The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
BACKGROUND - The cyanobacterial circadian program exerts genome-wide control of gene expression. KaiC undergoes rhythms of phosphorylation that are regulated by interactions with KaiA and KaiB. The phosphorylation status of KaiC is thought to mediate global transcription via output factors SasA, CikA, LabA, RpaA, and RpaB. Overexpression of kaiC has been reported to globally repress gene expression.
RESULTS - Here, we show that the positive circadian component KaiA upregulates "subjective dusk" genes and that its overexpression deactivates rhythmic gene expression without significantly affecting growth rates in constant light. We analyze the global patterns of expression that are regulated by KaiA versus KaiC and find in contrast to the previous report of KaiC repression that there is a "yin-yang" regulation of gene expression whereby kaiA overexpression activates "dusk genes" and represses "dawn genes," whereas kaiC overexpression complementarily activates dawn genes and represses dusk genes. Moreover, continuous induction of kaiA latched KaiABC-regulated gene expression to provide constitutively increased transcript levels of diverse endogenous and heterologous genes that are expressed in the predominant subjective dusk phase. In addition to analyzing KaiA regulation of endogenous gene expression, we apply these insights to the expression of heterologous proteins whose products are of potential value, namely human proinsulin, foreign luciferase, and exogenous hydrogenase.
CONCLUSIONS - Both KaiC and KaiA complementarily contribute to the regulation of circadian gene expression via yin-yang switching. Circadian patterns can be reprogrammed by overexpression of kaiA or kaiC to constitutively enhance gene expression, and this reprogramming can improve 24/7 production of heterologous proteins that are useful as pharmaceuticals or biofuels.
Copyright © 2013 Elsevier Ltd. All rights reserved.
The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P(i) (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the strikingly similar makeups of the active sites at the interfaces between α/β heterodimers of F1-ATPase and between monomeric subunits in the KaiCII hexamer. Several KaiCII residues play a critical role in the relative activities of kinase and ATP synthase, among them R385, which stabilizes the compact form and helps kinase action reach a plateau, and T426, a short-lived phosphorylation site that promotes and affects the order of dephosphorylation.
Circadian rhythms are endogenous cellular patterns that associate multiple physiological and molecular functions with time. The Neurospora circadian system contains at least three oscillators: the FRQ/WC-dependent circadian oscillator (FWO), whose core components are the FRQ, WC-1, WC-2, FRH, and FWD-1 proteins; the WC-dependent circadian oscillator (WC-FLO); and one or more FRQ/ WC-independent oscillators (FLO). Little is known about the distribution of homologs of the Neurospora clock proteins or about the molecular foundations of circadian rhythms across fungi. Here, we examined 64 diverse fungal proteomes for homologs of all five Neurospora clock proteins and retraced their evolutionary history. The FRH and FWD-1 proteins were likely present in the fungal ancestor. WC-1 and WC-2 homologs are absent from the early diverging chytrids and Microsporidia but are present in all other major clades. In contrast to the deep origins of these four clock proteins FRQ homologs are taxonomically restricted within Sordariomycetes, Leotiomycetes and Dothideomycetes. The large number of FRH and FWD-1 homologs identified and their lack of concordance with the fungal species phylogeny indicate that they likely underwent multiple rounds of duplications and losses. In contrast, the FRQ, WC-1 and WC-2 proteins exhibit relatively few duplications and losses. A notable exception is the 10 FRQ-like proteins in Fusarium oxysporum, which resulted from nine duplication events. Our results suggest that the machinery required for FWO oscillator function is taxonomically restricted within Ascomycetes. Although the WC proteins are widely distributed, the functional diversity of the few non-Neurospora circadian oscillators suggests that a WC-FLO oscillator is unlikely to fully explain the observed rhythms. The contrast between the diversity of circadian oscillators and the conservation of most of their machinery is likely best explained by considering the centrality of noncircadian functions in which RNA helicase (FRH), F-box (FWD-1), WC-1 and WC-2 (light-sensing) proteins participate in fungi and eukaryotes.
The daily biological clock regulates the timing of sleep and physiological processes that are of fundamental importance to human health, performance, and well-being. Environmental parameters of relevance to biological clocks include (1) daily fluctuations in light intensity and temperature, and (2) seasonal changes in photoperiod (day length) and temperature; these parameters vary dramatically as a function of latitude and locale. In wide-ranging species other than humans, natural selection has genetically optimized adaptiveness along latitudinal clines. Is there evidence for selection of clock gene alleles along latitudinal/photoperiod clines in humans? A number of polymorphisms in the human clock genes Per2, Per3, Clock, and AANAT have been reported as alleles that could be subject to selection. In addition, this investigation discovered several novel polymorphisms in the human Arntl and Arntl2 genes that may have functional impact upon the expression of these clock transcriptional factors. The frequency distribution of these clock gene polymorphisms is reported for diverse populations of African Americans, European Americans, Ghanaians, Han Chinese, and Papua New Guineans (including 5 subpopulations within Papua New Guinea). There are significant differences in the frequency distribution of clock gene alleles among these populations. Population genetic analyses indicate that these differences are likely to arise from genetic drift rather than from natural selection.
BACKGROUND - An interlocked transcriptional-translational feedback loop (TTFL) is thought to generate the mammalian circadian clockwork in both the central pacemaker residing in the hypothalamic suprachiasmatic nuclei and in peripheral tissues. The core circadian genes, including Period1 and Period2 (Per1 and Per2), Cryptochrome1 and Cryptochrome2 (Cry1 and Cry2), Bmal1, and Clock are indispensable components of this biological clockwork. The cycling of the PER and CRY clock proteins has been thought to be necessary to keep the mammalian clock ticking.
RESULTS - We provide a novel cell-permeant protein approach for manipulating cryptochrome protein levels to evaluate the current transcription and translation feedback model of the circadian clockwork. Cell-permeant cryptochrome proteins appear to be functional on the basis of several criteria, including the abilities to (1) rescue circadian properties in Cry1(-/-)Cry2(-/-) mouse fibroblasts, (2) act as transcriptional repressors, and (3) phase shift the circadian oscillator in Rat-1 fibroblasts. By using cell-permeant cryptochrome proteins, we demonstrate that cycling of CRY1, CRY2, and BMAL1 is not necessary for circadian-clock function in fibroblasts.
CONCLUSIONS - These results are not supportive of the current version of the transcription and translation feedback-loop model of the mammalian clock mechanism, in which cycling of the essential clock proteins CRY1 and CRY2 is thought to be necessary.
The central circadian pacemaker is located in the hypothalamus of mammals, but essentially the same oscillating system operates in peripheral tissues and even in immortalized cell lines. Using luciferase reporters that allow automated monitoring of circadian gene expression in mammalian fibroblasts, we report the collection and analysis of precise rhythmic data from these cells. We use these methods to analyze signaling pathways of peripheral tissues by studying the responses of Rat-1 fibroblasts to ten different compounds. To quantify these rhythms, which show significant variation and large non-stationarities (damping and baseline drifting), we developed a new fast Fourier transform-nonlinear least squares analysis procedure that specifically optimizes the quantification of amplitude for circadian rhythm data. This enhanced analysis method successfully distinguishes among the ten signaling compounds for their rhythm-inducing properties. We pursued detailed analyses of the responses to two of these compounds that induced the highest amplitude rhythms in fibroblasts, forskolin (an activator of adenylyl cyclase), and dexamethasone (an agonist of glucocorticoid receptors). Our quantitative analyses clearly indicate that the synchronization mechanisms by the cAMP and glucocorticoid pathways are different, implying that actions of different genes stimulated by these pathways lead to distinctive programs of circadian synchronization.
Circadian variation in plasminogen activator inhibitor-1 (PAI-1) production likely contributes to increased risk of myocardial infarction and decreased efficacy of thrombolytic therapy during the morning. In this study, we characterize the abilities of fundamental molecular components of intrinsic circadian clocks to regulate the human PAI-1 promoter in transfected endothelial cells. Both CLOCK:BMAL1 and CLOCK:BMAL2 heterodimers activate the PAI-1 promoter through requisite proximal (-565 to -560 bp) and distal (-680 to -675 bp) E-box enhancers. Although the distal E-box overlaps the 4G/5G polymorphism of the PAI-1 promoter, allelic variation at this site does not influence CLOCK:BMAL1-and CLOCK:BMAL2-mediated transactivation. Together, CLOCK:BMAL1 and CLOCK:BMAL2 make additive contributions to PAI-1 gene transcription. While the abilities of these heterodimers to activate gene expression differ by twofold, the susceptibilities of these circadian activators to inhibition by period and cryptochrome proteins are equivalent and redox independent. Given that BMAL1 and BMAL2 differ in their spatiotemporal distributions, such distinctions may allow intrinsic circadian clocks to modulate the amplitudes of their oscillators, while maintaining circadian periodicity. In this way, fundamental circadian clock components may drive circadian variation in PAI-1, which in turn influences the pathogenesis, timing, and treatment of acute atherothrombotic events.
The BMAL2 gene encodes a member of the basic helix-loop-helix PER-ARNT-SIM family of transcription factors, which control diverse physiological processes including circadian rhythms. We identified four novel human BMAL2 transcripts that differ by alternative splicing within their NH2-terminal regions. Divergent expression of these and previously reported transcripts was observed among human tissues. The functional consequences of alternative splicing for transcriptional activation by CLOCK:BMAL2 heterodimers were assessed using luciferase reporter gene constructs that contained one of three diurnally regulated promoters, namely, those of the mouse period1, mouse vasopressin, and human plasminogen activator inhibitor-1 genes. These studies revealed that alternative splicing generates BMAL2 isoforms possessing high, medium, low, or no transcriptional activity. Similar results were obtained with each promoter, suggesting that alternative splicing may influence the amplitudes of both central and peripheral oscillators. Indeed, alternative splicing of BMAL2 may provide tissues with a rheostat capable of regulating CLOCK:BMAL2 heterodimer function across a broad continuum of potential transcriptional activities to accommodate varied metabolic demands and physiological roles.