The circadian clock acts at the genomic level to coordinate internal behavioral and physiologic rhythms via the CLOCK-BMAL transcriptional heterodimer. Although the nuclear receptors REV-ERBα and β have been proposed to form an accessory feedback loop that contributes to clock function1,2, their precise roles and importance remain unresolved. To establish their regulatory potential we generated comparative cistromes of both REV-ERB isoforms, which revealed shared recognition at over 50% of their total sites and extensive overlap with the master circadian regulator BMAL1. While Rev-erbα has been shown to directly regulate Bmal1 expression1,2, the cistromic analysis reveals a direct connection between Bmal1 and Rev-erbα and β regulatory circuits than previously suspected. Genes within the intersection of the BMAL1, REV-ERBα and REV-ERBβ cistromes are highly enriched for both clock and metabolic functions. As predicted by the cistromic analysis, dual depletion of Rev-erbα/β function by creating double-knockout mice (DKOs) profoundly disrupted circadian expression of core circadian clock and lipid homeostatic gene networks. As a result, DKOs show strikingly altered circadian wheel-running behavior and deregulated lipid metabolism. These data now ally Rev-erbα/β with Per, Cry and other components of the principal feedback loop that drives circadian expression and suggest a more integral mechanism for the coordination of circadian rhythm and metabolism.

The molecular chaperone heat shock protein 90 (Hsp90) and its accessory cochaperones function by facilitating the structural maturation and complex assembly of client proteins, including steroid hormone receptors and selected kinases. By promoting the activity and stability of these signaling proteins, Hsp90 has emerged as a critical modulator in cell signaling. Here, we present evidence that Hsp90 chaperone activity is regulated by reversible acetylation and controlled by the deacetylase HDAC6. We show that HDAC6 functions as an Hsp90 deacetylase. Inactivation of HDAC6 leads to Hsp90 hyperacetylation, its dissociation from an essential cochaperone, p23, and a loss of chaperone activity. In HDAC6-deficient cells, Hsp90-dependent maturation of the glucocorticoid receptor (GR) is compromised, resulting in GR defective in ligand binding, nuclear translocation, and transcriptional activation. Our results identify Hsp90 as a target of HDAC6 and suggest reversible acetylation as a unique mechanism that regulates Hsp90 chaperone complex activity.

contributed equally to this workThe tumor suppressor p53 is activated in response to many types of cellular and environmental insults via mechanisms involving post-translational modi®cation.Here we demonstrate that, unlike phosphorylation, p53 invariably undergoes acetylation in cells exposed to a variety of stress-inducing agents including hypoxia, anti-metabolites, nuclear export inhibitor and actinomycin D treatment. In vivo, p53 acetylation is mediated by the p300 and CBP acetyltransferases. Overexpression of either p300 or CBP, but not an acetyltransferase-de®cient mutant, ef®ciently induces speci®c p53 acetylation. In contrast, MDM2, a negative regulator of p53, actively suppresses p300/CBPmediated p53 acetylation in vivo and in vitro. This inhibitory activity of MDM2 on p53 acetylation is in turn abrogated by tumor suppressor p19 ARF , indicating that regulation of acetylation is a central target of the p53±MDM2±p19 ARF feedback loop. Functionally, inhibition of deacetylation promotes p53 stability, suggesting that acetylation plays a positive role in the accumulation of p53 protein in stress response. Our results provide evidence that p300/CBP-mediated acetylation may be a universal and critical modi®-cation for p53 function. Keywords: acetylation/CBP/MDM2/p300/p53 IntroductionThe tumor suppressor p53 plays a critical role in human cancer formation. In response to a variety of stress signals, often associated with the progression of neoplastic diseases, p53 becomes activated and induces cell cycle arrest and/or programmed cell death (apoptosis). By eliminating damaged and potentially dangerous cells that might otherwise become cancerous, p53 suppresses tumor formation. In unstressed cells, p53 is latent and is maintained at low levels by targeted degradation mediated by its negative regulator, MDM2 (reviewed in Freedman et al., 1999). The critical role of MDM2 in regulating p53 is best illustrated by a study carried out in mice where inactivation of p53 was shown to completely rescue the embryonic lethality caused by the loss of MDM2 function (Montes de Oca Luna et al., 1995). MDM2 counteracts p53 tumor suppressor activity by physically binding to p53 and suppressing its transcriptional activity. MDM2 also functions as the p53 ubiquitin ligase and triggers its degradation (reviewed in Freedman et al., 1999). This latter activity requires the Ring ®nger domain located at the C-terminus of MDM2 (Fang et al., 2000), and may also involve the acetyltransferase p300, which binds both MDM2 and p53 (Grossman et al., 1998). Therefore, MDM2 negatively regulates p53 by at least two independent mechanisms.The activation and stabilization of p53 are thought to be mediated by speci®c protein modi®cations, with phosphorylation being the major focus of earlier studies (reviewed in Giaccia and Kastan, 1998;Appella and Anderson, 2000). Although the exact functions of speci®c phosphorylation events remain controversial, evidence indicates that they probably contribute to both the stabilization and activation of p53. For ex...

The class II deacetylase histone deacetylase 4 (HDAC4) negatively regulates the transcription factor MEF2. HDAC4 is believed to repress MEF2 transcriptional activity by binding to MEF2 and catalyzing local histone deacetylation. Here we report that HDAC4 also controls MEF2 by a novel SUMO E3 ligase activity. We show that HDAC4 interacts with the SUMO E2 conjugating enzyme Ubc9 and is itself sumoylated. The overexpression of HDAC4 leads to prominent MEF2 sumoylation in vivo, whereas recombinant HDAC4 stimulates MEF2 sumoylation in a reconstituted system in vitro. Importantly, HDAC4 promotes sumoylation on a lysine residue that is also subject to acetylation by a MEF2 coactivator, the acetyltransferase CBP, suggesting a possible interplay between acetylation and sumoylation in regulating MEF2 activity. Indeed, MEF2 acetylation is correlated with MEF2 activation and dynamically induced upon muscle cell differentiation, while sumoylation inhibits MEF2 transcriptional activity. Unexpectedly, we found that HDAC4 does not function as a MEF2 deacetylase. Instead, the NAD ؉ -dependent deacetylase SIRT1 can potently induce MEF2 deacetylation. Our studies reveal a novel regulation of MEF2 transcriptional activity by two distinct classes of deacetylases that affect MEF2 sumoylation and acetylation.Precise temporal and spatial gene expression is critical for the execution of the differentiation program and other important biological processes. This tight regulation of gene expression is achieved by the interplay of transcriptional activation and repression controlled by transcription factors that recruit specific cofactor complexes capable of modifying histones and the local chromatin structure (reviewed in references 12 and 29). Among the cofactors that are involved in transcriptional repression, members of the histone deacetylase (HDAC) family are the most well characterized. Numerous studies have reported the recruitment of HDAC members by transcription factors to repress gene expression. Based on the long-established correlation between histone acetylation and gene transcription, it is generally thought that HDAC members repress gene transcription by catalyzing local histone deacetylation (reviewed in reference 21). However, whether this mechanism is universally applicable to HDAC-mediated transcriptional repression is not known.Among HDAC family members involved in transcriptional regulation, HDAC4 and HDAC5 are closely related and belong to a subfamily that also includes HDAC7 and HDAC9 (reviewed in reference 37). This subfamily of HDACs share several unique properties. First, they all contain the N-terminal noncatalytic MITR (MEF2-interacting transcription repressor) homology domain (37). Second, they are all regulated by phosphorylation-dependent cytoplasmic-nuclear trafficking (18,23,45). Third, and most importantly, they are all critical regulators of MEF2, a family of transcription factors important in muscle cell differentiation and apoptosis (reviewed in reference 24). In skeletal muscle cells, MEF2 members coll...

In C2C12 myoblasts, endogenous histone deacetylase HDAC4 shuttles between cytoplasmic and nuclear compartments, supporting the hypothesis that its subcellular localization is dynamically regulated. However, upon differentiation, this dynamic equilibrium is disturbed and we find that HDAC4 accumulates in the nuclei of myotubes, suggesting a positive role of nuclear HDAC4 in muscle differentiation. Consistent with the notion of regulation of HDAC4 intracellular trafficking, we reveal that HDAC4 contains a modular structure consisting of a C-terminal autonomous nuclear export domain, which, in conjunction with an internal regulatory domain responsive to calcium/calmodulin-dependent protein kinase IV (CaMKIV), determines its subcellular localization. CaMKIV phosphorylates HDAC4 in vitro and promotes its nuclear-cytoplasmic shuttling in vivo. However, although 14-3-3 binding of HDAC4 has been proposed to be important for its cytoplasmic retention, we find this interaction to be independent of CaMKIV. Rather, the HDAC4⅐14-3-3 complex exists in the nucleus and is required to confer CaMKIV responsiveness. Our results suggest that the subcellular localization of HDAC4 is regulated by sequential phosphorylation events. The first event is catalyzed by a yet to be identified protein kinase that promotes 14-3-3 binding, and the second event, involving protein kinases such as CaMKIV, leads to efficient nuclear export of the HDAC4⅐14-3-3 complex.Accumulating evidence indicates that active transcriptional repression is an important component of many physiological events regulated at the level of gene expression, including muscle differentiation (1). The repression of transcription is manifest at the level of chromatin structure where histone deacetylases (HDACs) 1 are recruited to deacetylate histones and create a repressive chromatin structure (reviewed in Ref.2). Of the ten human HDACs identified so far (3), 2 HDAC4 and its closely related family member HDAC5 have been specifically implicated in regulating muscle differentiation ((1) and see below).The functional link between HDAC4/5 and muscle differentiation was first uncovered by the cloning of MITR, a transcriptional repressor identified as an interactive partner for myocyte enhancer factor 2 (MEF-2) transcription factor family members, which are important for muscle differentiation (4). MITR shows extensive homology to the non-catalytic N terminus of HDAC4 and -5 (4). Indeed both HDAC4 and HDAC5 interact with MEF-2. It was reported that overexpression of HDAC4 or HDAC5 represses MEF-2 transcriptional activity (5) and suppresses C2C12 myoblast differentiation (1). It was also found that the HDAC4/5⅐MEF-2 interaction and the effect of this complex on muscle differentiation could be reversed by a constitutively active form of a calcium/calmodulin-dependent protein kinase (CaMK) (6). However, the mechanism by which CaMK regulates HDAC4 and HDAC5 is not entirely clear.When ectopically expressed, HDAC4 can be found in either the nucleus or cytoplasm whereas the closely relat...

A primary cause of disease progression in type 2 diabetes (T2D) is β cell dysfunction due to inflammatory stress and insulin resistance. However, preventing β cell exhaustion under diabetic conditions is a major therapeutic challenge. Here, we identify the vitamin D receptor (VDR) as a key modulator of inflammation and β cell survival. Alternative recognition of an acetylated lysine in VDR by bromodomain proteins BRD7 and BRD9 directs association to PBAF and BAF chromatin remodeling complexes, respectively. Mechanistically, ligand promotes VDR association with PBAF to effect genome-wide changes in chromatin accessibility and enhancer landscape, resulting in an anti-inflammatory response. Importantly, pharmacological inhibition of BRD9 promotes PBAF-VDR association to restore β cell function and ameliorate hyperglycemia in murine T2D models. These studies reveal an unrecognized VDR-dependent transcriptional program underpinning β cell survival and identifies the VDR:PBAF/BAF association as a potential therapeutic target for T2D.

Summary Defects in circadian rhythm influence physiology and behavior with implications for the treatment of sleep disorders, metabolic disease and cancer. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms underpinning amplitude is limited. We show here that REV-ERBα, a core inhibitory component of clock transcription, is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7. By relieving REV-ERBα-dependent repression, FBXW7 provides an unrecognized mechanism for enhancing the amplitude of clock gene transcription. Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of REV-ERBα is necessary for FBXW7 recognition. Moreover, targeted hepatic disruption of FBXW7 alters circadian expression of core clock genes and perturbs whole body lipid and glucose levels. This CDK1-FBXW7 pathway controlling REV-ERBα repression defines an unexpected molecular mechanism for re-engaging the positive transcriptional arm of the clock, as well as a potential route to manipulate clock amplitude via small molecule CDK1 inhibition.

Nuclear hormone receptors (NRs) regulate physiology by sensing lipophilic ligands and adapting cellular transcription appropriately. A growing understanding of the impact of circadian clocks on mammalian transcription has sparked interest in the interregulation of transcriptional programs. Mammalian clocks are based on a transcriptional feedback loop featuring the transcriptional activators circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), and transcriptional repressors cryptochrome (CRY) and period (PER). CRY1 and CRY2 bind independently of other core clock factors to many genomic sites, which are enriched for NR recognition motifs. Here we report that CRY1/2 serve as corepressors for many NRs, indicating a new facet of circadian control of NR-mediated regulation of metabolism and physiology, and specifically contribute to diurnal modulation of drug metabolism.