Abstract
The mechanism of circadian oscillations in mammals is cell autonomous and is generated by a set of genes that form a transcriptional autoregulatory feedback loop. While these “clock genes” are well conserved among animals, their specific functions remain to be fully understood and their roles in central versus peripheral circadian oscillators remain to be defined. We utilized the in vivo inducible tetracycline-controlled transactivator (tTA) system to regulate Clock gene expression conditionally in a tissue-specific and temporally controlled manner. Through the use of Secretogranin II to drive tTA expression, suprachiasmatic nucleus– and brain-directed expression of a tetO::ClockΔ19 dominant-negative transgene lengthened the period of circadian locomotor rhythms in mice, whereas overexpression of a tetO::Clockwt wild-type transgene shortened the period. Low doses (10 μg/ml) of doxycycline (Dox) in the drinking water efficiently inactivated the tTA protein to silence the tetO transgenes and caused the circadian periodicity to return to a wild-type state. Importantly, low, but not high, doses of Dox were completely reversible and led to a rapid reactivation of the tetO transgenes. The rapid time course of tTA-regulated transgene expression demonstrates that the CLOCK protein is an excellent indicator for the kinetics of Dox-dependent induction/repression in the brain. Interestingly, the daily readout of circadian period in this system provides a real-time readout of the tTA transactivation state in vivo. In summary, the tTA system can manipulate circadian clock gene expression in a tissue-specific, conditional, and reversible manner in the central nervous system. The specific methods developed here should have general applicability for the study of brain and behavior in the mouse.
Highlights
Most organisms possess an endogenous circadian system that drives the daily timing of many physiological and behavioral processes
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 [6,7,8,9]
Significant progress has been made in unraveling the molecular mechanism of circadian clocks in mammals, previous work has focused on germline mutations and in vitro methods for analysis
Summary
Most organisms possess an endogenous circadian system that drives the daily timing of many physiological and behavioral processes. 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 [6,7,8,9]. The core constituents of the negative feedback loop are the Cry and Per genes, which are transcriptionally driven by CLOCK and BMAL1. As the negative elements turn over, CLOCK and BMAL1 renew their cycle of transcription of the Per and Cry genes
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