such as "Introduction", "Conclusion"..etc
Most organisms possess an endogenous circadian system that drives the daily timing of many physiological and behavioral processes. Genetic screens, spontaneous mutants, and gene-targeting approaches have been key in unraveling the essential set of genes underlying the circadian mechanism in mammals, Drosophila, and other model systems [1–4]. At the molecular and biochemical levels, a set of core clock genes govern positive and negative autoregulatory feedback loops of transcription and translation to form the core mechanism of the circadian clock in mammals [2,5]. The central oscillator is primarily driven by two bHLH-PAS transcription factors within the positive feedback loop, CLOCK and BMAL1, which heterodimerize and transactivate downstream clock and clock-controlled genes by binding to E-box elements that lie within their promoters [6–9]. The core constituents of the negative feedback loop are the Cry and Per genes, which are transcriptionally driven by CLOCK and BMAL1. PER and CRY proteins accumulate, associate with each other in the cytoplasm, translocate to the nucleus, and inhibit the CLOCK and BMAL1 activation of their own transcription . As the negative elements turn over, CLOCK and BMAL1 renew their cycle of transcription of the Per and Cry genes.
In mammals, nearly all cells in the body contain circadian oscillators organized in a hierarchical fashion, with a master pacemaker located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus [5,10,11]. The SCN is entrained to the 24-h daily light–dark cycle via retinal light input and, in turn, synchronizes and coordinates the rhythms of peripheral tissue clock cells [2,5]. In mammals, luciferase reporters of circadian genes [10,11] in conjunction with single cell imaging have been valuable in revealing self-sustained circadian oscillators in virtually every cell in the body [12–15]. These studies have shown that most peripheral organs and tissues can express circadian rhythms in isolation; however, inputs from the dominant circadian pacemaker in the SCN are essential in coordinating circadian rhythms in an intact animal [10,11,16,17]. For example, SCN transplant experiments have shown that SCN-lesioned arrhythmic animals and genetically arrhythmic mice take on the rhythm of the donor SCN [17–19]. Similarly, transplanted mouse embryonic fibroblasts exhibit a circadian period and phase characteristic of the host rhythm and phase . These findings have led to a widely accepted hierarchical model of the mammalian circadian system in which the SCN acts as pacemaker that drives and synchronizes peripheral circadian oscillators. Thus, understanding the physiological and functional relationships among central and peripheral clocks is essential; however, we still do not fully understand how the SCN governs peripheral oscillators to regulate circadian rhythms in physiology and behavior in multicellular organisms.
In nonmammalian systems, such as in Drosophila, analyses of the regulatory interactions of circadian genes led to the exploitation of novel tools to drive circadian genes, such as tissue-specific expression of transgenes (TGs) and reporters, which are valuable in elucidating the complexity of circadian system [21–23]. Conditional systems utilizing heat shock promoters have been developed in order to drive the temperature-dependent expression of circadian TGs [24–26]. Exogenous promoters in conjunction with the GAL4-UAS bipartite transgenic system have been valuable in expressing circadian TGs in subregions of the brain or distinct groups of circadian neurons, and even in ablation of discrete circadian neurons [27–30]. In mammals, however, other than ubiquitous inactivation of circadian genes by gene targeting techniques in embryonic stem cells or studies using the culture/explant-based system, the use of tissue-specific conditional regulation of circadian genes has not been reported [2,3]. Thus, to elucidate cellular and behavioral networks in the mammalian circadian system, more refined approaches are required, especially those affording temporal and spatial control of gene expression in vivo.
The ability to regulate TG expression in a conditional manner has made the tetracycline-controlled transactivator (tTA) system an attractive tool for use in mammalian systems. The tTA system was originally developed by Bujard and colleagues for the conditional expression of reporter genes in mammalian cells [31–34] and has been successfully applied in many experiments to study a variety of developmental processes and brain function in mammals [35–45]. The first component of this system contains a tissue-specific promoter that drives the expression of tTA, a fusion of the Escherichia coli tetracycline repressor sequence to the C-terminal transactivation domain of the herpes simplex virus VP16 gene that converts the repressor into a transcriptional activator. Expression of the target TG by tTA is achieved by introducing the TG of interest downstream of a minimal cytomegalovirus promoter sequence linked to multiple copies of the tet operator (tetO) sequence. Conditional and inducible regulation of the target TG is contingent on the ability of tTA to bind to the tetO sequences and activate transcription in a tetracycline-dependent manner. TG expression can be turned off with the administration of doxycycline (Dox), a tetracycline derivative, which prevents binding of tTA to the tetO sequence. Thus, unlike the site-specific recombinase Cre-loxP and Flp-FRT systems [46,47], which only allow conditional and/or tissue-specific inactivation of genes , the tTA system permits repeated cycles of conditional activation and inactivation of genes within the same animal. Hence, the tTA system provides truly conditional investigation of gene function. Despite the widespread use of the Tet system to manipulate various genes in a variety of tissues in mammals, only a few studies have used the system in the brain, and even fewer have used the system to study regulation of behavior [35,37–39,41–43,45,49]. A limitation of the tTA system in the study of brain function in vivo has been the slow induction of the TG (i.e., reversal) upon removal of standard dose of Dox .
Here we show that regulation of ClockΔ19 or Clockwt TG expression occurs with rapid kinetics of induction and repression, causing an immediate reversion of the mutant to wild-type (WT) phenotype, and vice versa, in a tissue-specific manner. We demonstrate that the CLOCK protein is an excellent indicator for the kinetics of Dox-dependent induction/repression in the brain. Using activity rhythms as an output, the circadian period length provides a daily readout of the transactivation state of the Tet system in vivo. The development of the tTA system for conditional TG expression in the brain/SCN will open new avenues of research to answer fundamental questions of mammalian circadian biology.
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