Joshua J. Gamsby

A Circadian Clock in Neurospora: How Genes and Proteins Cooperate to Produce a Sustained, Entrainable, and Compensated Biological Oscillator with a Period of about a Day

Neurospora has proven to be a tractable model system for understanding the molecular bases of circadian rhythms in eukaryotes. At the core of the circadian oscillatory system is a negative feedback loop in which two transcription factors, WC-1 and WC-2, act together to drive expression of the frq gene. WC-2 enters the promoter region of frq coincident with increases in frq expression and then exits when the cycle of transcription is over, whereas WC-1 can always be found there. FRQ promotes the phosphorylation of the WCs, thereby decreasing their activity, and phosphorylation of FRQ then leads to its turnover, allowing the cycle to reinitiate. By understanding the action of light and temperature on frq and FRQ expression, the molecular basis of circadian entrainment to environmental light and temperature cues can be understood, and recently a specific role for casein kinase 2 has been found in the mechanism underlying circadian temperature-compensation. These data promise molecular explanations for all of the canonical circadian properties of this model system, providing biochemical answers and regulatory logic that may be extended to more complex eukaryotes including humans.

Deregulated expression of LRBA facilitates cancer cell growth.

LRBA expression is induced by mitogens in lymphoid and myeloid cells. The Drosophila LRBA orthologue rugose/DAKAP550 is involved in Notch, Ras and EGFR pathways. These findings suggest that LRBA could play a role in cell types that have increased proliferative and survival capacity. Here, we show by microarray and real-time PCR analyses that LRBA is overexpressed in several different cancers relative to their normal tissue controls. We also show that LRBA promoter activity and endogenous LRBA mRNA levels are reduced by p53 and increased by E2F1, indicating that mutations in the tumor suppressors p53 and Rb could contribute to the deregulation of LRBA. Furthermore, inhibition of LRBA expression by RNA interference, or inhibition of its function by a dominant-negative mutant, leads to significant growth inhibition of cancer cells, demonstrating that deregulated expression of LRBA contributes to the altered growth properties of a cancer cell. Finally, we show that the phosphorylation of EGFR is affected by the dominant-negative mutant, suggesting LRBA plays a role in the mammalian EGFR pathway. These findings demonstrate that LRBA facilitates cancer cell growth and thus LRBA may represent a novel molecular target for cancer therapy.

The circadian Per1 and Per2 genes influence alcohol intake, reinforcement, and blood alcohol levels.

BACKGROUND Perturbations in the function of core circadian clock components such as the Period (Per) family of genes are associated with alcohol use disorder, and disruptions in circadian cycles may contribute to alcohol abuse and relapse. This study tested ethanol consumption, reinforcement, and metabolism in mice containing functional mutations in Per1 and/or Per2 genes on an ethanol-preferring background, C57BL/6J mice. METHODS Mice were tested in: (A) free-access intake with ascending concentrations of ethanol (2-16%, v/v), (B) conditioned place preference using ethanol (2g/kg for males; 2.5g/kg for females) vs. saline injections, (C) recovery of the righting reflex following a 4g/kg bolus of ethanol, and (D) blood ethanol levels 1h after a 2g/kg bolus of ethanol. RESULTS All Per mutant (mPer) mice showed increased ethanol intake and condition place preference compared to controls. There were also genotypic differences in blood ethanol concentration: in males, only mPer1 mice showed a significantly higher blood ethanol concentration than WT mice, but in females, all mPer mice showed higher blood ethanol levels than WT mice. CONCLUSIONS Mutation of either Per1 or Per2, as well as mutations of both genes, increases ethanol intake and reinforcement in an ethanol-preferring mouse model. In addition, this increase in ethanol seeking behavior seems to result both from a change in ethanol metabolism and a change in reward responding to ethanol, but not from any change in sensitivity to ethanols sedating effects.

A Phylogenetically Conserved DNA Damage Response Resets the Circadian Clock

The mammalian circadian clock influences the timing of many biological processes such as the sleep/wake cycle, metabolism, and cell division. Environmental cues such as light exposure can influence the timing of this system through the posttranslational modification of key components of the core molecular oscillator. We have previously shown that DNA damage can reset the circadian clock in a time-of-day—dependent manner in the filamentous fungus Neurospora crassa through the modulation of negative regulator FREQUENCY levels by PRD-4 (homologue of mammalian Chk2). We show that DNA damage, generated with either the radiomimetic drug methyl methane sulfonate or UV irradiation, in mouse embryonic fibroblasts isolated from PER2::LUC transgenic mice or in the NIH3T3 cell line, elicits similar responses. In addition to induction of phase advances, DNA damage caused a decrease in luciferase signal in PER2::LUC mouse embryonic fibroblast cells that is indicative of PER2 degradation. Finally, we show that the activity of the BMAL1 promoter is enhanced during DNA damage. These findings provide further evidence that the DNA damage-mediated response of the clock is conserved from lower eukaryotes to mammals.

Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.

A periodic luciferase reporter system from cell cycle–regulated promoters in synchronous U2OS cells measures periodic, cell cycle–regulated gene expression in live cells. This assay is used to identify Forkhead transcription factors that control periodic gene expression, and it identifies FOXK1 as an activator of key cell cycle genes.

Circadian Output, Input, and Intracellular Oscillators: Insights into the Circadian Systems of Single Cells

Circadian output comprises the business end of circadian systems in terms of adaptive significance. Work on Neurospora pioneered the molecular analysis of circadian output mechanisms, and insights from this model system continue to illuminate the pathways through which clocks control metabolism and overt rhythms. In Neurospora, virtually every strain examined in the context of rhythms bears the band allele that helps to clarify the overt rhythm in asexual development. Recent cloning of band showed it to be an allele of ras-1 and to affect a wide variety of signaling pathways yielding enhanced light responses and asexual development. These can be largely phenocopied by treatments that increase levels of intracellular reactive oxygen species. Although output is often unidirectional, analysis of the prd-4 gene provided an alternative paradigm in which output feeds back to affect input. prd-4 is an allele of checkpoint kinase-2 that bypasses the requirement for DNA damage to activate this kinase; FRQ is normally a substrate of activated Chk2, so in Chk2(PRD-4), FRQ is precociously phosphorylated and the clock cycles more quickly. Finally, recent adaptation of luciferase to fully function in Neurospora now allows the core FRQ/WCC feedback loop to be followed in real time under conditions where it no longer controls the overt rhythm in development. This ability can be used to describe the hierarchical relationships among FRQ-Less Oscillators (FLOs) and to see which are connected to the circadian system. The nitrate reductase oscillator appears to be connected, but the oscillator controlling the long-period rhythm elicited upon choline starvation appears completely disconnected from the circadian system; it can be seen to run with a very long noncompensated 60-120-hour period length under conditions where the circadian FRQ/WCC oscillator continues to cycle with a fully compensated circadian 22-hour period.

Modulation of clock gene expression by the transcriptional coregulator receptor interacting protein 140 (RIP140)

Circadian rhythms are generated in central and peripheral tissues by an intracellular oscillating timing mechanism known as the circadian clock. Several lines of evidence show a strong and bidirectional interplay between metabolism and circadian rhythms. Receptor interacting protein 140 (RIP140) is a coregulator for nuclear receptors and other transcription factors that represses catabolic pathways in metabolic tissues. Although RIP140 functions as a corepressor for most nuclear receptors, mounting evidence points to RIP140 as a dual coregulator that can repress or activate different sets of genes. Here, we demonstrate that RIP140 mRNA and protein levels are under circadian regulation and identify RIP140 as a modulator of clock gene expression, suggesting that RIP140 can participate in a feedback mechanism affecting the circadian clock. We show that the absence of RIP140 disturbs the basal levels of BMAL1 and other clock genes, reducing the amplitude of their oscillations. In addition, we demonstrate that RIP140 is recruited to retinoid-related orphan receptor (ROR) binding sites on the BMAL1 promoter, directly interacts with RORα, and increases transcription from the BMAL1 promoter in a RORα-dependent manner. These results indicate that RIP140 is not only involved in metabolic control but also acts as a coactivator for RORα, influencing clock gene expression.

Retinoic acid mediates long-paced oscillations in retinoid receptor activity: evidence for a potential role for RIP140.

Background Mechanisms that underlie oscillatory transcriptional activity of nuclear receptors (NRs) are incompletely understood. Evidence exists for rapid, cyclic recruitment of coregulatory complexes upon activation of nuclear receptors. RIP140 is a NR coregulator that represses the transactivation of agonist-bound nuclear receptors. Previously, we showed that RIP140 is inducible by all-trans retinoic acid (RA) and mediates limiting, negative-feedback regulation of retinoid signaling. Methodology and Findings Here we report that in the continued presence of RA, long-paced oscillations of retinoic acid receptor (RAR) activity occur with a period ranging from 24 to 35 hours. Endogenous expression of RIP140 and other RA-target genes also oscillate in the presence of RA. Cyclic retinoid receptor transactivation is ablated by constitutive overexpression of RIP140. Further, depletion of RIP140 disrupts cyclic expression of the RA target gene HOXA5. Evidence is provided that RIP140 may limit RAR signaling in a selective, non-redundant manner in contrast to the classic NR coregulators NCoR1 and SRC1 that are not RA-inducible, do not cycle, and may be partially redundant in limiting RAR activity. Finally, evidence is provided that RIP140 can repress and be induced by other nuclear receptors in a manner that suggests potential participation in other NR oscillations. Conclusions and Significance We provide evidence for novel, long-paced oscillatory retinoid receptor activity and hypothesize that this may be paced in part, by RIP140. Oscillatory NR activity may be involved in mediating hormone actions of physiological and pathological importance.

Chronic shifts in the length and phase of the light cycle increase intermittent alcohol drinking in C57BL/6J mice

Introduction: Shift workers—e.g., health care professionals, truck drivers, and factory workers—are forced to maintain daily cycles at odds with their natural circadian rhythms and as a consequence need to frequently readjust these cycles. This shift work-induced circadian desynchrony (CD) is associated with increased sleep disorders and with alcohol abuse. Nonetheless, it has proven difficult to model CD-induced changes in alcohol consumption in mouse models, which is an important step toward identifying the mechanisms by which CD increases alcohol intake. This study examined whether frequent changes in the light cycle could increase free access alcohol intake in a mouse line that readily consumes alcohol. Methods: Free access alcohol intake, water intake, and wheel-running activity patterns of male C57BL/6J mice were measured while the mice were maintained on a normal 12HR photoperiod for baseline data for 2 weeks. The mice were then exposed to an alternating photoperiod of 12 h and 18 h, with light onset advanced 8 h during the 18HR photoperiod. The photoperiods rotated every 3 days, for 21 days total. Results: The repeated pattern of phase advances and delays, with a concurrent change in the length of the photoperiod, shifted mice to a pattern of intermittent alcohol drinking without altering water intake. Wheel running activity demonstrated that mice were unable to reset their behavioral clocks during CD, showing constant, low-level activity with no peak in activity at the start of the dark phase and greater activity during the morning light phase. Conclusion: It is possible to model CD effects on alcohol intake in C57BL/6J mice using a pattern of phase shifts and changes in the photoperiod. Using this model, we demonstrate that mice begin intermittent drinking during CD, and this increase in alcohol intake does not correlate with an increase in overall activity or in overall fluid intake.

Disruption of normal circadian clock function in a mouse model of tauopathy

&NA; Disruption of normal circadian rhythm physiology is associated with neurodegenerative disease, which can lead to symptoms such as altered sleep cycles. In Alzheimers disease (AD), circadian dysfunction has been attributed to &bgr;‐amyloidosis. However, it is unclear whether tauopathy, another AD‐associated neuropathology, can disrupt the circadian clock. We have evaluated the status of the circadian clock in a mouse model of tauopathy (Tg4510). Tg4510 mice display a long free‐running period at an age when tauopathy is present, and show evidence of tauopathy in the suprachiasmatic nucleus (SCN) of the hypothalamus — the site of the master circadian clock. Additionally, cyclic expression of the core clock protein PER2 is disrupted in the hypothalamus of Tg4510 mice. Finally, disruption of the cyclic expression of PER2 and BMAL1, another core circadian clock protein, is evident in the Tg4510 hippocampus. These results demonstrate that tauopathy disrupts normal circadian clock function both at the behavioral and molecular levels, which may be attributed to the tauopathy‐induced neuropathology in the SCN. Furthermore, these results establish the Tg4510 mouse line as a model to study how tauopathy disrupts normal circadian rhythm biology. HighlightsAged Tg4510 mice display long free‐running period, which indicates a disrupted circadian clock.The suprachiasmatic nucleus (SCN) of aged Tg4510 mice shows evidence of tauopathy.Aged Tg4510 mice show a disrupted molecular clock in the hypothalamus and hippocampus when tauopathy is evident.