ABSTRACT
A ubiquitous feature of circadian clocks across life forms is its organization as a network of coupled cellular oscillators. Individual cellular oscillators of the network often exhibit a considerable degree of heterogeneity in their intrinsic periods. While the interaction of coupling and heterogeneity in circadian clock networks is hypothesized to influence clock’s entrainability, our knowledge of mechanisms governing network heterogeneity remains elusive. In this study, we aimed to explore the principles that underlie inter-cellular period variation in circadian clock networks (clonal period-heterogeneity). To this end, we employed a laboratory selection approach and derived a panel of 25 clonal cell populations exhibiting circadian periods ranging from 22 h to 28 h. We report that while a single parent clone can produce progeny clones with a wide distribution of circadian periods, heterogeneity is not entirely stochastically driven but has a strong heritable component. By quantifying the expression of 20 circadian clock and clock-associated genes across our panel, we found that inheritance of different expression patterns in at least three clock genes might govern clonal period-heterogeneity in circadian clock networks. Furthermore, we provide preliminary evidence suggesting that epigenetic variation might underlie such gene expression variation.
Footnotes
Summary of revision: The previous version of the manuscript reported Principal Component Analysis based identification of gene expression differences in 5 of the 20 circadian clock genes that might govern clonal heterogeneity in circadian period. In the revised version, we use RNAi mediated gene silencing to knockdown each of the five candidate genes and demonstrate that 3 of the 5 previously identified genes indeed govern clonal heterogeneity. Furthermore, we also provide preliminary experimental evidence suggesting a role epigenetic regulation in governing clonal heterogeneity of circadian period. In conclusion, our study reports that the heterogeneity in periods observed within circadian clock networks in mammals is not stochastically driven but has a heritable basis and that this is likely to be a multi-gene trait. We identified that differential regulation of three E-box associated transcription factors might govern period-heterogeneity in circadian clock networks and provide preliminary evidence that epigenetically regulated gene expression differences may underlie clonal period-heterogeneity Specific changes: 1.Figures 2 and 3 reorganized to fit in data from new experiments. 2.New figures added to supplemental information. 3.Two new sections added to Materials and Methods. 4.Results and discussion section updated based on new experimental evidence. 5.Funding source details updated. 6. New references added to support the updated discussion section based on new results.