Identification of heme as the ligand for the orphan nuclear receptors REV-ERBalpha and REV-ERBbeta.
ABSTRACT: The nuclear receptors REV-ERBalpha (encoded by NR1D1) and REV-ERBbeta (NR1D2) have remained orphans owing to the lack of identified physiological ligands. Here we show that heme is a physiological ligand of both receptors. Heme associates with the ligand-binding domains of the REV-ERB receptors with a 1:1 stoichiometry and enhances the thermal stability of the proteins. Results from experiments of heme depletion in mammalian cells indicate that heme binding to REV-ERB causes the recruitment of the co-repressor NCoR, leading to repression of target genes including BMAL1 (official symbol ARNTL), an essential component of the circadian oscillator. Heme extends the known types of ligands used by the human nuclear receptor family beyond the endocrine hormones and dietary lipids described so far. Our results further indicate that heme regulation of REV-ERBs may link the control of metabolism and the mammalian clock.
Project description:Heme is a ligand for the human nuclear receptors (NR) REV-ERBalpha and REV-ERBbeta, which are transcriptional repressors that play important roles in circadian rhythm, lipid and glucose metabolism, and diseases such as diabetes, atherosclerosis, inflammation, and cancer. Here we show that transcription repression mediated by heme-bound REV-ERBs is reversed by the addition of nitric oxide (NO), and that the heme and NO effects are mediated by the C-terminal ligand-binding domain (LBD). A 1.9 A crystal structure of the REV-ERBbeta LBD, in complex with the oxidized Fe(III) form of heme, shows that heme binds in a prototypical NR ligand-binding pocket, where the heme iron is coordinately bound by histidine 568 and cysteine 384. Under reducing conditions, spectroscopic studies of the heme-REV-ERBbeta complex reveal that the Fe(II) form of the LBD transitions between penta-coordinated and hexa-coordinated structural states, neither of which possess the Cys384 bond observed in the oxidized state. In addition, the Fe(II) LBD is also able to bind either NO or CO, revealing a total of at least six structural states of the protein. The binding of known co-repressors is shown to be highly dependent upon these various liganded states. REV-ERBs are thus highly dynamic receptors that are responsive not only to heme, but also to redox and gas. Taken together, these findings suggest new mechanisms for the systemic coordination of molecular clocks and metabolism. They also raise the possibility for gas-based therapies for the many disorders associated with REV-ERB biological functions.
Project description:Repression of gene transcription by the nuclear receptor Rev-erbalpha plays an integral role in the core molecular circadian clock. We report the crystal structure of a nuclear receptor-co-repressor (N-CoR) interaction domain 1 (ID1) peptide bound to truncated human Rev-erbalpha ligand-binding domain (LBD). The ID1 peptide forms an unprecedented antiparallel beta-sheet with Rev-erbalpha, as well as an alpha-helix similar to that seen in nuclear receptor ID2 crystal structures but out of register by four residues. Comparison with the structure of Rev-erbbeta bound to heme indicates that ID1 peptide and heme induce substantially different conformational changes in the LBD. Although heme is involved in Rev-erb repression, the structure suggests that Rev-erbalpha could also mediate repression via ID1 binding in the absence of heme. The previously uncharacterized secondary structure induced by ID1 peptide binding advances our understanding of nuclear receptor-co-repressor interactions.
Project description:Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERB? (NR1D1) and REV-ERB? (NR1D2), but how heme regulates REV-ERB activity remains unclear. Cellular studies indicate that heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription. However, fluorescence-based biochemical assays suggest that heme displaces NCoR; here, we show that this is due to a heme-dependent artifact. Using ITC and NMR spectroscopy, we show that heme binding remodels the thermodynamic interaction profile of NCoR receptor interaction domain (RID) binding to REV-ERB? ligand-binding domain (LBD). We solved two crystal structures of REV-ERB? LBD cobound to heme and NCoR peptides, revealing the heme-dependent NCoR binding mode. ITC and chemical cross-linking mass spectrometry reveals a 2:1 LBD:RID stoichiometry, consistent with cellular studies showing that NCoR-dependent repression of REV-ERB transcription occurs on dimeric DNA response elements. Our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.
Project description:The mammalian circadian clockwork is composed of a core PER/CRY feedback loop and additional interlocking loops. In particular, the ROR/REV/Bmal1 loop, consisting of ROR activators and REV-ERB repressors that regulate Bmal1 expression, is thought to "stabilize" core clock function. However, due to functional redundancy and pleiotropic effects of gene deletions, the role of the ROR/REV/Bmal1 loop has not been accurately defined. In this study, we examined cell-autonomous circadian oscillations using combined gene knockout and RNA interference and demonstrated that REV-ERBalpha and beta are functionally redundant and are required for rhythmic Bmal1 expression. In contrast, the RORs contribute to Bmal1 amplitude but are dispensable for Bmal1 rhythm. We provide direct in vivo genetic evidence that the REV-ERBs also participate in combinatorial regulation of Cry1 and Rorc expression, leading to their phase-delay relative to Rev-erbalpha. Thus, the REV-ERBs play a more prominent role than the RORs in the basic clock mechanism. The cellular genetic approach permitted testing of the robustness of the intracellular core clock function. We showed that cells deficient in both REV-ERBalpha and beta function, or those expressing constitutive BMAL1, were still able to generate and maintain normal Per2 rhythmicity. Our findings thus underscore the resilience of the intracellular clock mechanism and provide important insights into the transcriptional topologies underlying the circadian clock. Since REV-ERB function and Bmal1 mRNA/protein cycling are not necessary for basic clock function, we propose that the major role of the ROR/REV/Bmal1 loop and its constituents is to control rhythmic transcription of clock output genes.
Project description:REV-ERB? and REV-ERB? are members of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors that play important roles in the regulation of circadian physiology, metabolism, and immune function. Although the REV-ERBs were originally characterized as orphan receptors, recent studies have demonstrated that they function as receptors for heme. Here, we demonstrate that cobalt protoporphyrin IX (CoPP) and zinc protoporphyrin IX (ZnPP) are ligands that bind directly to the REV-ERBs. However, instead of mimicking the agonist action of heme, CoPP and ZnPP function as antagonists of REV-ERB function. This was unexpected because the only distinction between these ligands is the metal ion that is coordinated. To understand the structural basis by which REV-ERB? can differentiate between a porphyrin agonist and antagonist, we characterized the interaction between REV-ERB? with heme, CoPP, and ZnPP using biochemical and structural approaches, including x-ray crystallography and NMR. The crystal structure of CoPP-bound REV-ERB? indicates only minor conformational changes induced by CoPP compared with heme, including the porphyrin ring of CoPP, which adopts a planar conformation as opposed to the puckered conformation observed in the heme-bound REV-ERB? crystal structure. Thus, subtle changes in the porphyrin metal center and ring conformation may influence the agonist versus antagonist action of porphyrins and when considered with other studies suggest that gas binding to the iron metal center heme may drive alterations in REV-ERB activity.
Project description:Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), but how heme regulates REV-ERB activity remains unclear. While cellular studies indicate heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription, fluorescence-based biochemical assays and crystal structures suggest that heme displaces NCoR. Here, we found that heme artifactually influences detection of NCoR interaction in fluorescence-based assays. Using fluorescence-independent methods, including isothermal titration calorimetry, NMR spectroscopy, and XL-MS, we determined that heme remodels the thermodynamic profile of NCoR binding to REV-ERBβ ligand-binding domain (LBD) and directly increases LBD binding affinity for an NCoR interaction motif. We further report two crystal structures of REV-ERBβ LBD cobound to heme and NCoR peptides, which reveal the heme-dependent NCoR binding mode. By resolving previous contradictory biochemical, structural, and cellular studies, our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.
Project description:The circadian clock regulates behavioural and physiological processes in a 24-h cycle. The nuclear receptors REV-ERB? and REV-ERB? are involved in the cell-autonomous circadian transcriptional/translational feedback loops as transcriptional repressors. A number of studies have also demonstrated a pivotal role of REV-ERBs in regulation of metabolic, neuronal, and inflammatory functions including bile acid metabolism, lipid metabolism, and production of inflammatory cytokines. Given the multifunctional role of REV-ERBs, it is important to elucidate the mechanism through which REV-ERBs exert their functions. To this end, we established a Rev-erb?/Rev-erb? double-knockout mouse embryonic stem (ES) cell model and analyzed the circadian clock and clock-controlled output gene expressions. A comprehensive mRNA-seq analysis revealed that the double knockout of both Rev-erb? and Rev-erb? does not abrogate expression rhythms of E-box-regulated core clock genes but drastically changes a diverse set of other rhythmically-expressed output genes. Of note, REV-ERB?/? deficiency does not compromise circadian expression rhythms of PER2, while REV-ERB target genes, Bmal1 and Npas2, are significantly upregulated. This study highlight the relevance of REV-ERBs as pivotal output mediators of the mammalian circadian clock.
Project description:The circadian clock imposes daily rhythms in cell proliferation, metabolism, inflammation and DNA damage response. Perturbations of these processes are hallmarks of cancer and chronic circadian rhythm disruption predisposes individuals to tumour development. This raises the hypothesis that pharmacological modulation of the circadian machinery may be an effective therapeutic strategy for combating cancer. REV-ERBs, the nuclear hormone receptors REV-ERB? (also known as NR1D1) and REV-ERB? (also known as NR1D2), are essential components of the circadian clock. Here we show that two agonists of REV-ERBs-SR9009 and SR9011-are specifically lethal to cancer cells and oncogene-induced senescent cells, including melanocytic naevi, and have no effect on the viability of normal cells or tissues. The anticancer activity of SR9009 and SR9011 affects a number of oncogenic drivers (such as HRAS, BRAF, PIK3CA and others) and persists in the absence of p53 and under hypoxic conditions. The regulation of autophagy and de novo lipogenesis by SR9009 and SR9011 has a critical role in evoking an apoptotic response in malignant cells. Notably, the selective anticancer properties of these REV-ERB agonists impair glioblastoma growth in vivo and improve survival without causing overt toxicity in mice. These results indicate that pharmacological modulation of circadian regulators is an effective antitumour strategy, identifying a class of anticancer agents with a wide therapeutic window. We propose that REV-ERB agonists are inhibitors of autophagy and de novo lipogenesis, with selective activity towards malignant and benign neoplasms.
Project description:Rev-erb? and ? are nuclear receptors that function as transcriptional repressors of genes involved in regulating circadian rhythms, glucose, and cholesterol metabolism and the inflammatory response. Given these key functions, Rev-erbs are important drug targets for treatment of a number of human pathologies, including cancer, heart disease, and type II diabetes. Transcriptional repression by the Rev-erbs involves direct competition with transcriptional activators for target sites, but also recruitment by the Rev-erbs of the NCoR corepressor protein. Interestingly, Rev-erbs do not appear to interact functionally with a very similar corepressor, Smrt. Transcriptional repression by Rev-erbs is thought to occur in response to the binding of heme, although structural, and ligand binding studies in vitro show that heme and corepressor binding are antagonistic. We carried out systematic studies of the ligand and corepressor interactions to address the molecular basis for corepressor specificity and the energetic consequences of ligand binding using a variety of biophysical approaches. Highly quantitative fluorescence anisotropy assays in competition mode revealed that the Rev-erb specificity for the NCoR corepressor lies in the first two residues of the ?-strand in Interaction Domain 1 of NCoR. Our studies confirmed and quantitated the strong antagonism of heme and corepressor binding and significant stabilization of the corepressor complex by a synthetic ligand in vitro. We propose a model which reconciles the contradictory observations concerning the effects of heme binding in vitro and in live cells.
Project description:The nuclear receptor Rev-erb? regulates circadian rhythm and metabolism, but its effects are modest and it has been considered to be a secondary regulator of the cell-autonomous clock. Here we report that depletion of Rev-erb? together with closely related Rev-erb? has dramatic effects on the cell-autonomous clock as well as hepatic lipid metabolism. Mouse embryonic fibroblasts were rendered arrhythmic by depletion of both Rev-erbs. In mouse livers, Rev-erb? mRNA and protein levels oscillate with a diurnal pattern similar to that of Rev-erb?, and both Rev-erbs are recruited to a remarkably similar set of binding sites across the genome, enriched near metabolic genes. Depletion of both Rev-erbs in liver synergistically derepresses several metabolic genes as well as genes that control the positive limb of the molecular clock. Moreover, deficiency of both Rev-erbs causes marked hepatic steatosis, in contrast to relatively subtle changes upon loss of either subtype alone. These findings establish the two Rev-erbs as major regulators of both clock function and metabolism, displaying a level of subtype collaboration that is unusual among nuclear receptors but common among core clock proteins, protecting the organism from major perturbations in circadian and metabolic physiology.