Rev-erb is a ubiquitously expressed orphan nuclear receptor which functions as a constitutive transcriptional repressor and is expressed in vertebrates according to a robust circadian rhythm. We report here that two Rev-erb mRNA isoforms, namely Rev-erb 1 and Rev-erb 2, are generated through alternative promoter usage and that both show a circadian expression pattern in an in vitro system using serum-shocked fibroblasts. Both promoter regions P1 (Rev-erb 1) and P2 (Rev-erb 2) contain several E-box DNA sequences which function as response elements for the core circadian-clock components: CLOCK and BMAL1. The CLOCK-BMAL1 heterodimer stimulates the activity of both P1 and P2 promoters in transient transfection assay by 3-6-fold. This activation was inhibited by the overexpression of CRY1, a component of the negative limb of the circadian transcriptional loop. Critical E-box elements were mapped within both promoters. This regulation is conserved in vertebrates since we found that the CLOCK-BMAL1 heterodimer also regulates the zebrafish Rev-erb gene. In line with these data Rev-erb circadian expression was strongly impaired in the livers of Clock mutant mice and in the pineal glands of zebrafish embryos treated with Clock and Bmal1 antisense oligonucleotides. Together these data demonstrate that CLOCK is a critical regulator of Rev-erb circadian gene expression in evolutionarily distant vertebrates and suggest a role for Rev-erb in the circadian clock output.

In mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play a key role in inhibiting pluripotency-maintaining genes and activating some major actors of cell differentiation.To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with RAR/RXR genomic redistribution during the differentiation process. In-depth analysis of RAR/RXR binding sites occupancy dynamics and composition show that in undifferentiated cells, RAR/RXR interact with genomic regions characterized by binding of pluripotency-associated factors and high prevalence of the non-canonical DR0-containing RA response element. By contrast, in differentiated cells, RAR/RXR bound regions are enriched in functional Sox17 binding sites and are characterized with a higher frequency of the canonical DR5 motif. Our data offer an unprecedentedly detailed view on the action of RA in triggering pluripotent cell differentiation and demonstrate that RAR/RXR action is mediated via two different sets of regulatory regions tightly associated with cell differentiation status.

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...

Adiponectin is an adipocytokine that plays important roles in glucose and lipid homeostasis. Adiponectin binds to two types of transmembrane receptors: Adiponectin receptor (AdipoR) type 1 and 2. We isolated and characterized the two adiponectin genes (adiponectin A and B) and the three adiponectin receptors in zebrafish. In adult, adiponectin A is only detected in the kidney while adiponectin B mRNAs are widely expressed and are detected in the liver, adipose tissue, muscle, and brain. The receptors are found in many tissues such as the brain, gut, liver, adipose tissue, kidney, and ovary. Interestingly, we detect embryonic synexpression of all genes in the pharyngeal region. We observed that adiponectin B expression in adult liver is reduced while the expression of the receptors is increased in fasted fish. These data indicate that the upstream members of the Adiponectin pathway have complex expression patterns and are regulated by food deprivation in zebrafish.