1e6 for MS scans and 5e4 for MS/MS scans. Lock mass option was applied for internal calibration in all runs using background ions from protonated decamethylcyclopentasiloxane (m/z 371.10124). For the Orbitrap Fusion, both MS and MS/MS scans were acquired in the Orbitrap analyzer with a resolution of 60,000 for MS scans and 15,000 for MS/MS scans. HCD fragmentation with 35% normalized collision energy was applied. A Top Speed data-dependent MS/MS method applying HCD and ETD fragmentation from the same precursor with a fixed cycle time of 3 seconds was used. Dynamic exclusion was applied with a repeat count of 1 and an exclusion duration of 120 seconds; singly charged precursors were excluded from selection. Minimum signal threshold for precursor selection was set to 50,000. Predictive AGC was used with AGC a target value of 5e5 for MS scans and 5e4 for MS/MS scans. EASY-IC was used for internal calibration.
Mascot Distiller 2.5 was used for raw data processing and for generating peak lists, essentially with standard settings for the Orbitrap (high/high settings). Mascot Server 2.5 was used for database searching with the following parameters: peptide mass tolerance: 7 ppm, MS/MS mass tolerance: 0.02 Da, enzyme: "semi-trypsin" for tryptic digests and "none" for elastase digests; fixed modifications: carbamidomethyl (C); variable modifications: Gln->pyroGlu (Nterm Q), oxidation (M), acetyl (protein N-term), phosphorylation (STY), HexNAc (ST). Searches containing both HCD and ETD spectra (Fusion) were searched separately for either b and y ions (HCD) or c and z ions (ETD). For ETD-searches different modifications definitions (without neutral losses) for phosphorylation and HexNAc were applied. Separate Mascot searches were performed for light peptides (quantitation: "none") and heavy peptides (quantitation: 15 N-labeling). Database searching was performed against a small custom database containing 187 of the most abundant proteins identified in these samples before in a first round search (without PTMs) against UniProt Drosophila database. This was necessary to limit search space and processing times.
The results from the different Mascot searches (different time-points, proteases and fragmentation techniques) were merged (separately for light and heavy peptides) and filtered for phosphorylated and HexNAc-modified peptides using a custom software tool (A. Schlosser). A Mascot score cut-off of 15 and a delta score cut-off of 10 [62] were applied, and only "rank 1" peptides were accepted. For one modification site and one type of modification, only the peptide spectrum match (PSM) with the highest score was kept, all other PSMs were filtered out. All remaining spectra were verified manually, e.g. by checking the presence of modification specific marker ions. HexNAc-modified peptides were only accepted when at least one of the HexNAc-specific fragment ions (204, 186 and 168) [63] was present in the corresponding HCD spectra. After manually filtering, all remaining peptides were exported to generate a summary of the results (

Affinity purification, sample and data processing for label-free mass spectrometry analysis
All flies were reared on standard Drosophila medium (Bloomington Drosophila Stock Center standard recipe). Entrainment and collection of flies at the appropriate time-points were described as above. Roughly 4ml of fly heads were grinded into fine powder using chilled ceramic mortar and pestle and mixed in 30ml of lysis buffer (20mM HEPES pH 7.5, 1mM DTT, 1x protease inhibitor). Homogenate was dounced and poured over a cell strainer as described above prior to centrifugation at 7000xg for 45 minutes at 4˚C to separate nuclear and cytoplasmic lysates, repeated once. Lower layer (pellet) as the nuclear fraction from both spins was resuspended in 10ml Nuclear Extraction buffer (20mM HEPES pH 7.5, 10% Glycerol, 350mM NaCl, 0.1% Triton X-100, 1mM DTT, 1mM MgCl 2 , 0.5mM EDTA, 1x protease inhibitor, 10mM NaF) with the addition of MG132 (Sigma) and DNAse (Promega). Upper layer (supernatant) as the cytoplasmic fraction was supplemented with Lysis buffer with the addition of MG132 (Sigma) and DNAse (Promega). Nuclear and cytoplasmic fractions were incubated at 4˚C for 30 minutes over a nutator. After incubation, nuclear fraction was diluted to 150mM NaCl with Lysis buffer. Nuclear and cytoplasmic fractions were centrifuged at 27,000rpm for 15 minutes at 4˚C. Supernatant of nuclear and cytoplasmic samples was recovered before incubation with 200μl α-FLAG M2 resin (Sigma) at 4˚C overnight. Beads were washed three times with Lysis buffer for 15 minutes each and subsequently eluted in 200μl of 3XFLAG peptide (Sigma) at a dilution of 250μg/ml at room temperature for 15 minutes. Eluates were resolved on a Tris-Tricine gel and PER bands were excised for protease digestion and mass spectrometry as described in [64]. The label-free MS proteomics data for PER phosphorylation site mapping have been deposited into ProteomeXchange (PXD008281) (ProteomeXchange: http://pro teomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD008281), MassIVE repository (MSV000081736) (MassIVE: https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=384c7750 b3004b7eac91054935a4e038), and Chorus repository (Project ID 1424) (https://chorusproject. org/anonymous/download/experiment/1e0023a15da84e51bb18c55146104b32).

Measuring daily locomotor activity rhythms in flies
Locomotor activity rhythms were measured as previously described [30]. 3-4-day old male flies were collected and subjected to 12hr light:12hr dark (LD) cycles at 25˚C for four days followed by seven days of constant darkness (DD) to measure free-running period using the Drosophila Activity Monitor System (DAMS) (TriKinetics, Waltham, MA). Data analysis was performed using FaasX as described in [30].

Transcriptional reporter assay in S2 cell culture
Luciferase reporter assays were performed as described [3,37]. Measurements of luciferase activity were performed using the Dual-Glo luciferase assay system (Promega, Madison, WI) according to the manufacturer's recommendation on a TriStar LD 941 microplate reader (Berthold Technologies, Oak Ridge, TN).

Cycloheximide chase experiment in S2 cell culture
pAc-per-V5(WT) or mutant variants were transfected into S2 cells with pMT-FLAG-ogt or an empty plasmid. 20 hours after transfection, ogt expression was induced for 16 hours. Cycloheximide was then added to a final concentration of 10 μg/ml. Cells were harvested and lysed with EB2 [20] at the times indicated. Proteins were analyzed by western blotting as detailed below and in [65].

Coimmunoprecipitation (co-IP) in S2 cells and in flies
Co-IP assays using protein extracts from S2 cells and fly heads were performed as described [58,65] with modifications. Proteins were extracted using modified RIPA buffer with the addition of 100μM PUGNAc to preserve O-GlcNAcylation of proteins prior to input analysis by western blotting or Co-IP with appropriate antibodies. Samples were pre-cleared using sepharose beads (Sigma) to reduce nonspecific binding. For co-IP in S2 cells, CLK IP samples were incubated with 15μl α-V5 resin (Sigma) and negative non-specific control IP samples were incubated with 15μl α-HA resin (Sigma). For co-IP in fly heads, CLK IP samples were incubated with 4μl α-CLK antibody (Santa Cruz Biotechnology, Dallas, TX) for 3 hours prior to incubation with 20μl gamma sepharose beads (GE, Pittsburgh, PA) for 1 hour; PER IP samples were incubated with 20μl α-HA resin (Sigma). Immune complexes were resolved by SDS-PAGE as described [58,65]. IP signal intensity was normalized to intensity of the bait protein.
These values were then converted as relative to the peak value of the dataset (peak = 1). Representative data shown are averages of normalized PER or CLK interactions from at least three independent experiments.

Quantitative RT-PCR to analyze gene expression in fly heads and abdominal fat bodies
Total RNA was extracted from fly heads and abdominal fat bodies using TRI-Reagent (Sigma). cDNA synthesis from total RNA and real-time PCR analysis was performed as previously described [58,65]. For isolating abdominal fat bodies, flies were collected at the indicated time-points on the first day of DD and immediately transferred in TRI-Reagent (Sigma) for 40 minutes at room temperature with agitation following dissection in RNAlater buffer (Thermo Fischer Scientific). At least 16 flies were dissected for each genotype and time-point. After dissection, fat bodies were rinsed with nuclease-free water twice and resuspended in TRI-Reagent prior to RNA isolation.

Chromatin immunoprecipitation (ChIP)
ChIP assays were essentially performed as described [58]. qPCR of an intergenic region (FBgn0003638) of the Drosophila genome representing background CLK binding was subtracted from input samples. Technical triplicates for the qPCR step were performed for each of the three biological replicates. Two-tailed t-tests were used to determine statistical differences between genotypes at each time-point.

Immunohistochemistry
Adult fly brain immunohistochemistry was performed as described previously [65] with modifications. Briefly, adult flies were entrained in LD cycles for 3 days and collected at the appropriate time-point following incubation with fixative solution (4% paraformaldehyde, 0.2% Triton X-100 in PBS) for at least 40 minutes in the dark at room temperature with gentle rotation. Fixative solution was removed, and then wash solution (0.2% Triton X-100 in PBS) was added to transfer flies into an embryo dish. Brains were dissected using #5 Rubis nano tweezers (Electron Microscopy Sciences, Hatfield, PA). Approximately 10 brains were dissected for each time-point. After dissection, brains were incubated in fixative solution at room temperature for 40 minutes with gentle rotation. Brains were rinsed quickly with wash solution three times prior to three slow washes in wash solution for 10 minutes each with gentle rotation. Wash solution was removed, and blocking solution (0.2% Triton X-100, 5% horse serum in PBS) was added to the brains for 40 minutes at 4˚C with gentle rotation. Brains were then incubated with new blocking solution at 4˚C overnight with primary antibodies at the following dilutions: α-HA 3F10 antibody (Roche) (1:100), and α-PDF antibody (Developmental Studies Hybridoma Bank, Iowa City, IA) (1:100). After~18 hr, brains were rinsed quickly with wash solution three times prior to four slow washes with wash solution for 10 minutes each with gentle rotation. Brains were then incubated in secondary antibodies in blocking solution overnight. Secondary antibodies used at the following dilutions were Dylight88-conjugated αrat (1:100) and Alexa647-conjugated α-mouse (1:100) (Thermo Fischer Scientific). After~18 hr, brains were rinsed quickly with wash solution three times prior to four slow washes with wash solution for 10 minutes each with gentle rotation. Brains were rinsed quickly with PBS following incubation with 85% glycerol for 15 minutes. Brains were mounted on microscope slides in VectaShield mounting medium (Vector Laboratories, Burlingame, CA) under a #1.5 (17-mm) coverslip. Confocal images were obtained with an Olympus FV1000 Confocal Microscope (Olympus Life Science, Center Valley, PA) and processed with the FV1000 software (Olympus Life Science). Scoring of PER subcellular localization was performed as previously described [66,67]. At least five brains were used for each genotype and time-point. For statistical analysis, scored LN v s from each brain served as one biological replicate. Two-tailed t-tests were used to determine statistical differences between genotypes at each time-point. Error bars indicate ± SEM at individual time-point. Asterisks denote significance difference ( � P-value < 0.05, �� P-value < 0.01, ��� P-value < 0.001) in relative food consumption at the highest feeding period (ZT0-2) compared to other feeding times (ZT8-10, ZT12-14, ZT16-18, and ZT20-22) for (A) mixed populations of male and females (black asterisk) or (B) separately housed males (grey asterisk) or females (black asterisk). Rhythmicity of feeding activity in females was confirmed by JTK-cycle (P < 0.05). (TIF) S1