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

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Circadian rhythms in metazoan eukaryotes are controlled by an endogenous molecular clock. It functions in many locations, including subsets of brain neurons (clock neurons) within the central nervous system. Although the molecular clock relies on transcription/translation feedback loops, posttranscriptional regulation also plays an important role. Here, we show that the abundant Drosophila melanogaster microRNA mir-276a regulates molecular and behavioral rhythms by inhibiting expression of the important clock gene timeless (tim). Misregulation of mir-276a in clock neurons alters tim expression and increases arrhythmicity under standard constant darkness (DD) conditions. mir-276a expression itself appears to be light-regulated because its levels oscillate under 24-h light-dark (LD) cycles but not in DD. mir-276a is regulated by the transcription activator Chorion factor 2 in flies and in tissue-culture cells. Evidence from flies mutated using the clustered, regularly interspaced, short palindromic repeats (CRISPR) tool shows that mir-276a inhibits tim expression: Deleting the mir276a-binding site in the tim 3′ UTR causes elevated levels of TIM and ∼50% arrhythmicity. We suggest that this pathway contributes to the more robust rhythms observed under light/dark LD conditions than under DD conditions. microRNA | circadian rhythms | light regulation | CRISPR

Section: Discussionmentioning

confidence: 99%

mir-276a strengthens Drosophila circadian rhythms by regulating timeless expression

Xiao

Rosbash

2016

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“…The primers for tim were taken from ref. 78, and the primers for the housekeeping gene gapdh were from ref. 79.…”

Section: Methodsmentioning

confidence: 99%

Temperature compensation and temperature sensation in the circadian clock

Kidd

Young

Siggia

2015

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.circadian clock | temperature compensation | mathematical models C ircadian rhythms are daily oscillations in gene expression and protein concentration that regulate sleep (1, 2), metabolism (3,4), and a host of other biological processes (5-8). Circadian oscillations are known to be present in nearly all animals and plants, as well as some fungi and bacteria (9). In all organisms in which circadian oscillations have been observed, circadian oscillations satisfy three defining properties (10). First, circadian oscillations are self-sustained and spontaneously maintain a period of about 24 h. Second, the circadian rhythm is sensitive to light and temperature and can be synchronized to external oscillations in the quantity of either. Third, the period of the circadian oscillation is "temperature-compensated"; that is, the endogenous period of the oscillation is relatively insensitive to temperature.The genetic basis of circadian clocks has been elucidated in considerable detail over the past two decades, particularly in the fruitfly Drosophila melanogaster, the mouse Mus musculus, and the bread mold Neurospora crassa (11). In Drosophila, a self-sustained circadian oscillation in neurons is generated when a pair of proteins, PERIOD (PER) and TIMELESS (TIM), dimerize and, after some delay, translocate into the nucleus, where the proteins repress their own transcription by inactivating a transcription factor dimer composed of the proteins CLOCK and CYCLE. Light sensitivity is mediated by the blue light photoreceptor CRYPTOCHROME (CRY), which upon activation binds TIMELESS and promotes TIMELESS degradation. A remarkably similar mechanism underlies circadian clocks in mouse and Neurospora. The circadian clock of cyanobacteria has also bee...

“…In flies, this mechanism consists of the CLOCK and CYCLE heterodimer (CLK/CYC), which drives the rhythmic transcription of its repressor protein genes period and timeless (9). After translation, the proteins PER and TIM decrease their own transcription by inhibiting the activity of CLK/ CYC (10)(11)(12). A set of largely orthologous proteins (CLK, BMAL1, PER, and CRYPTOCHROME) functions similarly in mammals.…”

Section: Nas-seq | Chromatin Dynamicsmentioning

confidence: 99%

Nascent-Seq analysis of Drosophila cycling gene expression

Rodriguez

Tang

Khodor

et al. 2013

Self Cite

100

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.Nas-seq | chromatin dynamics T he circadian clock controls the rhythmic expression of thousands of genes. These mRNAs mediate the oscillation of numerous biochemical, physiological, and behavioral functions. Although inconsistency among microarray datasets has made it difficult to identify a definitive set of mRNA cyclers in Drosophila, there is good agreement on a small number of robust cycling mRNAs (see below). Moreover, it appears that a much larger number of cycling mRNAs exist within circadian neurons (1), and thousands of mRNAs have been shown to oscillate within individual tissues in mammals (2-5).Cycling mRNAs are dependent on a functional pacemaker (6, 7), but most probably are regulated only indirectly by the core circadian clock (8). In flies, this mechanism consists of the CLOCK and CYCLE heterodimer (CLK/CYC), which drives the rhythmic transcription of its repressor protein genes period and timeless (9). After translation, the proteins PER and TIM decrease their own transcription by inhibiting the activity of CLK/ CYC (10-12). A set of largely orthologous proteins (CLK, BMAL1, PER, and CRYPTOCHROME) functions similarly in mammals. Although posttranslational regulation of these transcription factors makes a major contribution to core clock function (13-19), there is evidence that transcriptional regulation also is important for circadian timing. For example, flies with enhanced CLK/CYC-mediated transcription have unusually high levels of per mRNA and significantly shorter periods (20).The core clock model drives the assumption that the large population of cycling mRNAs-the hundreds or thousands of output mRNAs under circadian regulation-is also under transcriptional regulation. However, it is not known w...