Circadian Clock - Systems Biology Approaches To Elucidate Oscillating Mechanisms

Systems Biology Approaches To Elucidate Oscillating Mechanisms

Modern experimental approaches using systems biology have identified many novel components in biological clocks that suggest an integrative view on how organisms maintain circadian oscillation.

Recently, Baggs et al. developed a novel strategy termed "Gene Dosage Network Analysis" (GDNA) to describe network features in the human circadian clock that contribute to an organism's robustness against genetic perturbations. In their study, the authors used small interfering RNA (siRNA) to induce dose-dependent changes in gene expression of clock components within immortalized human osteosarcoma U2OS cells in order to build gene association networks consistent with known biochemical constraints in the mammalian circadian clock. Employing multiple doses of siRNA powered their quantitative RT-PCR analysis to uncover several network features of the circadian clock, including proportional responses of gene expression, signal propagation through interacting modules, and compensation through gene expression changes.

Proportional responses in downstream gene expression following siRNA-induced perturbation revealed levels of expression that were actively altered with respect to the gene being knocked down. For example, when Bmal1 was knocked down in a dose-dependent manner, Rev-ErbA alpha and Rev-ErbA beta mRNA levels were shown to decrease in a linear, proportional manner. This supported previous findings that Bma1 directly activates Rev-erb genes and further suggests Bma1 as a strong contributor to Rev-erb expression.

In addition, the GDNA method provided a framework to study biological relay mechanisms in circadian networks through which modules communicate changes in gene expression. The authors observed signal propagation through interactions between activators and repressors, and uncovered unidirectional paralog compensation among several clock gene repressors—for example, when PER1 is depleted, there is an increase in Rev-erbs, which in turn propagates a signal to decrease expression in BMAL1, the target of the Rev-erb repressors.

By examining knockdown of several transcriptional repressors, GDNA also revealed paralog compensation where gene paralogs were upregulated through an active mechanism by which gene function is replaced following knockdown in a nonredunant manner—that is, one component is sufficient to sustain function. These results further suggested that a clock network utilizes active compensatory mechanisms rather than simple redundancy to confer robustness and maintain function. In essence, the authors proposed that the observed network features act in concert as a genetic buffering system to maintain clock function in the face of genetic and environmental perturbation. Following this logic, we may use genomics to explore network features in the circadian oscillator.

Another study conducted by Zhang et al. also employed a genome-wide small interfering RNA screen in U2OS cell line to identify additional clock genes and modifiers using luciferase reporter gene expression. Knockdown of nearly 1000 genes reduced rhythm amplitude. The authors found and confirmed hundreds of potent effects on period length or increased amplitude in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling pathway, the cell cycle, and folate metabolism. Coupled with data demonstrating that many of these pathways are clock-regulated, Zhang et al. postulated that the clock is interconnected with many aspects of cellular function.

A systems biology approach may relate circadian rhythms to cellular phenomena that were not originally considered regulators of circadian oscillation.