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

Abstract

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.

Hexosamine biosynthetic pathway integrates circadian and metabolic signals to regulate daily rhythms in protein O-linked N-acetylglucosaminylation

“…A number of key circadian clock proteins have previously been observed to exhibit daily rhythms in O-GlcNAcylation that modulate their time-of-day-specific functions (Kim et al, 2012;Kaasik et al, 2013;Li et al, 2013;Li et al, 2019). We hypothesized that this phenomenon may be more widespread and could serve as an important mechanism that regulates daily rhythms in protein structure and function.…”

“…Complementary DNA synthesis and real-time quantitative PCR were performed as previously described (Kwok et al, 2015;Li et al, 2019). Expression of target genes was normalized to noncycling cbp20 (Table S2).…”

“…In previous studies, we and others have shown that a number of key clock transcription factors in flies and mice exhibit daily O-GlcNAcylation rhythms that regulate their subcellular localization, activity, and stability (Kim et al, 2012;Kaasik et al 2013;Li et al, 2013;Li et al, 2019). However, it is not clear whether cellular proteins beyond the circadian oscillator also exhibit daily O-GlcNAcylation rhythms.…”

New Drosophila Circadian Clock Mutants Affecting Temperature Compensation Induced by Targeted Mutagenesis of Timeless

“…Additional interconnected transcription/translational feedback loops that contribute to the circadian system were described in Drosophila as well as other insects. The PER/TIM feedback loop model was established and further refined through a combination of immunocytochemistry (ICC) (Siwicki et al, 1988), time-course expression profiling (Hardin et al, 1990, 1992), protein biochemical approaches addressing phosphorylation (Edery et al, 1994; Chiu et al, 2011), glycosylation (Li et al, 2019), protein coexpression in Drosophila Schneider cell culture (Saez and Young, 1996; Nawathean and Rosbash, 2004; Meyer et al, 2006), and yeast two-hybrid experiments (Rutila et al, 1996). But the key starting point in the per and tim research was the identification of mutants in extensive genetic screens using either chemical mutagens (Konopka and Benzer, 1971; Konopka et al, 1994; Rothenfluh et al, 2000a), or P-element mobilization (Sehgal et al, 1994).…”

“…The protein–protein interaction between PER and TIM is a complex and dynamic event (Meyer et al, 2006), including PER homodimerization (Landskron et al, 2009), multiple sequential phosphorylations (Martinek et al, 2001; Ko et al, 2010; Chiu et al, 2011), dephosphorylations (Sathyanarayanan et al, 2004; Fang et al, 2007), and possibly additional posttranslational modifications (Li et al, 2019). A key feature of the negative feedback loop in Drosophila is the ∼ 6 h delay that exists between the cytoplasmic accumulation and nuclear translocation of PER and TIM.…”