The mammalian circadian clock is a cell-autonomous system that drives oscillations in behavior and physiology in anticipation of daily environmental change. These oscillations manifest through interactions of the central pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus with local circadian clocks present in most mammalian tissues [1
]. Many clock components have been identified that act to generate circadian transcriptional oscillations through a regulatory system comprised of negative feedback loops [2
]. Studies in mouse models indicate that relatively few molecular perturbations of clock components (e.g., knockout or mutant animals) lead to complete loss of oscillator function as assessed by locomotor activity or circadian gene expression in isolated tissues [2
]. This suggests that the molecular clockwork constitutes a regulatory module that is phenotypically robust, i.e., resistant to and/or buffered against genetic perturbations such as gene loss, deletion, or mutation [4
]. Consistent with these findings, it often takes deletion of multiple factors (often gene paralogs) to disrupt behavioral and/or cellular rhythms (summarized in ).
Effects of Gene Knockout or Knockdown in Animal Models and U-2 OS Cells
The majority of identified clock components function as transcriptional activators or repressors. These proteins, along with other components that modulate protein stability and nuclear translocation, create two interlocking transcriptional feedback loops [2
]. At the core of the oscillator are two transcriptional activators, CLOCK and BMAL1, which heterodimerize and bind to E-box elements in the promoters of target genes, including two families of transcriptional repressors, the PERIOD (Per1
, and Per3
), and CRYPTOCHROME (Cry1
) proteins, as well as in other genes that regulate outputs mediated by the clock. Upon accumulation in the cytoplasm, PER and CRY proteins enter the nucleus and inhibit their own expression by repressing CLOCK/BMAL1-mediated transcription [2
]. Multiple proteins regulate stability and/or nuclear accumulation of clock components, including casein kinase I family members (CSNK1D and CSNK1E) and the F-box and leucine-rich repeat protein 3 (FBXL3) [5
]. A second stabilizing feedback loop interlocks with the primary loop and regulates Bmal1
expression positively by retinoic acid receptor–related orphan receptors (ROR) transcriptional activators and negatively by REV-ERB transcriptional repressors through binding to retinoic acid-related orphan receptor elements (ROREs) in the Bmal1
]. Paralogs of several clock genes exist, such as Bmal2
(paralogs of Bmal1
, respectively), which display similar biochemical functions yet may regulate unique target genes due to differential spatial expression [14
Cellular models of clock function such as immortalized cell lines (e.g., NIH3T3 cells), mouse embryonic fibroblasts (MEFs), and dissociated cells derived from the SCN, recapitulate robust oscillator function yet are free from neural, humoral, and behavioral cues that influence the clock in the intact organism [18
]. Many important properties of circadian behavior are maintained in these isolated autonomous oscillators. For example, MEFs derived from knockout mice of the transcriptional repressors Cry1
maintain defects in period length consistent with the short- and long-period locomotor behavior rhythms observed in the respective knockout mice [20
]. These cellular models are ideal for genetic perturbation experiments, as clock gene dosage can be altered over a wide range of concentrations. The consequences of these perturbations can be measured using kinetic imaging, biochemistry, and gene expression analysis.
Here, we present comprehensive genetic perturbation analysis of a new model of the autonomous human circadian clock. Using this system, we analyzed the functional consequences of decreasing expression of individual and combinations of circadian clock components in a dose-dependent fashion using RNA interference (RNAi). Effects on cellular oscillations were monitored using kinetic imaging, and changes in gene expression of other clock components were assessed by quantitative real-time PCR (RT-PCR). Using these data and applying biochemical constraints, we constructed gene interaction networks to describe, visualize, and mine the perturbation-induced changes for emerging network features. Disruption of gene function using previous methods, e.g., comparing knockout versus wild-type animals, lacked the statistical power necessary for uncovering such changes due to a lower number of gene doses (zero, one, or two copies). Using this new method, we uncovered several novel features of the circadian clock, including proportional response of gene expression, where levels of expression are altered actively and in a proportional fashion with respect to the gene being knocked down. By measuring the responses of most clock factors following perturbation of a single gene, we also observed signal propagation through interacting activator and repressor modules. Furthermore, our method uncovered unidirectional paralog compensation among several clock gene repressors, providing the first example to our knowledge of multiple paralogs regulated in this fashion in a single pathway. We propose that the features we uncovered provide mechanisms to buffer the clock against genetic perturbation, and ultimately contribute to the extraordinary robustness of the oscillator.