Jerome S. Menet

The Drosophila Circadian Network Is a Seasonal Timer

Previous work in Drosophila has defined two populations of circadian brain neurons, morning cells (M-cells) and evening cells (E-cells), both of which keep circadian time and regulate morning and evening activity, respectively. It has long been speculated that a multiple oscillator circadian network in animals underlies the behavioral and physiological pattern variability caused by seasonal fluctuations of photoperiod. We have manipulated separately the circadian photoentrainment pathway within E- and M-cells and show that E-cells process light information and function as master clocks in the presence of light. M-cells in contrast need darkness to cycle autonomously and dominate the network. The results indicate that the network switches control between these two centers as a function of photoperiod. Together with the different entraining properties of the two clock centers, the results suggest that the functional organization of the network underlies the behavioral adjustment to variations in daylength and season.

Nascent-Seq reveals novel features of mouse circadian transcriptional regulation

A substantial fraction of the metazoan transcriptome undergoes circadian oscillations in many cells and tissues. Based on the transcription feedback loops important for circadian timekeeping, it is commonly assumed that this mRNA cycling reflects widespread transcriptional regulation. To address this issue, we directly measured the circadian dynamics of mouse liver transcription using Nascent-Seq (genome-wide sequencing of nascent RNA). Although many genes are rhythmically transcribed, many rhythmic mRNAs manifest poor transcriptional rhythms, indicating a prominent contribution of post-transcriptional regulation to circadian mRNA expression. This analysis of rhythmic transcription also showed that the rhythmic DNA binding profile of the transcription factors CLOCK and BMAL1 does not determine the transcriptional phase of most target genes. This likely reflects gene-specific collaborations of CLK:BMAL1 with other transcription factors. These insights from Nascent-Seq indicate that it should have broad applicability to many other gene expression regulatory issues. DOI: http://dx.doi.org/10.7554/eLife.00011.001

Dynamic PER repression mechanisms in the Drosophila circadian clock: from on-DNA to off-DNA

Transcriptional feedback loops are central to the generation and maintenance of circadian rhythms. In animal systems as well as Neurospora, transcriptional repression is believed to occur by catalytic post-translational events. We report here in the Drosophila model two different mechanisms by which the circadian repressor PERIOD (PER) inhibits CLOCK/CYCLE (CLK/CYC)-mediated transcription. First, PER is recruited to circadian promoters, which leads to the nighttime decrease of CLK/CYC activity. This decrease is proportional to PER levels on DNA, and PER recruitment probably occurs via CLK. Then CLK is released from DNA and sequestered in a strong, approximately 1:1 PER-CLK off-DNA complex. The data indicate that the PER levels bound to CLK change dynamically and are important for repression, first on-DNA and then off-DNA. They also suggest that these mechanisms occur upstream of post-translational events, and that elements of this two-step mechanism likely apply to mammals.

Photoperiod differentially regulates clock genes’ expression in the suprachiasmatic nucleus of Syrian hamster

The suprachiasmatic nuclei (SCN) contain the master circadian pacemaker in mammals. Generation and maintenance of circadian oscillations involve clock genes which interact to form transcriptional/translational loops and constitute the molecular basis of the clock. There is some evidence that the SCN clock can integrate variations in day length, i.e. photoperiod. However, the effects of photoperiod on clock-gene expression remain largely unknown. We here report the expression pattern of Period (Per) 1, Per2, Per3, Cryptochrome (Cry) 1, Cry2, Bmal1 and Clock genes in the SCN of Syrian hamsters when kept under long (LP) and short (SP) photoperiods. Our data show that photoperiod differentially affects the expression of all clock genes studied. Among the components of the negative limb of the feedback loop, Per1, Per2, Per3, Cry2 but not Cry1 genes show a shortened duration of their peak expression under SP compared with LP. Moreover, mRNA expression of Per1, Per3 and Cry1 are phase advanced in SP compared with LP. Per3 shows an mRNA peak of higher amplitude under SP conditions whereas Per1 and Per2 peak amplitudes are unaffected by photoperiod changes. Bmal1 expression is phase advanced without a change of duration in SP compared with LP. Furthermore, the expression of Clock is rhythmic under SP whereas no rhythm is observed under LP. These results, which provide further evidence that the core clock mechanisms of the SCN integrate photoperiod, are discussed in the context of the existing molecular model.

CLOCK:BMAL1 is a pioneer-like transcription factor

The mammalian circadian clock relies on the master genes CLOCK and BMAL1 to drive rhythmic gene expression and regulate biological functions under circadian control. Here we show that rhythmic CLOCK:BMAL1 DNA binding promotes rhythmic chromatin opening. Mechanisms include CLOCK:BMAL1 binding to nucleosomes and rhythmic chromatin modification; e.g., incorporation of the histone variant H2A.Z. This rhythmic chromatin remodeling mediates the rhythmic binding of other transcription factors adjacent to CLOCK:BMAL1, suggesting that the activity of these other transcription factors contributes to the genome-wide CLOCK:BMAL1 heterogeneous transcriptional output. These data therefore indicate that the clock regulation of transcription relies on the rhythmic regulation of chromatin accessibility and suggest that the concept of pioneer function extends to acute gene regulation.

Nascent-Seq Indicates Widespread Cotranscriptional RNA Editing in Drosophila

The RNA editing enzyme ADAR chemically modifies adenosine (A) to inosine (I), which is interpreted by the ribosome as a guanosine. Here we assess cotranscriptional A-to-I editing in Drosophila by isolating nascent RNA from adult fly heads and subjecting samples to high throughput sequencing. There are a large number of edited sites within nascent exons. Nascent RNA from an ADAR-null strain was also sequenced, indicating that almost all A-to-I events require ADAR. Moreover, mRNA editing levels correlate with editing levels within the cognate nascent RNA sequence, indicating that the extent of editing is set cotranscriptionally. Surprisingly, the nascent data also identify an excess of intronic over exonic editing sites. These intronic sites occur preferentially within introns that are poorly spliced cotranscriptionally, suggesting a link between editing and splicing. We conclude that ADAR-mediated editing is more widespread than previously indicated and largely occurs cotranscriptionally.

The circadian clock stops ticking during deep hibernation in the European hamster

Hibernation is a fascinating, yet enigmatic, physiological phenomenon during which body temperature and metabolism are reduced to save energy. During the harsh season, this strategy allows substantial energy saving by reducing body temperature and metabolism. Accordingly, biological processes are considerably slowed down and reduced to a minimum. However, the persistence of a temperature-compensated, functional biological clock in hibernating mammals has long been debated. Here, we show that the master circadian clock no longer displays 24-h molecular oscillations in hibernating European hamsters. The clock genes Per1, Per2, and Bmal1 and the clock-controlled gene arginine vasopressin were constantly expressed in the suprachiasmatic nucleus during deep torpor, as assessed by radioactive in situ hybridization. Finally, the melatonin rhythm-generating enzyme, arylalkylamine N-acetyltransferase, whose rhythmic expression in the pineal gland is controlled by the master circadian clock, no longer exhibits day/night changes of expression but constantly elevated mRNA levels over 24 h. Overall, these data provide strong evidence that in the European hamster the molecular circadian clock is arrested during hibernation and stops delivering rhythmic output signals.

Nascent-Seq analysis of Drosophila cycling gene expression

Significance Statement Rhythmic mRNA expression is a hallmark of circadian rhythms. To address this regulation in Drosophila, we used high throughput sequencing to compare nascent RNA and mRNA levels in fly heads at different times of day. This indicated that some genes undergo rhythmic transcription with no impact on mRNA, whereas a few strongly rhythmic mRNAs have weak transcriptional oscillations. A substantial number of rhythmic genes manifest more robust mRNA cycling than transcriptional cycling, indicating that post-transcriptional regulation plays a widespread role in boosting circadian gene expression amplitude. Our study highlights the importance of directly assaying transcription to understand gene regulation. Rhythmic mRNA expression is a hallmark of circadian biology and has been described in numerous experimental systems including mammals. A small number of core clock gene mRNAs and a much larger number of output mRNAs are under circadian control. The rhythmic expression of core clock genes is regulated at the transcriptional level, and this regulation is important for the timekeeping mechanism. However, the relative contribution of transcriptional and posttranscriptional regulation to global circadian mRNA oscillations is unknown. To address this issue in Drosophila, we isolated nascent RNA from adult fly heads collected at different time points and subjected it to high-throughput sequencing. mRNA was isolated and sequenced in parallel. Some genes had cycling nascent RNAs with no cycling mRNA, caused, most likely, by light-mediated read-through transcription. Most genes with cycling mRNAs had significant nascent RNA cycling amplitudes, indicating a prominent role for circadian transcriptional regulation. However, a considerable fraction had higher mRNA amplitudes than nascent RNA amplitudes. The same comparison for core clock gene mRNAs gives rise to a qualitatively similar conclusion. The data therefore indicate a significant quantitative contribution of posttranscriptional regulation to mRNA cycling.

Circadian Transcription Contributes to Core Period Determination in Drosophila

The Clock–Cycle (CLK–CYC) heterodimer constitutes a key circadian transcription complex in Drosophila. CYC has a DNA-binding domain but lacks an activation domain. Previous experiments also indicate that most of the transcriptional activity of CLK–CYC derives from the glutamine-rich region of its partner CLK. To address the role of transcription in core circadian timekeeping, we have analyzed the effects of a CYC–viral protein 16 (VP16) fusion protein in the Drosophila system. The addition of this potent and well-studied viral transcriptional activator (VP16) to CYC imparts to the CLK–CYC-VP16 complex strongly enhanced transcriptional activity relative to that of CLK–CYC. This increase is manifested in flies expressing CYC-VP16 as well as in S2 cells. These flies also have increased levels of CLK–CYC direct target gene mRNAs as well as a short period, implicating circadian transcription in period determination. A more detailed examination of reporter gene expression in CYC-VP16–expressing flies suggests that the short period is due at least in part to a more rapid transcriptional phase. Importantly, the behavioral effects require a period (per) promoter and are therefore unlikely to be merely a consequence of generally higher PER levels. This indicates that the CLK–CYC-VP16 behavioral effects are a consequence of increased per transcription. All of this also suggests that the timing of transcriptional activation and not the activation itself is the key event responsible for the behavioral effects observed in CYC-VP16-expressing flies. The results taken together indicate that circadian transcription contributes to core circadian function in Drosophila.

When brain clocks lose track of time: cause or consequence of neuropsychiatric disorders.

Patients suffering from neuropsychiatric disorders often exhibit a loss of regulation of their biological rhythms which leads to altered sleep/wake cycle, body temperature rhythm and hormonal rhythms. Whereas these symptoms have long been considered to result from the pathology of the underlying disease, increasing evidence now indicates that the circadian system may be more directly involved in the etiology of psychiatric disorders. This emerging view originated with the discovery that the genes involved in the generation of biological rhythms are expressed in many brain structures where clocks function-and perhaps malfunction. It is also due to the interesting phenotypes of clock mutant mice. Here we summarize recent reports showing that alteration of circadian clocks within key brain regions associated with neuropsychiatric disorders may be an underlying cause of the development of mental illness. We discuss how these alterations take place at both systems and molecular levels.