Circadian Regulation of Diverse Gene Products Revealed by mRNA Expression Profiling of Synchronized Fibroblasts

Abstract

Genes under a 24-h regulation period may represent drug targets relevant to diseases involving circadian dysfunctions. As a testing model of the circadian clock system, we have used synchronized rat fibroblasts that are known to express at least six genes in a circadian fashion. We have determined the expression patterns of 9957 transcripts every 4 h over a total period of 76 h using high density oligonucleotide microarrays. The spectral analysis of our mRNA profiling data indicated that ϳ2% (85 genes) of all expressed genes followed a robust circadian pattern. We have confirmed the circadian expression of previously known clock or clockdriven genes, and we identified 81 novel circadian genes. The majority of the circadian-regulated gene products are known and are involved in diverse cellular functions. We have classified these circadian genes in seven clusters according to their phase of cycling. Our pathway analysis of the mRNA profiling data strongly suggests a direct link between circadian rhythm and cell cycle.The circadian rhythm allows organisms to anticipate daily environmental changes, and evolutionary conserved molecular clock and oscillator mechanisms have been observed in plants, fungi, and mammals (1). Circadian oscillators are generated by a small number of clock proteins that are regulated by selfsustained transcriptional-translational feedback loops, which drive the rhythmic expression of "clock-controlled" output genes. In mammals, the central pacemaker is located in the suprachiasmatic nucleus (SCN) 1 of the hypothalamus and is adjusted and entrained to the 24-h environmental light/dark cycle by photic stimuli via the retina. Surprisingly, confluent cultured fibroblast cells show an identical (2) circadian clock mechanism following synchronization with a 50% v/v serum shock treatment (3) and can be used as a simple model to study circadian gene expression. The discovery of a circadian clock in peripheral cells strongly suggests that this rhythmic mechanism controls basic cellular functions. So far the extent of gene expression following a circadian rhythm in cultured fibroblasts is still unknown, as well as its overall nature. We report on fibroblast experiments using high density oligonucleotide microarray expression profiling (GeneChip®, Affymetrix) to determine the expression levels of 9957 rat genes over a 76-h period after synchronization. EXPERIMENTAL PROCEDURESSample Preparation and Chip Hybridization-Rat 3Y1 embryonic fibroblasts (passage 30) (4) were grown in Dulbecco's modified Eagle's medium with 5% v/v fetal calf serum (Roche Molecular Biochemicals), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin (Life Technologies, Inc.) at 37°C in 7.5% CO 2 and were plated (in duplicate) on 15-cm dishes at 3 ϫ 10 6 cells/plate and grown in the same medium for 7 days. The cells were confluent for 4 days before serum synchronization. At this time (t ϭ 0 h), cells were treated with 50% v/v horse serum (Roche Molecular Biochemicals) for 2 h and then kept in serum-free Dulbecco's mo...

Analysis of daily and circadian gene expression in the rat pineal gland

“…In general, it appears that about 10 % of the genes are circadianly expressed in the liver, heart, and kidneys (Akhtar et al, 2002;Kita et al, 2002;Panda et al, 2002;Storch et al, 2002;Ueda et al, 2002). Interestingly, in the fibroblasts it has been reported that fewer than 100 genes (or about 2% of the genes present in these cells) are rhythmically expressed (Duffield et al, 2002;Grundschober et al, 2001). The fact that fibroblasts showed much less rhythmic genes expression than the SCN and/or other tissues, but are similar to what has been observed in the pineal (Humpries et al 2002, and this study) suggests that the reduction in the number of rhythmically regulated genes may be due to the reduction in metabolic and physiological activities in fibroblasts and pinealocytes in comparison with cells in other organs and tissues.…”

“…In the recent years, the DNA microarray technique has been successfully used to study circadian gene expression in the SCN, liver, heart, kidney, and fibroblasts (Grundschober et al, 2001;Akhtar et al, 2002;Duffield et al, 2002;Kita et al, 2002;Panda et al, 2002;Storch et al, 2002;Ueda et al, 2002). The picture that is emerging from these studies indicates that in each tissue or organ a certain number of genes (approximately 2-10 %) are under circadian control.…”

Persistent twenty-four hour changes in liver and bone marrow despite suprachiasmatic nuclei ablation in mice

“…The ability of peripheral oscillators to maintain circadian rhythms in the absence of a central pacemaker has been further demonstrated as cultured fibroblasts displayed sustained rhythms in gene expression (3,17) and cultured murine bone marrow progenitors displayed sustained rhythms in response to hematopoietic growth factors (8). Nevertheless, these rhythms tended to fade away unless a stimulation (glucocorticoid, serum shock) or an environmental 24-h cycle (temperature, light) was introduced after a few days in culture, suggesting the need for a regular resetting of free-running peripheral oscillators in order for their coordination to be maintained (2,3,9,21,49).…”

“…We selected cell cycle phase distribution in bone marrow and dihydropyrimidine dehydrogenase (DPD) mRNA expression in liver as determinants of 5-fluorouracil tolerability (17,40,45,51) and reduced glutathione (GSH) in liver as a determinant of platinum complex tolerability (4,6,7,28).…”

Pharmacological modulators of the circadian clock as potential therapeutic drugs

“…Components of a molecular clock underlying rhythmic gene expression have been characterized in neurons of the suprachiasmatic nucleus (SCN) [23,24], in liver [23][24][25][26][27], heart [26], synchronized fibroblasts [28], adipose tissue [29], adrenal gland [30], skeletal muscle [31], pituitary [32] and pineal [33] glands, calvarial bone [34], as well as in epithelial cells of lung [35,36] and gastrointestinal tract [37][38][39]. In order to ensure proper temporal organization in metabolism and behavior at the level of an organism, the multiple tissue-specific transcriptional oscillations have to be synchronized with the natural 24-hr cycle.…”