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Steve A Kay

Publications and source records attributed to Steve A Kay.

At least 19 recordsLinked to original sources
PubMed2006

A constitutive shade-avoidance mutant implicates TIR-NBS-LRR proteins in Arabidopsis photomorphogenic development.

In plants, light signals caused by the presence of neighbors accelerate stem growth and flowering and induce a more erect position of the leaves, a developmental strategy known as shade-avoidance syndrome. In addition, mutations in the photoreceptors that mediate shade-avoidance responses enhance disease susceptibility in Arabidopsis thaliana. Here, we describe the Arabidopsis constitutive shade-avoidance1 (csa1) mutant, which shows a shade-avoidance phenotype in the absence of shade and enhanced growth of a bacterial pathogen. The csa1 mutant has a T-DNA inserted within the second exon of a Toll/Interleukin1 receptor-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) gene, which leads to the production of a truncated mRNA. Arabidopsis plants transformed with the truncated TIR-NBS-LRR gene recapitulate the mutant phenotype, indicating that csa1 is a dominant-negative mutation that interferes with phytochrome signaling. TIR-NBS-LRR proteins have been implicated in defense responses in plants. RPS4, the closest homolog of CSA1, confers resistance to Pseudomonas syringae and complements the csa1 mutant phenotype, indicating that responses to pathogens and neighbors share core-signaling components in Arabidopsis. In Drosophila melanogaster and Caenorhabditis elegans, TIR domain proteins are implicated in both development and immunity. Thus, the dual role of the TIR domain is conserved across kingdoms.

Animals↗
PubMed2006

A novel computational model of the circadian clock in Arabidopsis that incorporates PRR7 and PRR9.

In plants, as in animals, the core mechanism to retain rhythmic gene expression relies on the interaction of multiple feedback loops. In recent years, molecular genetic techniques have revealed a complex network of clock components in Arabidopsis. To gain insight into the dynamics of these interactions, new components need to be integrated into the mathematical model of the plant clock. Our approach accelerates the iterative process of model identification, to incorporate new components, and to systematically test different proposed structural hypotheses. Recent studies indicate that the pseudo-response regulators PRR7 and PRR9 play a key role in the core clock of Arabidopsis. We incorporate PRR7 and PRR9 into an existing model involving the transcription factors TIMING OF CAB (TOC1), LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED (CCA1). We propose candidate models based on experimental hypotheses and identify the computational models with the application of an optimization routine. Validation is accomplished through systematic analysis of various mutant phenotypes. We introduce and apply sensitivity analysis as a novel tool for analyzing and distinguishing the characteristics of proposed architectures, which also allows for further validation of the hypothesized structures.

Arabidopsis↗
PubMed2006

Photoperiodic control of flowering: not only by coincidence.

The timing of floral transition has a direct impact on reproductive success. One of the most important environmental factors that affect the transition is the change in day length (photoperiod). Classical experiments imply that plants monitor photoperiods in the leaf, and transmit that information coded within an elusive signal dubbed florigen to the apex to reprogram development. Recent advances in Arabidopsis research indicate that the core of the day-length measurement mechanism lies in the circadian regulation of CONSTANS (CO) expression and the subsequent photoperiodic induction of the expression of FLOWERING LOCUS T (FT) gene, which might encode a major component of florigen. In this review, we introduce current perspectives on how, when and where the floral signal is generated.

Flowers↗
PubMed2006

Arabidopsis FHY3 specifically gates phytochrome signaling to the circadian clock.

Circadian gating of light signaling limits the timing of maximum responsiveness to light to specific times of day. The fhy3 (for far-red elongated hypocotyl3) mutant of Arabidopsis thaliana is involved in independently gating signaling from a group of photoreceptors to an individual response. fhy3 shows an enhanced response to red light during seedling deetiolation. Analysis of two independent fhy3 alleles links enhanced inhibition of hypocotyl elongation in response to red light with an arrhythmic pattern of hypocotyl elongation. Both alleles also show disrupted rhythmicity of central-clock and clock-output gene expression in constant red light. fhy3 exhibits aberrant phase advances under red light pulses during the subjective day. Release-from-light experiments demonstrate clock disruption in fhy3 during the early part of the subjective day in constant red light, suggesting that FHY3 is important in gating red light signaling for clock resetting. The FHY3 gating function appears crucial in the early part of the day for the maintenance of rhythmicity under these conditions. However, unlike previously described Arabidopsis gating mutants that gate all light signaling, gating of direct red light-induced gene expression in fhy3 is unaffected. FHY3 appears to be a novel gating factor, specifically in gating red light signaling to the clock during daytime.

Arabidopsis↗
PubMed2006

Circadian rhythms lit up in Chlamydomonas.

Recent work on the circadian clock of the unicellular green alga Chlamydomonas reinhardtii strengthens its standing as a convenient model system for circadian study. It was shown to be amenable to molecular engineering using a luciferase-based real-time reporter for circadian rhythms. Together with the completed draft genomic sequence, the new system opens the door for genome-scale forward and reverse genetic analysis.

Animals↗
PubMed2006

Feedback repression is required for mammalian circadian clock function.

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1 resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function.

3T3 Cells↗
PubMed2006

Second messenger and Ras/MAPK signalling pathways regulate CLOCK/CYCLE-dependent transcription.

The heterodimeric complex of the transcription factors CLOCK (CLK) and CYCLE (CYC) constitutes the positive element of the circadian clock in Drosophila and mammals. Phosphorylation of clock proteins represents an essential mechanism for promotion and control of the molecular oscillator. However, the kinases and signalling pathways that regulate CLK/CYC function remain largely elusive. In the present study we performed a chemical screen of kinase inhibitors in a cell culture reporter assay to identify functional regulators of CLK/CYC-dependent gene expression. These studies and analysis of constitutively active forms of kinases revealed that cyclic nucleotide/protein kinase A (PKA), calcium/calmodulin-dependent kinase (CaMK) II and Ras/mitogen-activated protein kinase (MAPK) regulate CLK/CYC activity. In vitro phosphorylation analysis showed a direct phosphorylation of CLK by CaMK II and p42 MAPK [extracellular signal-regulated kinase (ERK) 2], suggesting that these kinases regulate CLK/CYC-dependent transcription by direct phosphorylation of CLK.

ARNTL Transcription Factors↗
PubMed2005

LUX ARRHYTHMO encodes a Myb domain protein essential for circadian rhythms.

In higher plants, the circadian clock orchestrates fundamental processes such as light signaling and the transition to flowering. We isolated mutants of the circadian clock from an Arabidopsis thaliana mutagenized reporter line by screening for seedlings with long hypocotyl phenotypes and subsequently assaying for abnormal clock-regulated CAB2::LUC expression. This screen identified five mutant alleles of a clock gene, LUX ARRHYTHMO (LUX), that significantly affect amplitude and robustness of rhythms in both constant white light and dark conditions. In addition, the transition from vegetative to floral development is accelerated and hypocotyl elongation is accentuated in these mutants under light:dark cycles. We genetically mapped the mutations by bulk segregant analysis with high-density oligonucleotide array genotyping to a small putative Myb transcription factor related to other clock components and response regulators in Arabidopsis. The negative arm of the Arabidopsis circadian clock, CIRCADIAN CLOCK ASSOCIATED (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), is repressed in the lux mutants, whereas TIMING OF CAB2 EXPRESSION (TOC1) is activated. We demonstrate that CCA1 and LHY bind to the evening element motif in the LUX promoter, which strongly suggests that these proteins repress LUX expression, as they do TOC1. The data are also consistent with LUX being necessary for activation of CCA1 and LHY expression.

Arabidopsis↗
PubMed2005

FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis.

The temporal control of CONSTANS (CO) expression and activity is a key mechanism in photoperiodic flowering in Arabidopsis. FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) protein regulates CO transcription, although the molecular mechanism is unknown. We demonstrate here that FKF1 controls the stability of a Dof transcription factor, CYCLING DOF FACTOR 1 (CDF1). FKF1 physically interacts with CDF1, and CDF1 protein is more stable in fkf1 mutants. Plants with elevated levels of CDF1 flower late and have reduced expression of CO. CDF1 and CO are expressed in the same tissues, and CDF1 binds to the CO promoter. Thus, FKF1 controls daily CO expression in part by degrading CDF1, a repressor of CO transcription.

Amino Acid Sequence↗
PubMed2005

Positive and negative factors confer phase-specific circadian regulation of transcription in Arabidopsis.

The circadian clock exerts a major influence on transcriptional regulation in plants and other organisms. We have previously identified a motif called the evening element (EE) that is overrepresented in the promoters of evening-phased genes. Here, we demonstrate that multimerized EEs are necessary and sufficient to confer evening-phased circadian regulation. Although flanking sequences are not required for EE function, they can modulate EE activity. One flanking sequence, taken from the PSEUDORESPONSE REGULATOR 9 promoter, itself confers dawn-phased rhythms and has allowed us to define a new clock promoter motif (the morning element [ME]). Scanning mutagenesis reveals that both activators and repressors of gene expression act through the ME and EE. Although our experiments confirm that CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are likely to act as repressors via the EE, they also show that they have an unexpected positive effect on EE-mediated gene expression as well. We have identified a clock-regulated activity in plant extracts that binds specifically to the EE and has a phase consistent with it being an activator of expression through the EE. This activity is reduced in CCA1/LHY null plants, suggesting it may itself be part of a circadian feedback loop and perhaps explaining the reduction in EE activity in these double mutant plants.

3' Flanking Region↗
PubMed2005

Rapid array mapping of circadian clock and developmental mutations in Arabidopsis.

Classical forward genetics, the identification of genes responsible for mutant phenotypes, remains an important part of functional characterization of the genome. With the advent of extensive genome sequence, phenotyping and genotyping remain the critical limiting variables in the process of map-based cloning. Here, we reduce the genotyping problem by hybridizing labeled genomic DNA to the Affymetrix Arabidopsis (Arabidopsis thaliana) ATH1 GeneChip. Genotyping was carried out on the scale of detecting greater than 8,000 single feature polymorphisms from over 200,000 loci in a single assay. By combining this technique with bulk segregant analysis, several high heritability development and circadian clock traits were mapped. The mapping accuracy using bulk pools of 26 to 100 F(2) individuals ranged from 0.22 to 1.96 Mb of the mutations revealing mutant alleles of EARLY FLOWERING 3, EARLY FLOWERING 4, TIMING OF CAB EXPRESSION 1, and ASYMMETRIC LEAVES 1. While direct detection of small mutations, such as an ethyl-methane sulfonate derived single base substitutions, is limited by array coverage and sensitivity, large deletions such as those that can be caused by fast neutrons are easily detected. We demonstrate this by resolving two deletions, the 77-kb flavin-binding, kelch repeat, f-box 1 and the 7-kb cryptochrome2-1 deletions, via direct hybridization of mutant DNA to ATH1 expression arrays.

Arabidopsis↗
PubMed2005

Overlapping and distinct roles of PRR7 and PRR9 in the Arabidopsis circadian clock.

The core mechanism of the circadian oscillators described to date rely on transcriptional negative feedback loops with a delay between the negative and the positive components . In plants, the first suggested regulatory loop involves the transcription factors CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and the pseudo-response regulator TIMING OF CAB EXPRESSION 1 (TOC1/PRR1). TOC1 is a member of the Arabidopsis circadian-regulated PRR gene family . Analysis of single and double mutants in PRR7 and PRR9 indicates that these morning-expressed genes play a dual role in the circadian clock, being involved in the transmission of light signals to the clock and in the regulation of the central oscillator. Furthermore, CCA1 and LHY had a positive effect on PRR7 and PRR9 expression levels, indicating that they might form part of an additional regulatory feedback loop. We propose that the Arabidopsis circadian oscillator is composed of several interlocking positive and negative feedback loops, a feature of clock regulation that appears broadly conserved between plants, fungi, and animals.

Arabidopsis↗
PubMed2005

Bioluminescence imaging in living organisms.

Luciferase enzymes catalyze the emission of light from a substrate -- a phenomenon known as bioluminescence -- and have been employed as reporters of many biological functions. Luminescent reporters are much dimmer than fluorescent reporters, and therefore provide relatively modest spatial and temporal resolution. Yet, they are generally more sensitive and less toxic, making them particularly useful for long-term longitudinal studies of living cells, tissues and whole animals. Bioluminescence imaging has proven useful for detecting protein-protein interactions, for tracking cells in vivo, and for monitoring the transcriptional and post-transcriptional regulation of specific genes. Recent applications have included longitudinal monitoring of tumor progression in vivo, and monitoring circadian rhythms with single-cell resolution.

Animals↗
PubMed2004

Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression.

Circadian (ca. 24 hr) oscillations in expression of mammalian "clock genes" are found not only in the suprachiasmatic nucleus (SCN), the central circadian pacemaker, but also in peripheral tissues. Under constant conditions in vitro, however, rhythms of peripheral tissue explants or immortalized cells damp partially or completely. It is unknown whether this reflects an inability of peripheral cells to sustain rhythms, as SCN neurons can, or a loss of synchrony among cells. Using bioluminescence imaging of Rat-1 fibroblasts transfected with a Bmal1::luc plasmid and primary fibroblasts dissociated from mPer2(Luciferase-SV40) knockin mice, we monitored single-cell circadian rhythms of clock gene expression for 1-2 weeks. We found that single fibroblasts can oscillate robustly and independently with undiminished amplitude and diverse circadian periods. Cells were partially synchronized by medium changes at the start of an experiment, but due to different intrinsic periods, their phases became randomly distributed after several days. Closely spaced cells in the same culture did not have similar phases, implying a lack of functional coupling among cells. Thus, like SCN neurons, single fibroblasts can function as independent circadian oscillators; however, lack of oscillator coupling in dissociated cell cultures leads to a loss of synchrony among individual cells and damping of the ensemble rhythm at the population level.

Animals↗
PubMed2004

A functional genomics strategy reveals Rora as a component of the mammalian circadian clock.

The mammalian circadian clock plays an integral role in timing rhythmic physiology and behavior, such as locomotor activity, with anticipated daily environmental changes. The master oscillator resides within the suprachiasmatic nucleus (SCN), which can maintain circadian rhythms in the absence of synchronizing light input. Here, we describe a genomics-based approach to identify circadian activators of Bmal1, itself a key transcriptional activator that is necessary for core oscillator function. Using cell-based functional assays, as well as behavioral and molecular analyses, we identified Rora as an activator of Bmal1 transcription within the SCN. Rora is required for normal Bmal1 expression and consolidation of daily locomotor activity and is regulated by the core clock in the SCN. These results suggest that opposing activities of the orphan nuclear receptors Rora and Rev-erb alpha, which represses Bmal1 expression, are important in the maintenance of circadian clock function.

ARNTL Transcription Factors↗
PubMed2004

Universality and flexibility in gene expression from bacteria to human.

Highly parallel experimental biology is offering opportunities to not just accomplish work more easily, but to explore for underlying governing principles. Recent analysis of the large-scale organization of gene expression has revealed its complex and dynamic nature. However, the underlying dynamics that generate complex gene expression and cellular organization are not yet understood. To comprehensively and quantitatively elucidate these underlying gene expression dynamics, we have analyzed genome-wide gene expression in many experimental conditions in Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Mus musculus, and Homo sapiens. Here we demonstrate that the gene expression dynamics follows the same and surprisingly simple principle from E. coli to human, where gene expression changes are proportional to their expression levels, and show that this "proportional" dynamics or "rich-travel-more" mechanism can regenerate the observed complex and dynamic organization of the transcriptome. These findings provide a universal principle in the regulation of gene expression, show how complex and dynamic organization can emerge from simple underlying dynamics, and demonstrate the flexibility of transcription across a wide range of expression levels.

Animals↗
PubMed2003

A PERIOD inhibitor buffer introduces a delay mechanism for CLK/CYC-activated transcription.

We investigated the functions of clock genes period (per) and timeless (tim) in establishing negative feedback on circadian transcription factors clock/cycle (Clk/cyc) in Drosophila. We show that PER protein persists for several hours after rapid degradation of TIM in the morning. We observed in cell culture that isolated PER inhibits CLK/CYC-activated transcription in the absence of TIM and we further demonstrated for the first time in vivo that PER accumulation in a tim loss-of-function mutant background causes efficient inhibition of CLK/CYC-dependent transcription. These results identify PER to be the main inhibitor for CLK/CYC and they suggest a delay mechanism during early morning, when PER protein, after degradation of TIM, forms an inhibitor buffer for CLK/CYC that attenuates the restart of the next cycle of CLK/CYC-activated transcription. While TIM likely enhances the inhibition of CLK/CYC by PER in the dark, our results suggest a reduction of PER-mediated inhibition by TIM in light.

Animals↗