Editing Circadian clock (section)

== 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.<ref name= "Zhang"/><ref name="Baggs"/>

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.<ref name="Baggs"/> 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 PCR]] 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 [[small interfering RNA|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 Bmal1 directly activates Rev-erb genes and further suggests Bmal1 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.<ref name="Baggs"/> 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 the 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 non-redundant 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.<ref name="Baggs"/> 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.<ref name="Zhang"/> Knockdown of nearly 1000 genes reduced rhythm amplitude. The authors found and confirmed hundreds of potent effects on [[periodic function|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 were 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.  For example, a 2014 workshop<ref name="workshop">{{cite web |url=http://www.nhlbi.nih.gov/research/reports/2014-circadian-clock-lung-health.htm |title=NHLBI Workshop: "Circadian Clock at the Interface of Lung Health and Disease" April 28-29, 2014 Executive Summary |author=<!--Staff writer(s); no by-line.--> |date=September 2014 |publisher=National Heart, Lung, and Blood Institute |access-date=20 September 2014 |archive-url=https://web.archive.org/web/20141004183349/http://www.nhlbi.nih.gov/research/reports/2014-circadian-clock-lung-health.htm |archive-date=4 October 2014 |url-status=dead }}</ref> at [[NHLBI]] assessed newer circadian genomic findings and discussed the interface between the body clock and many different cellular processes.