Roles of peripheral clocks: lessons from the fly.

To adapt to and anticipate rhythmic changes in the environment such as daily light-dark and temperature cycles, internal timekeeping mechanisms called biological clocks evolved in a diverse set of organisms, from unicellular bacteria to humans. These biological clocks play critical roles in organisms' fitness and survival by temporally aligning physiological and behavioral processes to the external cues. The central clock is located in a small subset of neurons in the brain and drives daily activity rhythms, whereas most peripheral tissues harbor their own clock systems, which generate metabolic and physiological rhythms. Since the discovery of Drosophila melanogaster clock mutants in the early 1970s, the fruit fly has become an extensively studied model organism to investigate the mechanism and functions of circadian clocks. In this review, we primarily focus on D. melanogaster to survey key discoveries and progresses made over the past two decades in our understanding of peripheral clocks. We discuss physiological roles and molecular mechanisms of peripheral clocks in several different peripheral tissues of the fly.

Perception of Daily Time: Insights from the Fruit Flies.

We create mental maps of the space that surrounds us; our brains also compute time-in particular, the time of day. Visual, thermal, social, and other cues tune the clock-like timekeeper. Consequently, the internal clock synchronizes with the external day-night cycles. In fact, daylength itself varies, causing the change of seasons and forcing our brain clock to accommodate layers of plasticity. However, the core of the clock, i.e., its molecular underpinnings, are highly resistant to perturbations, while the way animals adapt to the daily and annual time shows tremendous biological diversity. How can this be achieved? In this review, we will focus on 75 pairs of clock neurons in the Drosophila brain to understand how a small neural network perceives and responds to the time of the day, and the time of the year.

The Paralogous Krüppel-like Factors 9 and 13 Regulate the Mammalian Cellular Circadian Clock Output Gene Dbp.

An intricate transcription-translation feedback loop (TTFL) governs cellular circadian rhythms in mammals. Here, we report that the zinc finger transcription factor Krüppel-like factor 9 (KLF9) is regulated by this TTFL, it associates in chromatin at the core circadian clock and clock-output genes, and it acts to modulate transcription of the clock-output gene Dbp. Our earlier genome-wide analysis of the mouse hippocampus-derived cell line HT22 showed that KLF9 associates in chromatin with Per1, Per3, Dbp, Tef, Bhlhe40, Bhlhe41, Nr1d1, and Nr1d2. Of the 3514 KLF9 peaks identified in HT22 cells, 1028 contain E-box sequences to which the transcriptional activators CLOCK and BMAL1 may bind, a frequency significantly greater than expected by chance. Klf9 mRNA showed circadian oscillation in synchronized HT22 cells, mouse hippocampus, and liver. At the clock-output gene Dbp, KLF9 exhibited circadian rhythmicity in its association in chromatin in HT22 cells and hippocampus. Forced expression of KLF9 in HT22 cells repressed basal Dbp transcription and strongly inhibited CLOCK+BMAL1-dependent transcriptional activation of a transfected Dbp reporter. Mutational analysis showed that this action of KLF9 depended on 2 intact KLF9-binding motifs within the Dbp locus that are in close proximity to E-boxes. Knockout of Klf9 or the paralogous gene Klf13 using CRISPR/Cas9 genome editing in HT22 cells had no effect on Dbp expression, but combined knockout of both genes strongly impaired circadian Dbp mRNA oscillation. Like KLF9, KLF13 also showed association in chromatin with clock- and clock-output genes, and forced expression of KLF13 inhibited the actions of CLOCK+BMAL1 on Dbp transcription. Our results suggest novel and partly overlapping roles for KLF9 and KLF13 in modulating cellular circadian clock output by a mechanism involving direct interaction with the core TTFL.

Atomic force microscopy analysis of SasA-KaiC complex formation involved in information transfer from the KaiABC clock machinery to the output pathway in cyanobacteria.

The cyanobacterial clock oscillator is composed of three clock proteins: KaiA, KaiB and KaiC. SasA, a KaiC-binding EnvZ-like orthodox histidine kinase involved in the main clock output pathway, exists mainly as a trimer (SasA3mer ) and occasionally as a hexamer (SasA6mer ) in vitro. Previously, the molecular mass of the SasA-KaiCDD complex, where KaiCDD is a mutant KaiC with two Asp substitutions at the two phosphorylation sites, has been estimated by gel-filtration chromatography to be larger than 670 kDa. This value disagrees with the theoretical estimation of 480 kDa for a SasA3mer -KaiC hexamer (KaiC6mer ) complex with a 1:1 molecular ratio. To clarify the structure of the SasA-KaiC complex, we analyzed KaiCDD with 0.1 mmol/L ATP and 5 mmol/L MgCl2 (Mg-ATP), SasA and a mixture containing SasA and KaiCDD6mer with Mg-ATP by atomic force microscopy (AFM). KaiCDD images were classified into two types with height distribution corresponding to KaiCDD monomer (KaiCDD1mer ) and KaiCDD6mer , respectively. SasA images were classified into two types with height corresponding to SasA3mer and SasA6mer , respectively. The AFM images of the SasA-KaiCDD mixture indicated not only KaiCDD1mer , KaiCDD6mer , SasA3mer and SasA6mer , but also wider area "islands," suggesting the presence of a polymerized form of the SasA-KaiCDD complex.

OsPRR37 confers an expanded regulation of the diurnal rhythms of the transcriptome and photoperiodic flowering pathways in rice.

The circadian clock enables organisms to rapidly adapt to the ever-changing environmental conditions that are caused by daily light/dark cycles. Circadian clock genes universally affect key agricultural traits, particularly flowering time. Here, we show that OsPRR37, a circadian clock gene, delays rice flowering time in an expression level-dependent manner. Using high-throughput mRNA sequencing on an OsPRR37 overexpressing transgenic line (OsPRR37-OE5) and the recipient parent Guangluai4 that contains the loss-of-function Osprr37, we identify 14,992 genes that display diurnal rhythms, which account for 52.9% of the transcriptome. Overexpressing OsPRR37 weakens the transcriptomic rhythms and alters the phases of rhythmic genes. In total, 3,210 differentially expressed genes (DEGs) are identified, among which 1,863 rhythmic DEGs show a correlation between the change of absolute amplitudes and the mean expression levels. We further reveal that OsPRR37 functions as a transcriptional repressor to repress the expression levels and amplitudes of day-phased clock genes. More importantly, OsPRR37 confers expanded regulation on the evening-phased rhythmic DEGs by repressing the morning-phased rhythmic DEGs. Further study shows that OsPRR37 expands its regulation on flowering pathways by repressing Ehd1. Thus, our results demonstrate an expanded regulation mechanism of the circadian clock on the diurnal rhythms of the transcriptome.

Clock Gene Dysregulation Induced by Chronic ER Stress Disrupts ß-cell Function.

In Wfs1-/-Ay/a islets, in association with endoplasmic reticulum (ER) stress, D-site-binding protein (Dbp) expression decreased and Nuclear Factor IL-3 (Nfil3)/E4 Promoter-binding protein 4 (E4bp4) expression increased, leading to reduced DBP transcriptional activity. Similar alterations were observed with chemically-induced ER stress. Transgenic mice expressing E4BP4 under the control of the mouse insulin I gene promoter (MIP), in which E4BP4 in ß-cells is expected to compete with DBP for D-box, displayed remarkable glucose intolerance with severely impaired insulin secretion. Basal ATP/ADP ratios in MIP-E4BP4 islets were elevated without the circadian oscillations observed in wild-type islets. Neither elevation of the ATP/ADP ratio nor an intracellular Ca2+ response was observed after glucose stimulation. RNA expressions of genes involved in insulin secretion gradually increase in wild-type islets early in the feeding period. In MIP-E4BP4 islets, however, these increases were not observed. Thus, molecular clock output DBP transcriptional activity, susceptible to ER stress, plays pivotal roles in ß-cell priming for insulin release by regulating ß-cell metabolism and gene expressions. Because ER stress is also involved in the ß-cell failure in more common Type-2 diabetes, understanding the currently identified ER stress-associated mechanisms warrants novel therapeutic and preventive strategies for both rare form and common diabetes.

Differential arousal regulation by prokineticin 2 signaling in the nocturnal mouse and the diurnal monkey.

The temporal organization of activity/rest or sleep/wake rhythms for mammals is regulated by the interaction of light/dark cycle and circadian clocks. The neural and molecular mechanisms that confine the active phase to either day or night period for the diurnal and the nocturnal mammals are unclear. Here we report that prokineticin 2, previously shown as a circadian clock output molecule, is expressed in the intrinsically photosensitive retinal ganglion cells, and the expression of prokineticin 2 in the intrinsically photosensitive retinal ganglion cells is oscillatory in a clock-dependent manner. We further show that the prokineticin 2 signaling is required for the activity and arousal suppression by light in the mouse. Between the nocturnal mouse and the diurnal monkey, a signaling receptor for prokineticin 2 is differentially expressed in the retinorecipient suprachiasmatic nucleus and the superior colliculus, brain projection targets of the intrinsically photosensitive retinal ganglion cells. Blockade with a selective antagonist reveals the respectively inhibitory and stimulatory effect of prokineticin 2 signaling on the arousal levels for the nocturnal mouse and the diurnal monkey. Thus, the mammalian diurnality or nocturnality is likely determined by the differential signaling of prokineticin 2 from the intrinsically photosensitive retinal ganglion cells onto their retinorecipient brain targets.