Microarrays: determining the balance of cellular transcription.
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Acyltransferases↗
Biomedical subjects
S A Kay
Publications and source records attributed to S A Kay.
Fluorescent differential display identifies circadian clock-regulated genes in Arabidopsis thaliana.
Circadian rhythms in gene expression were first observed in plants more than 13 years ago, but the underlying mechanism controlling rhythmic gene expression is still not understood. The isolation of novel circadian clock-controlled genes (ccgs) is likely to provide new tools for studying circadian rhythms. Fluorescent differential display (FDD) was used to screen Arabidopsis thaliana mRNAs for cycling transcripts. Seventy PCR primer pairs were screened, and 17 different cycling bands were observed out of an estimated 10,500 bands screened. The identities of 10 bands were determined, and the rhythmic gene expression was confirmed using northern blot analysis. The 10 cycling bands represent 7 different genes, 6 of which are present in the databases and 1 that does not match anything in current databases. The rhythmic expression of the 7 genes is composed of four distinct phases of clock regulation. The results demonstrate that FDD can be used to isolate ccgs. The genes identified in this screen range from known A. thaliana ccgs, as well as genes shown to be clock controlled in other plant species, to a novel gene that may encode a pioneer protein. Further study of these ccgs is likely to increase our understanding of circadian-regulated gene expression.
Arabidopsis↗
The period E-box is sufficient to drive circadian oscillation of transcription in vivo.
The minimum element from the Drosophila period promoter capable of driving in vivo cycling mRNA is the 69 bp circadian regulatory sequence (CRS). In cell culture, an 18 bp E-box element from the period promoter is regulated by five genes that are involved in the regulation of circadian expression in flies. This E-box is a target for transcriptional activation by bHLH-PAS proteins dCLOCK (dCLK) and CYCLE (CYC), this activation is inhibited by PERIOD (PER) and TIMELESS (TIM) together, and inhibition of dCLK/CYC by PER and TIM is blocked by CRYPTOCHROME (CRY) in the presence of light. Here, the same 18 bp E-box region generated rhythmic expression of luciferase in flies under both light-dark cycling and constant conditions. Flies heterozygous for the Clke(jrk) mutation maintained rhythmic expression from the E-box although at a lower level than wild type. Homozygous mutant Clk(jrk) animals had drastically lowered and arrhythmic expression. In a per01 background, expression from the E-box was high and not rhythmic. Transcription mediated by the per E-box was restricted to the same spatial pattern as the CRS. The per E-box DNA element and cognate binding proteins can confer per-like temporal and spatial expression. This demonstrates in vivo that the known circadian genes that form the core of the circadian oscillator in Drosophila integrate their activities at a single DNA element.
Animals↗
Specific sequences outside the E-box are required for proper per expression and behavioral rescue.
A 69 bp circadian regulatory sequence (CRS) upstream of the per gene is sufficient to drive circadian transcription, mediate proper spatial expression, and rescue behavioral rhythmicity in per01 flies. Within the CRS, an E-box is required for transcriptional activation by two basic-helix-loop-helix (bHLH) PERARNT-SIM (PAS) transcription factors, dCLOCK (dCLK) and CYCLE (CYC). To define sequences within the CRS that are required for spatial expression, circadian expression, and behavioral rhythmicity, a series of mutants that alter blocks of 3 to 12 nucleotides across the entire CRS were used to drive lacZ or per expression in vivo. As expected, the E-box within the CRS is necessary for high-level expression and behavioral rhythmicity, but sequences outside the E-box are also required for transcriptional activation, proper spatial expression, and behavioral rhythmicity. These results indicate that the dCLK-CYC target site extends beyond the E-box and that factors other than dCLK and CYC modulate per transcription.
ARNTL Transcription Factors↗
Control of circadian rhythms and photoperiodic flowering by the Arabidopsis GIGANTEA gene.
Photoperiodic responses in plants include flowering that is day-length-dependent. Mutations in the Arabidopsis thaliana GIGANTEA (GI) gene cause photoperiod-insensitive flowering and alteration of circadian rhythms. The GI gene encodes a protein containing six putative transmembrane domains. Circadian expression patterns of the GI gene and the clock-associated genes, LHY and CCA1, are altered in gi mutants, showing that GI is required for maintaining circadian amplitude and appropriate period length of these genes. The gi-1 mutation also affects light signaling to the clock, which suggests that GI participates in a feedback loop of the plant circadian system.
Arabidopsis↗
Light-dependent sequestration of TIMELESS by CRYPTOCHROME.
Most organisms have circadian clocks consisting of negative feedback loops of gene regulation that facilitate adaptation to cycles of light and darkness. In this study, CRYPTOCHROME (CRY), a protein involved in circadian photoperception in Drosophila, is shown to block the function of PERIOD/TIMELESS (PER/TIM) heterodimeric complexes in a light-dependent fashion. TIM degradation does not occur under these conditions; thus, TIM degradation is uncoupled from abrogation of its function by light. CRY and TIM are part of the same complex and directly interact in yeast in a light-dependent fashion. PER/TIM and CRY influence the subcellular distribution of these protein complexes, which reside primarily in the nucleus after the perception of a light signal. Thus, CRY acts as a circadian photoreceptor by directly interacting with core components of the circadian clock.
Animals↗
Light-dependent translocation of a phytochrome B-GFP fusion protein to the nucleus in transgenic Arabidopsis.
Phytochrome is a ubiquitous photoreceptor of plants and is encoded by a small multigene family. We have shown recently that a functional nuclear localization signal may reside within the COOH-terminal region of a major member of the family, phytochrome B (phyB) (Sakamoto, K., and A. Nagatani. 1996. Plant J. 10:859-868). In the present study, a fusion protein consisting of full-length phyB and the green fluorescent protein (GFP) was overexpressed in the phyB mutant of Arabidopsis to examine subcellular localization of phyB in intact tissues. The resulting transgenic lines exhibited pleiotropic phenotypes reported previously for phyB overexpressing plants, suggesting that the fusion protein is biologically active. Immunoblot analysis with anti-phyB and anti-GFP monoclonal antibodies confirmed that the fusion protein accumulated to high levels in these lines. Fluorescence microscopy of the seedlings revealed that the phyB-GFP fusion protein was localized to the nucleus in light grown tissues. Interestingly, the fusion protein formed speckles in the nucleus. Analysis of confocal optical sections confirmed that the speckles were distributed within the nucleus. In contrast, phyB-GFP fluorescence was observed throughout the cell in dark-grown seedlings. Therefore, phyB translocates to specific sites within the nucleus upon photoreceptor activation.
Arabidopsis↗
Synchronous real-time reporting of multiple cellular events.
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Animals↗
Cryptochromes--bringing the blues to circadian rhythms.
Cryptochromes are blue/UV-A-absorbing photoreceptor proteins discovered originally in plants and so named because their nature proved elusive in over a century of research. Now we know that the photoreceptor essential for proper seedling establishment in blue light has homologues in the animal kingdom - in insects, in mice and in humans. In recent months, evidence has emerged pointing to a common role for cryptochromes in all of these organisms in entraining the circadian clock, a biochemical timing mechanism running within cells, synchronizing metabolism to the daily light-dark cycle and having consequences on a much larger scale in the regulation of behaviour such as the sleep-wake cycle.
Animals↗
The ins and outs of circadian regulated gene expression.
The circadian rhythms we observe originate from the circadian regulation of gene expression. Common control points, like transcription and protein phosphorylation, are used to effect this regulation, both within the clock and in output pathways. Recent work has advanced our understanding of these processes in plants and other models. Also, specific photoreceptors that mediate light entrainment of the clock have been identified in Arabidopsis.
Arabidopsis↗
The cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila.
A new rhythm mutation was isolated based on its elimination of per-controlled luciferase cycling. Levels of period or timeless clock gene products in the mutant are flat in daily light-dark cycles or constant darkness (although PER and TIM oscillate normally in temperature cycles). Consistent with the fact that light normally suppresses TIM, cryb is an apparent null mutation in a gene encoding Drosophila's version of the blue light receptor cryptochrome. Behaviorally, cryb exhibits poor synchronization to light-dark cycles in genetic backgrounds that cause external blindness or demand several hours of daily rhythm resets, and it shows no response to brief light pulses. cryb flies are rhythmic in constant darkness, correlating with robust PER and TIM cycling in certain pacemaker neurons.
Animals↗
Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock.
Circadian clocks are synchronized by environmental cues such as light. Photoreceptor-deficient Arabidopsis thaliana mutants were used to measure the effect of light fluence rate on circadian period in plants. Phytochrome B is the primary high-intensity red light photoreceptor for circadian control, and phytochrome A acts under low-intensity red light. Cryptochrome 1 and phytochrome A both act to transmit low-fluence blue light to the clock. Cryptochrome 1 mediates high-intensity blue light signals for period length control. The presence of cryptochromes in both plants and animals suggests that circadian input pathways have been conserved throughout evolution.
Arabidopsis↗
Closing the circadian loop: CLOCK-induced transcription of its own inhibitors per and tim.
The circadian oscillator generates a rhythmic output with a period of about 24 hours. Despite extensive studies in several model systems, the biochemical mode of action has not yet been demonstrated for any of its components. Here, the Drosophila CLOCK protein was shown to induce transcription of the circadian rhythm genes period and timeless. dCLOCK functioned as a heterodimer with a Drosophila homolog of BMAL1. These proteins acted through an E-box sequence in the period promoter. The timeless promoter contains an 18-base pair element encompassing an E-box, which was sufficient to confer dCLOCK responsiveness to a reporter gene. PERIOD and TIMELESS proteins blocked dCLOCK's ability to transactivate their promoters via the E-box. Thus, dCLOCK drives expression of period and timeless, which in turn inhibit dCLOCK's activity and close the circadian loop.
ARNTL Transcription Factors↗
Photoactive yellow protein: a structural prototype for the three-dimensional fold of the PAS domain superfamily.
PAS domains are found in diverse proteins throughout all three kingdoms of life, where they apparently function in sensing and signal transduction. Although a wealth of useful sequence and functional information has become recently available, these data have not been integrated into a three-dimensional (3D) framework. The very early evolutionary development and diverse functions of PAS domains have made sequence analysis and modeling of this protein superfamily challenging. Limited sequence similarities between the approximately 50-residue PAS repeats and one region of the bacterial blue-light photosensor photoactive yellow protein (PYP), for which ground-state and light-activated crystallographic structures have been determined to high resolution, originally were identified in sequence searches using consensus sequence probes from PAS-containing proteins. Here, we found that by changing a few residues particular to PYP function, the modified PYP sequence probe also could select PAS protein sequences. By mapping a typical approximately 150-residue PAS domain sequence onto the entire crystallographic structure of PYP, we show that the PAS sequence similarities and differences are consistent with a shared 3D fold (the PAS/PYP module) with obvious potential for a ligand-binding cavity. Thus, PYP appears to prototypically exhibit all the major structural and functional features characteristic of the PAS domain superfamily: the shared PAS/PYP modular domain fold of approximately 125-150 residues, a sensor function often linked to ligand or cofactor (chromophore) binding, and signal transduction capability governed by heterodimeric assembly (to the downstream partner of PYP). This 3D PAS/PYP module provides a structural model to guide experimental testing of hypotheses regarding ligand-binding, dimerization, and signal transduction.
Amino Acid Sequence↗
An Arabidopsis mutant hypersensitive to red and far-red light signals.
A new mutant called psi2 (for phytochrome signaling) was isolated by screening for elevated activity of a chlorophyll a/b binding protein-luciferase (CAB2-LUC) transgene in Arabidopsis. This mutant exhibited hypersensitive induction of CAB1, CAB2, and the small subunit of ribulose-1,5-bisphosphate carboxylase (RBCS) promoters in the very low fluence range of red light and a hypersensitive response in hypocotyl growth in continuous red light of higher fluences. In addition, at high- but not low-light fluence rates, the mutant showed light-dependent superinduction of the pathogen-related protein gene PR-1a and developed spontaneous necrotic lesions in the absence of any pathogen. Expression of genes responding to various hormone and environmental stress pathways in the mutant was not significantly different from that of the wild type. Analysis of double mutants demonstrated that the effects of the psi2 mutation are dependent on both phytochromes phyA and phyB. The mutation is recessive and maps to the bottom of chromosome 5. Together, our results suggest that PSI2 specifically and negatively regulates both phyA and phyB phototransduction pathways. The induction of cell death by deregulated signaling pathways observed in psi2 is reminiscent of retinal degenerative diseases in animals and humans.
Arabidopsis↗
The short-period mutant, toc1-1, alters circadian clock regulation of multiple outputs throughout development in Arabidopsis thaliana.
The coordination of developmental and physiological events with environmental signals is facilitated by the action of the circadian clock. Here we report a new set of circadian clock-controlled phenotypes for Arabidopsis thaliana. We use these markers together with the short-period mutant, toc1-1, and the clock-controlled cab2::luciferase reporter gene to assess the nature of the circadian clock throughout development and to suggest the position of TOC1 within the circadian clock system. In dark-grown seedlings, the toc1-1 lesion conferred a short period to the cycling of cab2::luciferase luminescence, as previously found in light-grown plants, indicating that the circadian clocks in these two divergent developmental states share at least one component. Stomatal conductance rhythms were similarly approximately 3 hours shorter than wild type in toc1-1, suggesting that a cell-autonomous clockwork may be active in guard cells in 5- to 6-week-old leaves. The effect of daylength on flowering time in the C24 ecotype was diminished by toc1-1, and was nearly eliminated in the Landsberg erecta background where the plants flowered equally early in both short and long days. Throughout a 500-fold range of red light intensities, both the wild type and the mutant showed an inverse log-linear relationship of fluence rate to period, with a 2-3 hour shorter period for the mutant at all intensities. These results indicate that TOC1 acts on or within the clock independently of light input. Temperature entrainment appears normal in toc1-1, and the period-shortening effects of the mutant remain unchanged over a 20 degrees C temperature range. Taken together our results are consistent with the likelihood that TOC1 codes for an oscillator component rather than for an element of an input signaling pathway. In addition, the pervasive effect of toc1-1 on a variety of clock-controlled processes throughout development suggests that a single circadian system is primarily responsible for controlling most, if not all, circadian rhythms in the plant.
Arabidopsis↗
Independent photoreceptive circadian clocks throughout Drosophila.
Transgenic Drosophila that expressed either luciferase or green fluorescent protein driven from the promoter of the clock gene period were used to monitor the circadian clock in explanted head, thorax, and abdominal tissues. The tissues (including sensory bristles in the leg and wing) showed rhythmic bioluminescence, and the rhythms could be reset by light. The photoreceptive properties of the explanted tissues indicate that unidentified photoreceptors are likely to contribute to photic signal transduction to the clock. These results show that autonomous circadian oscillators are present throughout the body, and they suggest that individual cells in Drosophila are capable of supporting their own independent clocks.
Animals↗
Long-term monitoring of circadian rhythms in c-fos gene expression from suprachiasmatic nucleus cultures.
BACKGROUND: The AP-1 family of transcription factors has been implicated in the control of the expression of many genes in response to environmental signals. Previous studies have provided temporal profiles for c-fos expression by taking measurements from many animals at several points in time, but these studies provide limited information about dynamic changes in expression. Here, we have devised a method of continuously measuring c-fos expression. RESULTS: A transgenic mouse line expressing the human c-fos promoter linked to the firefly luciferase reporter gene (fos/luc) was generated to continuously monitor c-fos gene expression. A second transgenic mouse line expressing luciferase under the control of the cytomegalovirus promoter (CMV/luc) served as a control. Luminescence originating from identifiable brain regions was imaged from fos/luc brain slice cultures. Expression of the fos/luc transgene accurately reflected transcriptional responses of the endogenous c-fos gene. Dynamic changes in fos/luc expression in suprachiasmatic nuclei (SCN) explant cultures were monitored continuously, and luminescence showed almost 24 hour rhythms lasting up to five circadian cycles. In contrast, bioluminescence monitored from CMV/luc SCN explant cultures was not rhythmic. CONCLUSION: The fos/luc transgenic mouse will be useful for long-term, non-invasive monitoring of c-fos transcriptional responses to the changing cellular environment. Circadian rhythms in c-fos expression can be monitored non-invasively in real time from the SCN, clearly demonstrating that c-fos transcription is regulated by the circadian clock.
Animals↗