TIMELESS (TIM) was first identified as a molecular cog in the Drosophila circadian clock. Almost three decades of investigations have resulted in an insightful model describing the critical role of Drosophila TIM (dTIM) in circadian timekeeping in insects, including its function in mediating light entrainment and temperature compensation of the molecular clock. Furthermore, exciting discoveries on its sequence polymorphism and thermosensitive alternative RNA splicing have also established its role in regulating seasonal biology. Although mammalian TIM (mTIM), its mammalian paralog, was first identified as a potential circadian clock component in 1990s due to sequence similarity to dTIM, its role in clock regulation has been more controversial. Mammalian TIM has now been characterized as a DNA replication fork component and has been shown to promote fork progression and participate in cell cycle checkpoint signaling in response to DNA damage. Despite defective circadian rhythms displayed by mtim mutants, it remains controversial whether the regulation of circadian clocks by mTIM is direct, especially given the interconnection between the cell cycle and circadian clocks. In this review, we provide a historical perspective on the identification of animal tim genes, summarize the roles of TIM proteins in biological timing and genomic stability, and draw parallels between dTIM and mTIM despite apparent functional divergence.

O-linked β-N-acetylglucosamine (O-GlcNAcylation) is a dynamic post-translational modification that regulates thousands of proteins and almost all cellular processes. Aberrant O-GlcNAcylation has been associated with numerous diseases, including cancer, neurodegenerative diseases, cardiovascular diseases, and type 2 diabetes. O-GlcNAcylation is highly nutrient-sensitive since it is dependent on UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP). We previously observed daily rhythmicity of protein O-GlcNAcylation in a Drosophila model that is sensitive to the timing of food consumption. We showed that the circadian clock is pivotal in regulating daily O-GlcNAcylation rhythms given its control of the feeding-fasting cycle and hence nutrient availability. Interestingly, we reported that the circadian clock also modulates daily O-GlcNAcylation rhythm by regulating molecular mechanisms beyond the regulation of food consumption time. A large body of work now indicates that O-GlcNAcylation is likely a generalized cellular status effector as it responds to various cellular signals and conditions, such as ER stress, apoptosis, and infection. In this review, we summarize the metabolic regulation of protein O-GlcNAcylation through nutrient availability, HBP enzymes, and O-GlcNAc processing enzymes. We discuss the emerging roles of circadian clocks in regulating daily O-GlcNAcylation rhythm. Finally, we provide an overview of other cellular signals or conditions that impact O-GlcNAcylation. Many of these cellular pathways are themselves regulated by the clock and/or metabolism. Our review highlights the importance of maintaining optimal O-GlcNAc rhythm by restricting eating activity to the active period under physiological conditions and provides insights into potential therapeutic targets of O-GlcNAc homeostasis under pathological conditions.

Ambient temperature varies constantly. However, the period of circadian pacemakers is remarkably stable over a wide-range of ecologically- and physiologically-relevant temperatures, even though the kinetics of most biochemical reactions accelerates as temperature rises. This thermal buffering phenomenon, called temperature compensation, is a critical feature of circadian rhythms, but how it is achieved remains elusive. Here, we uncovered the important role played by the Drosophila PERIOD (PER) phosphodegron in temperature compensation. This phosphorylation hotspot is crucial for PER proteasomal degradation and is the functional homolog of mammalian PER2 S478 phosphodegron, which also impacts temperature compensation. Using CRISPR-Cas9, we introduced a series of mutations that altered three Serines of the PER phosphodegron. While all three Serine to Alanine substitutions lengthened period at all temperatures tested, temperature compensation was differentially affected. S44A and S45A substitutions caused undercompensation, while S47A resulted in overcompensation. These results thus reveal unexpected functional heterogeneity of phosphodegron residues in thermal compensation. Furthermore, mutations impairing phosphorylation of the per ^s phosphocluster showed undercompensation, consistent with its inhibitory role on S47 phosphorylation. We observed that S47A substitution caused increased accumulation of hyper-phosphorylated PER at warmer temperatures. This finding was corroborated by cell culture assays in which S47A slowed down phosphorylation-dependent PER degradation at high temperatures, causing PER degradation to be excessively temperature-compensated. Thus, our results point to a novel role of the PER phosphodegron in temperature compensation through temperature-dependent modulation of the abundance of hyper-phosphorylated PER. Our work reveals interesting mechanistic convergences and differences between mammalian and Drosophila temperature compensation of the circadian clock.

Circadian clock and chromatin-remodeling complexes are tightly intertwined systems that regulate rhythmic gene expression. The circadian clock promotes rhythmic expression, timely recruitment, and/or activation of chromatin remodelers, while chromatin remodelers regulate accessibility of clock transcription factors to the DNA to influence expression of clock genes. We previously reported that the BRAHMA (BRM) chromatin-remodeling complex promotes the repression of circadian gene expression in Drosophila. In this study, we investigated the mechanisms by which the circadian clock feeds back to modulate daily BRM activity. Using chromatin immunoprecipitation, we observed rhythmic BRM binding to clock gene promoters despite constitutive BRM protein expression, suggesting that factors other than protein abundance are responsible for rhythmic BRM occupancy at clock-controlled loci. Since we previously reported that BRM interacts with two key clock proteins, CLOCK (CLK) and TIMELESS (TIM), we examined their effect on BRM occupancy to the period (per) promoter. We observed reduced BRM binding to the DNA in clk null flies, suggesting that CLK is involved in enhancing BRM occupancy to initiate transcriptional repression at the conclusion of the activation phase. Additionally, we observed reduced BRM binding to the per promoter in flies overexpressing TIM, suggesting that TIM promotes BRM removal from DNA. These conclusions are further supported by elevated BRM binding to the per promoter in flies subjected to constant light and experiments in Drosophila tissue culture in which the levels of CLK and TIM are manipulated. In summary, this study provides new insights into the reciprocal regulation between the circadian clock and the BRM chromatin-remodeling complex.

Circadian clocks orchestrate daily rhythms in organismal physiology and behavior to promote optimal performance and fitness. In Drosophila, key pacemaker proteins PERIOD (PER) and TIMELESS (TIM) are progressively phosphorylated to perform phase-specific functions. Whereas PER phosphorylation has been extensively studied, systematic analysis of site-specific TIM phosphorylation is lacking. Here, we identified phosphorylation sites of PER-bound TIM by mass spectrometry, given the importance of TIM as a modulator of PER function in the pacemaker. Among the 12 TIM phosphorylation sites we identified, at least two of them are critical for circadian timekeeping as mutants expressing non-phosphorylatable mutations exhibit altered behavioral rhythms. In particular, we observed that CK2-dependent phosphorylation of TIM(S1404) promotes nuclear accumulation of PER-TIM heterodimers by inhibiting the interaction of TIM and nuclear export component, Exportin 1 (XPO1). We propose that proper level of nuclear PER-TIM accumulation is necessary to facilitate kinase recruitment for the regulation of daily phosphorylation rhythm and phase-specific transcriptional activity of CLOCK (CLK). Our results highlight the contribution of phosphorylation-dependent nuclear export of PER-TIM heterodimers to the maintenance of circadian periodicity and identify a new mechanism by which the negative elements of the circadian clock (PER-TIM) regulate the positive elements (CLK-CYC). Finally, because the molecular phenotype of tim(S1404A) non-phosphorylatable mutant exhibits remarkable similarity to that of a mutation in human timeless that underlies familial advanced sleep phase syndrome (FASPS), our results revealed an unexpected parallel between the functions of Drosophila and human TIM and may provide new insights into the molecular mechanisms underlying human FASPS.