Search

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding "big data" that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.

The cyanobacterial circadian oscillator, consisting of KaiA, KaiB, and KaiC proteins, drives global rhythms of gene expression and compaction of the chromosome and regulates the timing of cell division and natural transformation. While the KaiABC posttranslational oscillator can be reconstituted in vitro, the Kai-based oscillator is subject to several layers of regulation in vivo. Specifically, the oscillator proteins undergo changes in their subcellular localization patterns, where KaiA and KaiC are diffuse throughout the cell during the day and localized as a focus at or near the pole of the cell at night. Here, we report that the CI domain of KaiC, when in a hexameric state, is sufficient to target KaiC to the pole. Moreover, increased ATPase activity of KaiC correlates with enhanced polar localization. We identified proteins associated with KaiC in either a localized or diffuse state. We found that loss of Rbp2, found to be associated with localized KaiC, results in decreased incidence of KaiC localization and long-period circadian phenotypes. Rbp2 is an RNA-binding protein, and it appears that RNA-binding activity of Rbp2 is required to execute clock functions. These findings uncover previously unrecognized roles for Rbp2 in regulating the circadian clock and suggest that the proper localization of KaiC is required for a fully functional clock in vivo. [ABSTRACT FROM AUTHOR]

The authors analyzed the upstream regulatory region of purF, a gene that is expressed in a minority phase that peaks at dawn (class 2 circadian phasing) in Synechococcus elongates, to determine whether specific cis elements are responsible for this characteristic expression pattern. Fusions of various promoter-bearing fragments to luciferase reporter genes showed that normal class 2 phasing of purF expression was correlated with promoter strength. No specific cis element that is separable from the promoter was responsible for determining phase. Very weak promoter activity of unstable phasing was mapped to a 50-bp segment. Inclusion of sequences that flank this minimal promoter either upstream or downstream increased the promoter strength and stabilized the phase in class 2, but neither segment was individually necessary. Because the data suggested a role for the overall promoter context rather than a specific "phase element," the authors proposed that DNA topology is important in the phase determination of circadian gene expression in S. elongates. To test this hypothesis, they fused the well-characterized DNA topology-dependent Escherichia coli fis promoter to luciferase and showed that it acts as a class 2 promoter in S. elongates. [ABSTRACT FROM AUTHOR]