Circadian rhythms in the absence of the clock gene Bmal1

Circadian clocks exist in almost all levels of living organisms and play elementary roles in the persistence of regular physiological and behavioural processes. Canonical transcription/translation feedback loop models portray BMAL1 (ARNTL) as one of the principal drivers of circadian periodicity in mammalian systems. In this integrated multi-omics study, we demonstrate, for the first time, 24 hr circadian oscillations in the expression levels of several transcripts and proteins in dexamethasone-synchronized Bmal1-/- mouse fibroblast cells and liver tissue slices. Intriguingly, daily oscillations were also observed in phosphoproteome profiles in the absence of this core clock gene. Our findings reveal that Bmal1 knockout radically alters the expression and phosphorylation patterns of mice hepatic proteome, which possibly attributes to considerably different sets of rhythmic candidates compared to wild-type. It is, therefore, reasonable to accentuate that circadian rhythms are not obliterated in mammalian systems due to deletion of the core clock genes. Wild-type and Bmal1-/- MSF cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% (v/v) HyClone III Serum (Analab; Cat # SH30109.03), 1/100 Glutamax-I (Invitrogen; Cat # 35050-038), 1/100 Penicillin-Streptomycin (SIGMA; Cat # P4333) and 1/500 MycoZap™ (Lonza; Cat # VZA-2022) in multiple six-well plates until complete confluency (n=3, per time-point, per genotype). The confluent MSF cells were treated with 100nM (final concentration) of dexamethasone (DEX) for 15 min to synchronize the cells [Balsalobre et al. Science 2000]29. MSF cells were then washed three times with PBS (37°C) and were incubated in HEPES-buffered Medium; 1X DMEM powder (SIGMA; Cat # D5030), 0.35mg/mL sodium bicarbonate, 5mg/mL glucose, 0.01M HEPES, 10% (v/v) HyClone III Serum, 1/100 Glutamax-I, and 1/500 MycoZap™, pH 7.3 (adjusted with HCl) and osmolality 320 mOsm (adjusted with NaCl) at 37°C under DD cycle. 48 hrs after DEX treatment, MSF cells were harvested at every three or two hours interval for three days for transcriptomics analyses.

生物钟（Circadian clock）广泛存在于几乎所有层级的生物体中，对于维持机体规律性生理与行为过程发挥着核心作用。经典的转录/翻译反馈环路模型将BMAL1（ARNTL）定义为哺乳动物系统中调控昼夜节律周期的核心驱动因子之一。本项整合多组学研究首次证实，在地塞米松同步化的Bmal1基因敲除（Bmal1-/-）小鼠成纤维细胞与肝组织切片中，多个转录本与蛋白质的表达水平呈现24小时昼夜节律振荡。有趣的是，即便缺失该核心生物钟基因，磷酸化蛋白质组（phosphoproteome）的特征谱仍呈现每日振荡现象。本研究结果显示，Bmal1基因敲除会显著改变小鼠肝脏蛋白质组的表达与磷酸化模式，且相较于野生型（wild-type）小鼠，其节律性候选分子的组成存在显著差异。因此，我们有理由强调：哺乳动物体内的昼夜节律并不会因核心生物钟基因的缺失而完全消失。将野生型与Bmal1-/- MSF细胞接种于多块六孔板中，使用含10%（体积比）HyClone III血清（Analab；货号：SH30109.03）、1/100体积Glutamax-I（Invitrogen；货号：35050-038）、1/100体积青霉素-链霉素（SIGMA；货号：P4333）及1/500体积MycoZap™（Lonza；货号：VZA-2022）的高糖杜氏改良伊格尔培养基（Dulbecco’s Modified Eagle Medium, DMEM）进行培养，直至细胞完全汇合（每个基因型、每个时间点设置3个生物学重复）。待细胞完全汇合后，使用终浓度为100nM的地塞米松（dexamethasone, DEX）处理细胞15分钟以完成节律同步[Balsalobre等，《科学》，2000]²⁹。随后将MSF细胞用37℃的磷酸盐缓冲液（PBS）洗涤三次，并置于HEPES缓冲培养基中培养：该培养基含1×DMEM粉剂（SIGMA；货号：D5030）、0.35mg/mL碳酸氢钠、5mg/mL葡萄糖、0.01M HEPES、10%（体积比）HyClone III血清、1/100体积Glutamax-I及1/500体积MycoZap™，pH值调节至7.3（使用HCl调整），渗透压调整为320 mOsm（使用NaCl调整），培养条件为37℃、全黑暗（DD）循环。地塞米松处理48小时后，连续三天每隔3或2小时收集一次MSF细胞，用于转录组学分析。