Non-circadian expression masking clock-driven weak transcription rhythms in U2OS cells.

Julia Hoffmann,Michael Brunner,Felix Naef,Anton Shostak,Tamás Fischer,Laura Symul,Nicolas Cermakian

doi:10.1371/journal.pone.0102238

Julia Hoffmann, Michael Brunner + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0102238

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Abstract

U2OS cells harbor a circadian clock but express only a few rhythmic genes in constant conditions. We identified 3040 binding sites of the circadian regulators BMAL1, CLOCK and CRY1 in the U2OS genome. Most binding sites even in promoters do not correlate with detectable rhythmic transcript levels. Luciferase fusions reveal that the circadian clock supports robust but low amplitude transcription rhythms of representative promoters. However, rhythmic transcription of these potentially clock-controlled genes is masked by non-circadian transcription that overwrites the weaker contribution of the clock in constant conditions. Our data suggest that U2OS cells harbor an intrinsically rather weak circadian oscillator. The oscillator has the potential to regulate a large number of genes. The contribution of circadian versus non-circadian transcription is dependent on the metabolic state of the cell and may determine the apparent complexity of the circadian transcriptome.

Highlights

Circadian clocks are self-sustained oscillators that depend on interlocked transcriptional feedback loops
We show that BMAL1, CLOCK, and the circadian repressor CRY1 bind to about 3000 sites in the genome of U2OS cells, a number similar to the binding sites identified in mouse liver [19,23]
Using affinity purified antibodies (Figure S1A) we identified 3040 circadian regulator binding sites (CRBSs) in the genome of unsynchronized U2OS cells (Figure 1A)

Summary

Introduction

Circadian clocks are self-sustained oscillators that depend on interlocked transcriptional feedback loops. In a second loop ROR activators and REV-ERB repressors regulate rhythmic transcription of BMAL1 [5,6,7] and NPAS2 [8] and presumably CRY genes [7,9] by competing for ROR elements [10]. The central circadian pacemaker resides in the suprachiasmatic nucleus (SCN). It is entrained by the geophysical day/night cycle of the earth’s rotation through light input received via the retinohypothalamic tract [11]. Many organs and cell types contain a circadian clock [12,13,14,15] These peripheral clocks are synchronized by the SCN [16] and, in addition, by rhythmic cues independent of the SCN, such as entrainment by rhythmic feeding in case of the hepatic clock [17]

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