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.

Mammalian poly(ADP-ribose) polymerase (PARP) is a nuclear chromatin-associated protein with a molecular mass of 114 kDa that catalyzes the transfer of ADP-ribose units from NAD؉ to nuclear proteins that are located within chromatin. We report here the identification of a novel property of PARP as a modulator of nuclear receptor signalling. PARP bound directly to retinoid X receptors (RXR) and repressed liganddependent transcriptional activities mediated by heterodimers of RXR and thyroid hormone receptor (TR). The interacting surface is located in the DNA binding domain of RXR␣. Gel shift assays demonstrated that PARP bound to TR-RXR heterodimers on the response element. Overexpression of wild-type PARP selectively blocked nuclear receptor function in transient transfection experiments, while enzyme-defective mutant PARP did not show significant inhibition, suggesting that the essential role of poly(ADP-ribosyl) enzymatic activity is in gene regulation by nuclear receptors. Furthermore, PARP fused to the Gal4 DNA binding domain suppressed the transcriptional activity of the promoter harboring the Gal4 binding site. Thus, PARP has transcriptional repressor activity when recruited to the promoter. These results indicates that poly(ADPribosyl)ation is a negative cofactor in gene transcription, regulating a member of the nuclear receptor superfamily.Nuclear hormone receptors for steroids, retinoids, thyroid hormone, vitamin D 3 , and prostanoids comprise a large family of sequence-specific transcription factors. They play diverse roles in development, differentiation, and homeostasis (18) by modulating gene transcription. Retinoid X receptors (RXR) are members of a superfamily of nuclear hormone receptors and heterodimerize with a variety of other family members, including all-trans-retinoic acid receptor (RAR), thyroid hormone receptor (TR), and vitamin D receptor (VDR), indicating that RXR play a central role in ligand-dependent transcriptional regulation by nuclear receptors (15,35,37). These heterodimers bind to specific DNA sequences and directly regulate transcription of target genes in response to specific ligands. Nuclear receptors are thought to mediate their transcriptional effects in concert with coregulator proteins that modulate receptor interactions with components of the basal transcription machinery (3, 5, 6, 9, 11-14, 16, 30-32).The mechanism of transcriptional regulation by nuclear receptors has been a focus of intense study. The demonstration of direct interactions of receptors with basal transcription factors, such as TFIIB and TBP (19,20,22,23,34), suggests that liganded receptors may directly influence the function of the basal transcription machinery. However, these direct interaction models do not explain transcriptional squelching between receptors or the roles of receptor-associated cofactors. Negative transcriptional regulation by TR and RAR is mediated, in part, by their association with a class of silencing mediators termed 21,24,25,27). In addition, at least three distinct classes of re...

Peroxisome proliferators (e.g. clofibric acid) and thyroid hormone play an important role in the metabolism of lipids. These effectors display their action through their own nuclear receptors, peroxisome proliferatoractivated receptor (PPAR) and thyroid hormone receptor (TR). PPAR and TR are ligand-dependent, DNA binding, trans-acting transcriptional factors belonging to the erbA-related nuclear receptor superfamily. The present study focused on the convergence of the effectors on the peroxisome proliferator response element (PPRE). Transcriptional activation induced by PPAR through a PPRE was significantly suppressed by cotransfection of TR in transient transfection assays. The inhibition, however, was not affected by adding 3,5,3-triiodo-L-thyronine (T3). Furthermore, the inhibition was not observed in cells cotransfected with retinoic acid receptor or vitamin D3 receptor. The inhibitory action by TR was lost by introducing a mutation in the DNA binding domain of TR, indicating that competition for DNA binding is involved in the molecular basis of this functional interaction. Gel shift assays revealed that TRs, expressed in insect cells, specifically bound to the 32 P-labeled PPRE as heterodimers with the retinoid X receptor (RXR). Both PPAR and TR bind to PPRE, although only PPAR mediates transcriptional activation via PPRE. TR⅐RXR heterodimers are potential competitors with PPAR•RXR for binding to PPREs. It is concluded that PPAR-mediated gene expression is negatively controlled by TR at the level of PPAR binding to PPRE. We report here the novel action of thyroid hormone receptor in controlling gene expression through PPREs.Peroxisomes are cytoplasmic organelles that are important in mammalian lipid homeostasis (1). The structurally diverse xenobiotic peroxisome proliferators (PPs), 1 such as clofibrate, nafenopin, and WY-14,643 stimulate the proliferation of peroxisomes (2-5) and cause tumorigenic transformation of hepatic cells in rodents (6, 7). Some of these compounds have been used in man as hypolipidemic agents. PPs have been shown to induce peroxisomal and microsomal enzymes involved in lipid metabolism through activation of the peroxisome proliferatoractivated receptor (PPAR) (8, 9). The PPAR is a member of the nuclear receptor superfamily of ligand-dependent transcriptional factors and is structurally related to the subfamily of receptors that includes the thyroid hormone receptor (TR), retinoic acid receptor (RAR), and vitamin D3 receptor (VDR) (10). To date, three subtypes of PPARs have been identified in amphibians, rodents, and humans, PPAR␣,9,[11][12][13][14]. Further investigation revealed that natural fatty acids are also potent activators of PPAR␣ (14, 15), although no direct interaction of PPAR␣ with either PPs or fatty acids has been described so far. Recently, ligands for PPAR␥ have been identified that are potent inducers of adipogenesis in vivo. These include thiazolidine diones, a class of anti-diabetic drugs, and the arachidonic acid derivative 15-deoxy-D12, 14-prostaglandin J2 (16 ...

Abstract

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