Harmonics of Circadian Gene Transcription in Mammals

The circadian clock is a molecular and cellular oscillator found in most mammalian tissues that regulates rhythmic physiology and behavior. Numerous investigations have addressed the contribution of circadian rhythmicity to cellular, organ, and organismal physiology. We recently developed a method to look at transcriptional oscillations with unprecedented precision and accuracy using high-density time sampling. Here, we report a comparison of oscillating transcription from mouse liver, NIH3T3, and U2OS cells. Several surprising observations resulted from this study, including a 100-fold difference in the number of cycling transcripts in autonomous cellular models of the oscillator versus tissues harvested from intact mice. Strikingly, we found two clusters of genes that cycle at the second and third harmonic of circadian rhythmicity in liver, but not cultured cells. Validation experiments show that 12-hour oscillatory transcripts occur in several other peripheral tissues as well including heart, kidney, and lungs. These harmonics are lost ex vivo, as well as under restricted feeding conditions. Taken in sum, these studies illustrate the importance of time sampling with respect to multiple testing, suggest caution in use of autonomous cellular models to study clock output, and demonstrate the existence of harmonics of circadian gene expression in the mouse

Guidelines for Genome-Scale Analysis of Biological Rhythms

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

Guidelines for Genome-Scale Analysis of Biological Rhythms

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 is 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

A system for generating reversible knockout cell lines

AbstractA powerful use of gene editing technology is the generation of genetic-null (knockout) mutants of human immortalized cell lines. However, a problem that arises in the process of generating such edited lines is that to ensure edit homogeneity, clonal selection must often be performed and, given the genomic instability of immortalized cell lines and the high division number inherent to the clonal selection process, the resulting clone can exhibit genotypic and phenotypic differences from the parental line. Here, we present a system that allows for the generation of genetic null cell lines with an excisable cassette. This system allows for the generation of suitable controls without the need of further rounds of clonal selection.</jats:p

A high-throughput assay for siRNA-based circadian screens in human U2OS cells.

The advent of siRNA-based screens has revolutionized the efficiency by which functional components of biological processes are identified. A notable exception has been the field of mammalian circadian rhythms. Here, we outline a medium- to high-throughput siRNA-based approach that, in combination with real-time bioluminescence measurement of a circadian reporter gene, can be utilized to elucidate the effects of gene knockdown across several days in human cells

A High-Throughput Assay for siRNA-Based Circadian Screens in

The advent of siRNA-based screens has revolutionized the efficiency by which functional components of biological processes are identified. A notable exception has been the field of mammalian circadian rhythms. Here, we outline a medium- to high-throughput siRNA-based approach that, in combination with real-time bioluminescence measurement of a circadian reporter gene, can be utilized to elucidate the effects of gene knockdown across several days in human cells

Correction: JMJD5 links CRY1 function and proteasomal degradation.

[This corrects the article DOI: 10.1371/journal.pbio.2006145.]