Carl Hirschie Johnson

Peroxiredoxins are conserved markers of circadian rhythms

Cellular life emerged ∼3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles owing to the Earth’s rotation. The advantage conferred on organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation–reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterizing their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription–translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular timekeeping with redox homeostatic mechanisms after the Great Oxidation Event ∼2.5 billion years ago.

Entrainment of Circadian Programs

Of the three defining properties of circadian rhythmicity—persisting free‐running rhythm, temperature compensation, and entrainment—the last is often poorly understood by many chronobiologists. This paper gives an overview of entrainment. Where have we been? Where are we now? Whence should we be going? Particular emphasis is given to a discussion of the Discrete vs. Continuous models for entrainment. We provide an integrated mechanism for entrainment from a limit‐cycle perspective.

A Predictive Model for Transcriptional Control of Physiology in a Free Living Cell

The environment significantly influences the dynamic expression and assembly of all components encoded in the genome of an organism into functional biological networks. We have constructed a model for this process in Halobacterium salinarum NRC-1 through the data-driven discovery of regulatory and functional interrelationships among approximately 80% of its genes and key abiotic factors in its hypersaline environment. Using relative changes in 72 transcription factors and 9 environmental factors (EFs) this model accurately predicts dynamic transcriptional responses of all these genes in 147 newly collected experiments representing completely novel genetic backgrounds and environments-suggesting a remarkable degree of network completeness. Using this model we have constructed and tested hypotheses critical to this organisms interaction with its changing hypersaline environment. This study supports the claim that the high degree of connectivity within biological and EF networks will enable the construction of similar models for any organism from relatively modest numbers of experiments.

Cyanobacterial circadian clockwork: roles of KaiA, KaiB and the kaiBC promoter in regulating KaiC

Using model strains in which we ectopically express the cyanobacterial clock protein KaiC in cells from which the clock genes kaiA, kaiB and/or kaiC are deleted, we found that some features of circadian clocks in eukaryotic organisms are conserved in the clocks of prokaryotic cyanobacteria, but others are not. One unexpected difference is that the circadian autoregulatory feedback loop in cyanobacteria does not require specific clock gene promoters as it does in eukaryotes, because a heterologous promoter can functionally replace the kaiBC promoter. On the other hand, a similarity between eukaryotic clock proteins and the cyanobacterial KaiC protein is that KaiC is phosphorylated in vivo. The other essential clock proteins KaiA and KaiB modulate the status of KaiC phosphorylation; KaiA inhibits KaiC dephosphorylation and KaiB antagonizes this action of KaiA. Based upon an analysis of clock mutants, we conclude that the circadian period in cyanobacteria is determined by the phosphorylation status of KaiC and also by the degradation rate of KaiC. These observations are integrated into a model proposing rhythmic changes in chromosomal status.

Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm

The changes in the intracellular pH (pHi) of sea urchin sperm associated with motility initiation and acrosome reaction were investigated using uptake of two different probes; 9-aminoacridine and methylamine, as a qualitative index. Sperm suspended in Na+-free sea water were immotile and able to concentrate these amines 20-fold or greater indicating that pHi is more acidic than the external medium (pHo = 7.7). This uptake ratio was essentially constant over a wide range of probe and sperm concentrations. Discharge of the pH gradient with specific ionophores (nigericin, monensin, and tetrachlorosalicylanilide) or nonspecifically using low concentration of detergents (Triton X-100 and lysolecithin) all resulted in the release of the probes indicating they are indeed sensing the pH gradient across the sperm membrane. Addition of Na+ to sperm suspended in Na+-free sea water resulted in activation of motility with concomitant efflux of the probes indicating the alkalinization of pHi by 0.4-0.5 pH units. That this pHi change is the causal trigger of motility was suggested by experiments using NH4Cl and nigericin, which increased the pHi and resulted in activation of motility in the absence of Na+. When sperm were directly diluted into artificial sea water (motility activated), a slow reacidification of pHi was observed in one species of sea urchin (L. pictus) but not in the other (S. purpuratus). This acidification could be blocked by mitochondrial inhibitors, verapamil, or the removal of external calcium suggesting that the increase in metabolic activity stimulated by the influx of Ca2+ is responsible for the reacidification. Induction of acrosome reaction further alkalinized the pHi by about 0.16 pH units and was also followed by prolonged reacidification which correlated with the observed increase in Ca2+ uptake. Either mitochondrial agents or the removal of external Ca2+ could also block this pHi change suggesting a similar mechanism is involved.

Circadian disruption leads to insulin resistance and obesity

BACKGROUND Disruption of circadian (daily) timekeeping enhances the risk of metabolic syndrome, obesity, and type 2 diabetes. While clinical observations have suggested that insulin action is not constant throughout the 24 hr cycle, its magnitude and periodicity have not been assessed. Moreover, when circadian rhythmicity is absent or severely disrupted, it is not known whether insulin action will lock to the peak, nadir, or mean of the normal periodicity of insulin action. RESULTS We used hyperinsulinemic-euglycemic clamps to show a bona fide circadian rhythm of insulin action; mice are most resistant to insulin during their daily phase of relative inactivity. Moreover, clock-disrupted Bmal1-knockout mice are locked into the trough of insulin action and lack rhythmicity in insulin action and activity patterns. When rhythmicity is rescued in the Bmal1-knockout mice by expression of the paralogous gene Bmal2, insulin action and activity patterns are restored. When challenged with a high-fat diet, arhythmic mice (either Bmal1-knockout mice or wild-type mice made arhythmic by exposure to constant light) were obese prone. Adipose tissue explants obtained from high-fat-fed mice have their own periodicity that was longer than animals on a chow diet. CONCLUSIONS This study provides rigorous documentation for a circadian rhythm of insulin action and demonstrates that disturbing the natural rhythmicity of insulin action will disrupt the rhythmic internal environment of insulin sensitive tissue, thereby predisposing the animals to insulin resistance and obesity.

Forty Years of Prcs-What Have We Learned?

What are phase-response curves (PRCs)? How can they be measured? How should they be plotted? These questions and many other fascinating facets of PRCs are addressed in this review, including research topics in which phase-resetting data have provided crucial insights: entrainment, phototransduction, pacemaker mechanism, phase markers of the pacemaker, and gauges of oscillator amplitude. PRCs have enlightened us and will continue to be a valuable tool in clock research.

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecA/DnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecA/DnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

Regulation of the PAI-1 promoter by circadian clock components: differential activation by BMAL1 and BMAL2.

Circadian variation in plasminogen activator inhibitor-1 (PAI-1) production likely contributes to increased risk of myocardial infarction and decreased efficacy of thrombolytic therapy during the morning. In this study, we characterize the abilities of fundamental molecular components of intrinsic circadian clocks to regulate the human PAI-1 promoter in transfected endothelial cells. Both CLOCK:BMAL1 and CLOCK:BMAL2 heterodimers activate the PAI-1 promoter through requisite proximal (-565 to -560 bp) and distal (-680 to -675 bp) E-box enhancers. Although the distal E-box overlaps the 4G/5G polymorphism of the PAI-1 promoter, allelic variation at this site does not influence CLOCK:BMAL1-and CLOCK:BMAL2-mediated transactivation. Together, CLOCK:BMAL1 and CLOCK:BMAL2 make additive contributions to PAI-1 gene transcription. While the abilities of these heterodimers to activate gene expression differ by twofold, the susceptibilities of these circadian activators to inhibition by period and cryptochrome proteins are equivalent and redox independent. Given that BMAL1 and BMAL2 differ in their spatiotemporal distributions, such distinctions may allow intrinsic circadian clocks to modulate the amplitudes of their oscillators, while maintaining circadian periodicity. In this way, fundamental circadian clock components may drive circadian variation in PAI-1, which in turn influences the pathogenesis, timing, and treatment of acute atherothrombotic events.

Circadian gene expression in mammalian fibroblasts revealed by real-time luminescence reporting: Temperature compensation and damping

Mammalian cells such as rat-1 fibroblasts have been shown to exhibit daily oscillations in the expression of several gene transcripts in culture. After induction, these oscillations persist with a period of ≈24 h for several days. This characteristic suggests that the oscillations are controlled by a circadian clock, but the crucial criterion of temperature compensation has not been demonstrated for rat-1 fibroblasts. We have developed an automated assay of circadian expression of the mPer1 promoter in rat-1 fibroblasts that have been stably transfected with a luciferase reporter. Using this cell culture-based in vitro luminescent reporter assay, we found that the daily oscillation of mPer1 promoter activity in rat-1 cells is temperature compensated over the range of 28.5-36.5°C. This finding means that these oscillations are bona fide circadian rhythms. Moreover, the circadian clock of these homeothermic mammalian cells not only is temperature compensated but also is overcompensated such that it runs faster at cooler temperatures (Q10 of 0.85-0.88). The oscillations in rat-1 fibroblasts damp more rapidly at cooler temperatures, and damping is not due to cells becoming unhealthy because a second stimulus will reinitiate a robust rhythm. These data show that rat-1 cell cultures that are stably transfected with luminescence reporters are an excellent model system for studying circadian clocks at the cellular level in mammals.