Heritable gene expression variability and stochasticity govern clonal heterogeneity in circadian period

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.

A functional context for heterogeneity of the circadian clock in cells

“…The center panel depicts the same collection of curves but with the vertical axis scaled proportionally to the panel on the right, which shows a summation of the individual profiles shown to the left. This simply derived additive curve resembles, e.g., bioluminescence profiles showing clock regulated gene expression, such as shown in Nikhil and colleagues [4]. Actual expression curves should vary according to what is quantified-they could be broader or narrower depending on what is measured and on the distribution of component entrained phases.…”

“…A longer free-running rhythm usually leads to a later entrained phase relative to a shorter one. This has been shown on various levels, demonstrating the pervasiveness of the principles of circadian biology: not only do whole organisms show these properties, but so do the organs and cells within them [3,4].…”

“…Individual cells show a distribution of free-running periods and entrained phases when monitored using reporters of gene expression. A paper published in 2019 in the BioRxiv [16] and in a recent issue of PLOS Biology [4] addresses these issues. In addition, papers by Li and colleagues [17,18] report similar findings using different cell lines (immortalized mouse ear fibroblasts versus U-2 OS human osteosarcoma cells versus 3T3 cells), thus suggesting that this is a general property of cellular clocks.…”

“…In addition, papers by Li and colleagues [17,18] report similar findings using different cell lines (immortalized mouse ear fibroblasts versus U-2 OS human osteosarcoma cells versus 3T3 cells), thus suggesting that this is a general property of cellular clocks. Clonal lines could be subcloned to yield short or long period lines-from 22 to 28 h [4,16]. The circadian period of these subclones showed a degree of stability that was-to a modest extent-heritable.…”

Time-restricted feeding near dawn entrains long-term behavioral changes through the suprachiasmatic nucleus

“…Notably, this cluster corresponded to the observed high frequency of Ca 2+ spike, and might therefore represent SCN neuronal activity. To analyze which transcripts exerted the most significant influence on PC1 in this model, we used the cos 2 values to scale their relative importance (Nikhil et al, 2020). We shortlisted the top 214 genes that meet cos 2 > 0.5, and interestingly found that 142 genes among 214 genes had significantly different expression in the ZT0-4 TRF entrained SCN at CT1 samples compared with other groups (See detailed in methods).…”