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

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. Analysis of temporal expression profiles of 5708 expressed genes in synchronized U2OS cells. A 60k customized microarray was designed for 6356 genes, which corresponds to roughly one fourth of the human genome. 1373 genes were assigned to circadian regulator binding sites (CRBSs), 1503 genes were specifically selected in addition to a set of 3480 random genes. For each gene 10 independent probes in two microarray replicates were performed to increase reliability of the data.U2OS细胞拥有昼夜节律时钟（circadian clock），但在恒定培养条件下仅表达少量节律性基因。本研究在U2OS细胞基因组中鉴定出了昼夜节律调控因子BMAL1、CLOCK与CRY1的3040个结合位点。即便位于启动子（promoter）区域的绝大多数结合位点，也与可检测到的节律性转录本水平无显著相关性。荧光素酶融合（Luciferase fusion）实验证实，昼夜节律时钟可驱动代表性启动子产生稳健但振幅较低的转录节律。然而，在恒定培养条件下，这些潜在时钟调控基因的节律性转录会被非节律性转录掩盖——非节律性转录的强度覆盖了时钟的微弱调控作用。本研究数据表明，U2OS细胞内在的昼夜节律振荡器（circadian oscillator）实际强度较弱。该节律振荡器具备调控大量基因的潜力。节律性转录与非节律性转录的相对占比取决于细胞的代谢状态，这或许决定了节律转录组（circadian transcriptome）的表观复杂性。 本研究对同步化U2OS细胞中5708个表达基因的时间表达谱展开了分析。研究针对6356个基因设计了60k定制化微阵列（customized microarray），该基因数量约占人类基因组的四分之一。其中1373个基因被注释至昼夜节律调控因子结合位点（circadian regulator binding sites, CRBSs）；此外，除预设的3480个随机基因集外，还额外特异性筛选出1503个基因。为提升数据可靠性，每个基因设置10个独立探针，并开展了两次微阵列重复实验。