Abstract
Robust rhythms of abundances and phosphorylation profiles of PERIOD proteins were thought be the master rhythms that drive mammalian circadian clock functions. PER stability was proposed to ...be a major determinant of period length. In mammals, CK1 forms stable complexes with PER. Here we identify the PER residues essential for PER-CK1 interaction. In cells and in mice, their mutation abolishes PER phosphorylation and CLOCK hyperphosphorylation, resulting in PER stabilization, arrhythmic PER abundance and impaired negative feedback process, indicating that PER acts as the CK1 scaffold in circadian feedback mechanism. Surprisingly, the mutant mice exhibit robust short period locomotor activity and other physiological rhythms but low amplitude molecular rhythms. PER-CK1 interaction has two opposing roles in regulating CLOCK-BMAL1 activity. These results indicate that the circadian clock can function independently of PER phosphorylation and abundance rhythms due to another PER-CRY-dependent feedback mechanism and that period length can be uncoupled from PER stability.
Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and plays an important role in regulating gene expression levels. A major role of codon usage is thought to regulate ...protein expression levels by affecting mRNA translation efficiency, but the underlying mechanism is unclear. By analyzing ribosome profiling results, here we showed that codon usage regulates translation elongation rate and that rare codons are decoded more slowly than common codons in all codon families in Neurospora. Rare codons resulted in ribosome stalling in manners both dependent and independent of protein sequence context and caused premature translation termination. This mechanism was shown to be conserved in Drosophila cells. In both Neurospora and Drosophila cells, codon usage plays an important role in regulating mRNA translation efficiency. We found that the rare codon-dependent premature termination is mediated by the translation termination factor eRF1, which recognizes ribosomes stalled on rare sense codons. Silencing of eRF1 expression resulted in codon usage-dependent changes in protein expression. Together, these results establish a mechanism for how codon usage regulates mRNA translation efficiency.
In addition to responding to environmental entrainment with diurnal variation, metabolism is also tightly controlled by cell-autonomous circadian clock. Extensive studies have revealed key roles of ...transcription in circadian control. Post-transcriptional regulation for the rhythmic gating of metabolic enzymes remains elusive. Here, we show that arginine biosynthesis and subsequent ureagenesis are collectively regulated by CLOCK (circadian locomotor output cycles kaput) in circadian rhythms. Facilitated by BMAL1 (brain and muscle Arnt-like protein), CLOCK directly acetylates K165 and K176 of argininosuccinate synthase (ASS1) to inactivate ASS1, which catalyzes the rate-limiting step of arginine biosynthesis. ASS1 acetylation by CLOCK exhibits circadian oscillation in human cells and mouse liver, possibly caused by rhythmic interaction between CLOCK and ASS1, leading to the circadian regulation of ASS1 and ureagenesis. Furthermore, we also identified NADH dehydrogenase ubiquinone 1 alpha subcomplex subunit 9 (NDUFA9) and inosine-5′-monophosphate dehydrogenase 2 (IMPDH2) as acetylation substrates of CLOCK. Taken together, CLOCK modulates metabolic rhythmicity by acting as a rhythmic acetyl-transferase for metabolic enzymes.
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•CLOCK acetylates metabolic enzymes to modulate their activity•CLOCK drives the cycle of acetylation and activation of metabolic enzymes•Interaction between CLOCK and metabolic enzymes is rhythmic•Arginine biogenesis and ureagenesis are rhythmically controlled by ASS1 acetylation
Lin et al. reported that CLOCK acetylates ASS1 to inactivate its function in arginine biosynthesis. ASS1 acetylation by CLOCK exhibits rhythmicity, possibly caused by rhythmic interaction between CLOCK and ASS1, leading to the cycle of ASS1 activation. Two more enzymes, NDUFA9 and IMPDH2, were recognized as acetylation substrates of CLOCK.
Mechanisms underlying the vein development remain largely unknown. Tie2 signaling mediates endothelial cell (EC) survival and vascular maturation and its activating mutations are linked to venous ...malformations. Here we show that vein formation are disrupted in mouse skin and mesentery when Tie2 signals are diminished by targeted deletion of
either ubiquitously or specifically in embryonic ECs. Postnatal Tie2 attenuation resulted in the degeneration of newly formed veins followed by the formation of haemangioma-like vascular tufts in retina and venous tortuosity. Mechanistically, Tie2 insufficiency compromised venous EC identity, as indicated by a significant decrease of COUP-TFII protein level, a key regulator in venogenesis. Consistently, angiopoietin-1 stimulation increased COUP-TFII in cultured ECs, while Tie2 knockdown or blockade of Tie2 downstream PI3K/Akt pathway reduced COUP-TFII which could be reverted by the proteasome inhibition. Together, our results imply that Tie2 is essential for venous specification and maintenance via Akt mediated stabilization of COUP-TFII.
Many animals display morning and evening bimodal activities in the day/night cycle. However, little is known regarding the potential components involved in the regulation of bimodal behavioral ...rhythms in mammals. Here, we identified that the zinc finger protein gene
plays a crucial role in the regulation of bimodal activities in mice. Depletion of
in nerve system resulted in the loss of early evening activity, but the increase of morning activity. We found that
-deficient mice exhibited a pronounced decrease in the expression of
and resembled phenotypes of
and
-knockout mice. Injection of adeno-associated virus-double-floxed
in suprachiasmatic nucleus could partly restore evening activity in
(NS-ZB20KO) mice. Furthermore, loss of
in
loci, but intact in the suprachiasmatic nucleus, was not responsible for the unimodal activity of NS-ZB20KO mice. Our study provides evidence that ZBTB20-mediated PROKR2 signaling is critical for the evening behavioral rhythms.
In the core mammalian circadian negative feedback loop, the BMAL1-CLOCK complex activates the transcription of the genes Period (Per) and Cryptochrome (Cry). To close the negative feedback loop, the ...PER-CRY complex interacts with the BMAL1-CLOCK complex to repress its activity. These two processes are separated temporally to ensure clock function. Here, we show that histone deacetylase 3 (HDAC3) is a critical component of the circadian negative feedback loop by regulating both the activation and repression processes in a deacetylase activity-independent manner. Genetic depletion of Hdac3 results in low-amplitude circadian rhythms and dampened E-box-driven transcription. In subjective morning, HDAC3 is required for the efficient transcriptional activation process by regulating BMAL1 stability. In subjective night, however, HDAC3 blocks FBXL3-mediated CRY1 degradation and strongly promotes BMAL1 and CRY1 association. Therefore, these two opposing but temporally separated roles of HDAC3 in the negative feedback loop provide a mechanism for robust circadian gene expression.
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•HDAC3 is a critical component of the core mammalian circadian negative feedback loop•HDAC3 is required for efficient activation of E-box-driven gene expression•HDAC3 blocks CRY1 degradation and promotes BMAL1 and CRY1 association•Temporal separation of these opposing roles ensures robust circadian gene expression
Shi et al. report a deacetylase-independent function of HDAC3 in maintaining a robust circadian clock. HDAC3 modulates the function of several key components of the clock machinery and affects both the activation and suppression of transcription in the circadian cycle.
Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the ...physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.
Understanding the mechanism underlying the physiological divergence of species is a long-standing issue in evolutionary biology. The circadian clock is a highly conserved system existing in almost ...all organisms that regulates a wide range of physiological and behavioral events to adapt to the day-night cycle. Here, the interactions between hCK1ɛ/δ/DBT (Drosophila ortholog of CK1δ/ɛ) and serine-rich (SR) motifs from hPER2 (ortholog of Drosophila per) were reconstructed in a Drosophila circadian system. The results indicated that in Drosophila, the SR mutant form hPER2S662G does not recapitulate the mouse or human mutant phenotype. However, introducing hCK1δ (but not DBT) shortened the circadian period and restored the SR motif function. We found that hCK1δ is catalytically more efficient than DBT in phosphorylating the SR motif, which demonstrates that the evolution of CK1δ activity is required for SR motif modulation. Moreover, an abundance of phosphorylatable SR motifs and the striking emergence of putative SR motifs in vertebrate proteins were observed, which provides further evidence that the correlated evolution between kinase activity and its substrates set the stage for functional diversity in vertebrates. It is possible that such correlated evolution may serve as a biomarker associated with the adaptive benefits of diverse organisms. These results also provide a concrete example of how functional synthesis can be achieved through introducing evolutionary partners in vivo.
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