Abstract
A ubiquitous feature of the circadian clock across life forms is its organization as a network of cellular oscillators, with individual cellular oscillators within the network often exhibiting considerable heterogeneity in their intrinsic periods. The interaction of coupling and heterogeneity in circadian clock networks is hypothesized to influence clock's entrainability, but our knowledge of mechanisms governing period heterogeneity within circadian clock networks remains largely elusive. In this study, we aimed to explore the principles that underlie intercellular 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 to 28 h. We report that a single parent clone can produce progeny clones with a wide distribution of circadian periods, and this heterogeneity, in addition to being stochastically driven, has a heritable component. By quantifying the expression of 20 circadian clock and clock-associated genes across our clone panel, we found that inheritance of expression patterns in at least three clock genes might govern clonal period heterogeneity in circadian clock networks. Furthermore, we provide evidence suggesting that heritable epigenetic variation in gene expression regulation might underlie period heterogeneity.
Highlights
The majority of life forms on earth exhibit approximately 24-h behavioral and physiological rhythms generated by endogenous timekeeping mechanisms—circadian clocks
Both heritable and stochastic components contribute to clonal period heterogeneity Is the variation in period among individual circadian oscillator cells due to nonheritable stochastic noise? Or is there a heritable component? To test this, we single-cell cloned a “founding culture” of human U-2 OS cells harboring a BMAL1-luciferase reporter construct [32]
We observed that the short-period clonal line (SCL) exhibits an advanced phase of entrainment compared to long-period clonal line (LCL) by about 3.2 ± 1.3 h under T24 (12 hours of 37 ̊C and 33 ̊C each; S3 Fig top panel). This phase-difference further increases to about 5.8 ± 1.7 h under T26 (13 hours of 37 ̊C and 33 ̊C each; S3 Fig bottom panel) cycles, consistent with theoretical predictions [36]. These results indicate that (1) both heritable and stochastic components contribute to clonal period heterogeneity and (2) short circadian periods have a stronger heritable component compared to long periods, which are more strongly noise driven
Summary
The majority of life forms on earth exhibit approximately 24-h (circadian) behavioral and physiological rhythms generated by endogenous timekeeping mechanisms—circadian clocks. In addition to driving such endogenous rhythms, circadian clocks facilitate synchronization of organisms’ rhythms to daily and seasonal changes in the environment to enhance their survivability, thereby functioning as an adaptive mechanism [1]. The fundamental basis of circadian rhythm generation across all life forms are cell-autonomous molecular oscillators comprising evolutionarily conserved autoregulatory transcription-translation feedback loops [2]. In higher organisms, such cell-autonomous clocks often function as a network of coupled oscillators that, in unison, drive circadian rhythms [3].
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