scholarly journals Circadian Clock-Specific Roles for the Light Response Protein WHITE COLLAR-2

2001 ◽  
Vol 21 (8) ◽  
pp. 2619-2628 ◽  
Author(s):  
Michael A. Collett ◽  
Jay C. Dunlap ◽  
Jennifer J. Loros
Keyword(s):  
Circadian Clock ◽  
Temperature Compensation ◽  
Light Response ◽  
White Collar ◽  
Normal Activity ◽  
Relative Increase ◽  
Period Length ◽  
Mrna Levels ◽  
Temperature Dependent ◽  
Protein Levels

ABSTRACT To understand the role of white collar-2 in theNeurospora circadian clock, we examined alleles ofwc-2 thought to encode partially functional proteins. We found that wc-2 allele ER24 contained a conservative mutation in the zinc finger. This mutation results in reduced levels of circadian rhythm-critical clock gene products, frq mRNA and FRQ protein, and in a lengthened period of the circadian clock. In addition, this mutation altered a second canonical property of the clock, temperature compensation: as temperature increased, period length decreased substantially. This temperature compensation defect correlated with a temperature-dependent increase in overall FRQ protein levels, with the relative increase being greater in wc-2(ER24) than in wild type, while overall frq mRNA levels were largely unaltered by temperature. We suggest that this temperature-dependent increase in FRQ levels partially rescues the lowered levels of FRQ resulting from the wc-2 (ER24) defect, yielding a shorter period at higher temperatures. Thus, normal activity of the essential clock component WC-2, a positive regulator offrq, is critical for establishing period length and temperature compensation in this circadian system.

scholarly journals PERIOD phosphoclusters control temperature compensation of the Drosophila circadian clock

2021 ◽  
Author(s):  
Patrick Emery ◽  
Radhika Joshi ◽  
Yao Cai ◽  
Yomgliang Xia ◽  
Joanna Chiu
Keyword(s):  
Cell Culture ◽  
Circadian Clock ◽  
Temperature Compensation ◽  
Thermal Compensation ◽  
Functional Heterogeneity ◽  
Temperature Dependent ◽  
Critical Feature ◽  
Control Temperature ◽  
Functional Homolog

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. Using CRISPR-Cas9, we introduced a series of mutations that altered three Serines (S44, 45 and 47) belonging to the PER phosphodegron, the functional homolog of mammalian PER2’s S487 phosphodegron, which impacts temperature compensation. While all three Serine to Alanine substitutions lengthened period at all temperatures tested, temperature compensation was differentially affected. S44A and S45A substitutions caused decreased temperature compensation, 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 decreased thermal compensation, consistent with its inhibitory role on S47 phosphorylation. Interestingly,the S47A substitution caused increased accumulation of hyper-phosphorylated PER at warmer temperatures. This finding was corroborated by cell culture assays in which S47A caused excessive temperature compensation of phosphorylation-dependent PER degradation. Thus, we show a novel role of the PER phosphodegron in temperature compensation through temperature-dependent modulation of the abundance of hyper-phosphorylated PER. Our work also reveals interesting mechanistic convergences and differences between mammalian and Drosophila temperature compensation of the circadian clock.

scholarly journals Mutation of a PER2 phosphodegron perturbs the circadian phosphoswitch

2020 ◽  
Vol 117 (20) ◽  
pp. 10888-10896 ◽  
Author(s):  
Shusaku Masuda ◽  
Rajesh Narasimamurthy ◽  
Hikari Yoshitane ◽  
Jae Kyoung Kim ◽  
Yoshitaka Fukada ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Messenger Rna ◽  
Temperature Compensation ◽  
Mrna Levels ◽  
Circadian Period ◽  
Embryonic Fibroblasts ◽  
Casein Kinase 1 ◽  
Ubiquitin E3 Ligase ◽  
Three Phase

Casein kinase 1 (CK1) plays a central role in regulating the period of the circadian clock. In mammals, PER2 protein abundance is regulated by CK1-mediated phosphorylation and proteasomal degradation. On the other hand, recent studies have questioned whether the degradation of the core circadian machinery is a critical step in clock regulation. Prior cell-based studies found that CK1 phosphorylation of PER2 at Ser478 recruits the ubiquitin E3 ligase β-TrCP, leading to PER2 degradation. Creation of this phosphodegron is regulated by a phosphoswitch that is also implicated in temperature compensation. However, in vivo evidence that this phosphodegron influences circadian period is lacking. Here, we generated and analyzed PER2-Ser478Ala knock-in mice. The mice showed longer circadian period in behavioral analysis. Molecularly, mutant PER2 protein accumulated in both the nucleus and cytoplasm of the mouse liver, while Per2 messenger RNA (mRNA) levels were minimally affected. Nuclear PER1, CRY1, and CRY2 proteins also increased, probably due to stabilization of PER2-containing complexes. In mouse embryonic fibroblasts derived from PER2-Ser478Ala::LUC mice, three-phase decay and temperature compensation of the circadian period was perturbed. These data provide direct in vivo evidence for the importance of phosphorylation-regulated PER2 stability in the circadian clock and validate the phosphoswitch in a mouse model.

scholarly journals timrit Lengthens Circadian Period in a Temperature-Dependent Manner through Suppression of PERIOD Protein Cycling and Nuclear Localization

1999 ◽  
Vol 19 (6) ◽  
pp. 4343-4354 ◽  
Author(s):  
Akira Matsumoto ◽  
Kenji Tomioka ◽  
Yoshihiko Chiba ◽  
Teiichi Tanimura
Keyword(s):  
Nuclear Localization ◽  
Temperature Compensation ◽  
Feedback Loop ◽  
Gene Dosage ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Protein Levels ◽  
Free Running ◽  
Free Running Period ◽  
Posttranscriptional Level

ABSTRACT A fundamental feature of circadian clocks is temperature compensation of period. The free-running period of ritsu(timrit ) (a novel allele oftimeless [tim]) mutants is drastically lengthened in a temperature-dependent manner. PER and TIM protein levels become lower in timrit mutants as temperature becomes higher. This mutation reduces per mRNA but not tim mRNA abundance. PER constitutively driven by the rhodopsin1 promoter is lowered in ritmutants, indicating that timrit mainly affects the per feedback loop at a posttranscriptional level. An excess of per + gene dosage can ameliorate allrit phenotypes, including the weak nuclear localization of PER, suggesting that timrit affects circadian rhythms by reducing PER abundance and its subsequent transportation into nuclei as temperature increases.

scholarly journals FRQ-CK1 Interaction Underlies Temperature Compensation of the Neurospora Circadian Clock

mBio ◽  
2021 ◽  
Author(s):  
Yue Hu ◽  
Xiaolan Liu ◽  
Qiaojia Lu ◽  
Yulin Yang ◽  
Qun He ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Temperature Compensation ◽  
Period Length ◽  
Clock Proteins ◽  
Constant Period

Temperature compensation allows clocks to adapt to all seasons by having a relatively constant period length at different physiological temperatures, but the mechanism of temperature compensation is unclear. Stability of clock proteins was previously proposed to be a major factor that regulated temperature compensation.

scholarly journals Inhibition of αENaC expression and ENaC activity following blockade of the circadian clock-regulatory kinases CK1δ/ε

2012 ◽  
Vol 303 (7) ◽  
pp. F918-F927 ◽  
Author(s):  
Jacob Richards ◽  
Megan M. Greenlee ◽  
Lauren A. Jeffers ◽  
Kit-Yan Cheng ◽  
Laijing Guo ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Circadian Clock ◽  
Single Channel ◽  
Collecting Duct ◽  
Transcriptional Response ◽  
Mrna Levels ◽  
Cortical Collecting Duct ◽  
Nuclear Entry ◽  
Protein Levels ◽  
Clock Protein

Increasing evidence suggests that the circadian clock plays an important role in the control of renal function and blood pressure. We previously showed that the circadian clock protein Period (Per)1, positively regulates the expression of the rate limiting subunit of the renal epithelial sodium channel (αENaC), which contributes to blood pressure regulation. Casein kinases 1δ and 1ε (CK1δ/ε) are critical regulators of clock proteins. CK1δ/ε must phosphorylate the circadian clock protein Per1 in order for the latter to enter the nucleus. We used a commercially available CK1δ/ε inhibitor, PF670462, to test the effect of CK1δ/ε blockade and inhibited Per1 nuclear entry on αENaC in a model of the renal cortical collecting duct (mpkCCDc14 cells). CK1δ/ε blockade prevented Per1 and Clock from interacting with an E-box from the αENaC promoter. CK1δ/ε inhibition reduced αENaC mRNA levels by <60%. A similar decrease in αENaC mRNA was observed following siRNA-mediated CK1δ/ε knock-down. Inhibition of CK1δ/ε effectively prevented the transcriptional response of αENaC to aldosterone, suggesting an interaction between the circadian clock and aldosterone-mediated regulation of αENaC. CK1δ/ε inhibition significantly reduced αENaC but increased Caveolin-1 membrane protein levels; transepithelial current, a measure of ENaC activity, was decreased. Importantly, single channel analysis in amphibian renal cells demonstrated a dramatic decrease in the number of patches with observable ENaC current following CK1δ/ε inhibition. The present study shows for the first time that CK1δ/ε inhibition and impaired Per1 nuclear entry results in decreased αENaC expression and ENaC activity, providing further support for direct control of ENaC by the circadian clock.

scholarly journals Impact of the White Collar Photoreceptor WcoA on the Fusarium fujikuroi Transcriptome

2021 ◽  
Vol 11 ◽  
Author(s):  
Javier Pardo-Medina ◽  
Gabriel Gutiérrez ◽  
M. Carmen Limón ◽  
Javier Avalos
Keyword(s):  
Secondary Metabolites ◽  
Regulatory Protein ◽  
Antioxidant Properties ◽  
Wild Strain ◽  
Light Response ◽  
White Collar ◽  
Fusarium Fujikuroi ◽  
Mrna Levels ◽  
Transcriptional Responses ◽  
Central Function

The proteins of the White Collar 1 family (WC) constitute a major class of flavin photoreceptors, widely distributed in fungi, that work in cooperation with a WC 2 protein forming a regulatory complex. The WC complex was investigated in great detail in Neurospora crassa, a model fungus in photobiology studies, where it controls all its major photoresponses. The fungus Fusarium fujikuroi, a model system in the production of secondary metabolites, contains a single WC-1 gene called wcoA. The best-known light response in this fungus is the photoinduction of the synthesis of carotenoids, terpenoid pigments with antioxidant properties. Loss of WcoA in F. fujikuroi results in a drastic reduction in the mRNA levels of the carotenoid genes, and a diversity of morphological and metabolic changes, including alterations in the synthesis of several secondary metabolites, suggesting a complex regulatory role. To investigate the function of WcoA, the transcriptome of F. fujikuroi was analyzed in the dark and after 15-, 60- or 240-min illumination in a wild strain and in a formerly investigated wcoA insertional mutant. Using a threshold of four-fold change in transcript levels, 298 genes were activated and 160 were repressed in the wild strain under at least one of the light exposures. Different response patterns were observed among them, with genes exhibiting either fast, intermediate, and slow photoinduction, or intermediate or slow repression. All the fast and intermediate photoresponses, and most of the slow ones, were lost in the wcoA mutant. However, the wcoA mutation altered the expression of a much larger number of genes irrespective of illumination, reaching at least 16% of the annotated genes in this fungus. Such genes include many related to secondary metabolism, as well as others related to photobiology and other cellular functions, including the production of hydrophobins. As judged by the massive transcriptomic changes exhibited by the wcoA mutant in the dark, the results point to WcoA as a master regulatory protein in F. fujikuroi, in addition to a central function as the photoreceptor responsible for most of the transcriptional responses to light in this fungus.

scholarly journals Temperature compensation and temperature sensation in the circadian clock

2015 ◽  
Vol 112 (46) ◽  
pp. E6284-E6292 ◽  
Author(s):  
Philip B. Kidd ◽  
Michael W. Young ◽  
Eric D. Siggia
Keyword(s):  
Circadian Clock ◽  
Temperature Compensation ◽  
Temperature Oscillation ◽  
Circadian Clocks ◽  
Luciferase Reporter ◽  
Alternative Theory ◽  
Temperature Dependent ◽  
Western Blots ◽  
Temperature Changes ◽  
Clock Component

All known circadian clocks have an endogenous period that is remarkably insensitive to temperature, a property known as temperature compensation, while at the same time being readily entrained by a diurnal temperature oscillation. Although temperature compensation and entrainment are defining features of circadian clocks, their mechanisms remain poorly understood. Most models presume that multiple steps in the circadian cycle are temperature-dependent, thus facilitating temperature entrainment, but then insist that the effect of changes around the cycle sums to zero to enforce temperature compensation. An alternative theory proposes that the circadian oscillator evolved from an adaptive temperature sensor: a gene circuit that responds only to temperature changes. This theory implies that temperature changes should linearly rescale the amplitudes of clock component oscillations but leave phase relationships and shapes unchanged. We show using timeless luciferase reporter measurements and Western blots against TIMELESS protein that this prediction is satisfied by the Drosophila circadian clock. We also review evidence for pathways that couple temperature to the circadian clock, and show previously unidentified evidence for coupling between the Drosophila clock and the heat-shock pathway.

scholarly journals Mutation of a PER2 phosphodegron perturbs the circadian phosphoswitch

2019 ◽  
Author(s):  
Shusaku Masuda ◽  
Rajesh Narasimamurthy ◽  
Hikari Yoshitane ◽  
Jae Kyoung Kim ◽  
Yoshitaka Fukada ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Temperature Compensation ◽  
Protein Abundance ◽  
Mrna Levels ◽  
Circadian Period ◽  
Embryonic Fibroblasts ◽  
Casein Kinase 1 ◽  
Ubiquitin E3 Ligase ◽  
Three Phase

AbstractCasein kinase 1 (CK1) plays a central role in regulating the period of the circadian clock. In mammals, PER2 protein abundance is regulated by CK1-mediated phosphorylation and proteasomal degradation. On the other hand, recent studies have questioned whether the degradation of the core circadian machinery is a critical step in clock regulation. Prior cell-based studies found that CK1 phosphorylation of PER2 at Ser478 recruits the ubiquitin E3 ligase β-TrCP, leading to PER2 degradation. Creation of this phosphodegron is regulated by a phosphoswitch that is also implicated in temperature compensation. However, in vivo evidence that this phosphodegron influences circadian period is lacking. Here, we generated and analyzed PER2-Ser478Ala knock-in mice. The mice showed longer circadian period in behavioral analysis. Molecularly, mutant PER2 protein accumulated in both the nucleus and cytoplasm of the mouse liver, while Per2 mRNA levels were minimally affected. Nuclear PER1, CRY1 and CRY2 proteins also increased, probably due to stabilization of PER2-containing complexes. In mouse embryonic fibroblasts derived from PER2-Ser478Ala::LUC mice, three-phase decay and temperature compensation of the circadian period was perturbed. These data provide direct in vivo evidence for the importance of phosphorylation-regulated PER2 stability in the circadian clock and validate the phosphoswitch in a mouse model.

The Effects of Vitamin K1 and Warfarin on Prothrombin Expression in Human Hepatoblastoma (HepG2) Cells

1992 ◽  
Vol 68 (01) ◽  
pp. 040-047 ◽  
Author(s):  
C Scott Jamison ◽  
Bryan F Burkey ◽  
Sandra J Friezner Degen
Keyword(s):  
Total Protein ◽  
Hepg2 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Response Analysis ◽  
Secreted Protein ◽  
Mrna Levels ◽  
Vitamin K1 ◽  
Maximal Increase ◽  
Protein Levels ◽  
Warfarin Treatment

SummaryCultures of human hepatoblastoma (HepG2) cells were treated with vitamin K1 or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 µg vitamin K1 treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 µg/ml vitamin K1 treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with 35S-methionine and immunoprecipitation of 35S-labelled prothrombin demonstrated that, with vitamin K1 treatment (25 µg/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 µg/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K1 nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240–350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K1 and warfarin, in addition to effects on γ-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.

Sign in / Sign up

Export Citation Format

Share Document