Alterations of symbiosis between microbiota and intestinal epithelial cells (IEC) are associated with intestinal and systemic pathologies. Interactions between bacterial products (MAMPs) and Toll-like receptors (TLRs) are known to be mandatory for IEC homeostasis, but how TLRs may time homeostatic functions with circadian changes is unknown. Our functional and molecular dissections of the IEC circadian clock demonstrate that its integrity is required for microbiota-IEC dialog. In IEC, the antiphasic expression of the RORα activator and RevErbα repressor clock output regulators generates a circadian rhythmic TLR expression that converts the temporally arrhythmic microbiota signaling into circadian rhythmic JNK and IKKβ activities, which prevents RevErbα activation by PPARα that would disrupt the circadian clock. Moreover, through activation of AP1 and NF-κB, these activities, together with RORα and RevErbα, enable timing homeostatic functions of numerous genes with IEC circadian events. Interestingly, microbiota signaling deficiencies induce a prediabetic syndrome due to ileal corticosterone overproduction consequent to clock disruption.

The glucocorticoid (GC) receptor (GR), when liganded to GC, activates transcription through direct binding to simple (+)GRE DNA binding sequences (DBS). GC-induced direct repression via GR binding to complex "negative" GREs (nGREs) has been reported. However, GR-mediated transrepression was generally ascribed to indirect "tethered" interaction with other DNA-bound factors. We report that GC-induces direct transrepression via the binding of GR to simple DBS (IR nGREs) unrelated to (+)GRE. These DBS act on agonist-liganded GR, promoting the assembly of cis-acting GR-SMRT/NCoR repressing complexes. IR nGREs are present in over 1000 mouse/human ortholog genes, which are repressed by GC in vivo. Thus variations in the levels of a single ligand can coordinately turn genes on or off depending in their response element DBS, allowing an additional level of regulation in GR signaling. This mechanism suits GR signaling remarkably well, given that adrenal secretion of GC fluctuates in a circadian and stress-related fashion.

The light-entrained master central circadian clock (CC) located in the suprachiasmatic nucleus (SCN) not only controls the diurnal alternance of the active phase (the light period of the human lightdark cycle, but the mouse dark period) and the rest phase (the human dark period, but the mouse light period), but also synchronizes the ubiquitous peripheral CCs (PCCs) with these phases to maintain homeostasis. We recently elucidated in mice the molecular signals through which metabolic alterations induced on an unusual feeding schedule, taking place during the rest phase [i.e., restricted feeding (RF)], creates a 12-h PCC shift. Importantly, a previous study showed that the SCN CC is unaltered during RF, which creates a misalignment between the RF-shifted PCCs and the SCN CC-controlled phases of activity and rest. However, the molecular basis of SCN CC insensitivity to RF and its possible pathological consequences are mostly unknown. Here we deciphered, at the molecular level, how RF creates this misalignment. We demonstrate that the PPARα and glucagon receptors, the two instrumental transducers in the RF-induced shift of PCCs, are not expressed in the SCN, thereby preventing on RF a shift of the master SCN CC and creating the misalignment. Most importantly, this RF-induced misalignment leads to a misexpression (with respect to their normal physiological phase of expression) of numerous CC-controlled homeostatic genes, which in the long term generates in RF mice a number of metabolic pathologies including diabetes, obesity, and metabolic syndrome, which have been reported in humans engaged in shift work schedules.circadian clocks misalignment | shift work | diabetes | metabolic syndrome | mouse U nder physiological conditions, the light-entrained central master circadian clock (CC), which is located in the suprachiasmatic nucleus (SCN), synchronizes the ubiquitous peripheral CCs (PCCs) and generates a diurnal alternance of phases of activity and rest, both of which are at the origin of rhythmic variations of gene expression, which are essential to maintain metabolic and behavioral homeostasis (1-3). It is well established that shifting the feeding time in the mouse from the "active" to the "rest" phase [so-called restricted feeding (RF)] leads to a 12-h shift in the expression of PCC components (4). As the SCN CC is not affected during RF (4), this situation leads to a misalignment between the diurnal active and rest phases and the expression of PCC components. We recently unveiled in mice the origin and the identity of the molecular signals through which RF leads to this 12-h shift in the expression of PCC components (5). However, the molecular mechanisms that confer to the SCN CC an insensitivity to RF, as well as the consequences of the misalignment between the PCCs and the master SCN CC on homeostasis, are still largely unexplored (3, 6). In the present study, we elucidated, at the molecular level, how the SCN CC is protected against the RF-induced shift of PCCs, which is induced by metabolic alterations (5), a...

Each day, all organisms are subjected to changes in light intensity because of the Earth's rotation around its own axis. To anticipate this geo-physical variability, and to appropriately respond biochemically, most species, including mammals, have evolved an approximate 24-hour endogenous timing mechanism known as the circadian clock (CC). The 'clock' is self-sustained, cell autonomous and present in every cell type. At the core of the clock resides the CC-oscillator, an exquisitely crafted transcriptional-translational feedback system. Remarkably, components of the CC-oscillator not only maintain daily rhythmicity of their own synthesis, but also generate temporal variability in the expression levels of numerous target genes through transcriptional, post-transcriptional and post-translational mechanisms, thus, ensuring proper chronological coordination in the functioning of cells, tissues and organs, including the liver. Indeed, a variety of physiologically critical hepatic functions and cellular processes are CC-controlled. Thus, it is not surprising that modern lifestyle factors (e.g. travel and jet lag, night and rotating shift work), which force 'circadian misalignment', have emerged as major contributors to global health problems including obesity, non-alcoholic fatty liver disease and steatohepatitis. Herein, we provide an overview of the CC-dependent pathways which play critical roles in mediating several hepatic functions under physiological conditions, and whose deregulation is implicated in chronic liver diseases including non-alcoholic steatohepatitis and alcohol-related liver disease.

See Covering the Cover synopsis on 271. See editorial on page 300.

The HBx (X protein of hepatitis B virus) is a promiscuous transactivator implicated to play a key role in hepatocellular carcinoma. However, HBx-regulated molecular events leading to deregulation of cell cycle or establishment of a permissive environment for hepatocarcinogenesis are not fully understood. Our cell culture-based studies suggested that HBx had a profound effect on cell cycle progression even in the absence of serum. HBx presence led to an early and sustained level of cyclin-cdk2 complex during the cell cycle combined with increased protein kinase activity of cdk2 heralding an early proliferative signal. The increased cdk2 activity also led to an early proteasomal degradation of p27(Kip1) that could be reversed by HBx-specific RNA interference and blocked by a chemical inhibitor of cdk2 or the T187A mutant of p27. Further, our co-immunoprecipitation and in vitro binding studies with recombinant proteins suggested a direct interaction between HBx and the cyclin E/A-cdk2 complex. Interference with different signalling cascades known to be activated by HBx suggested a constitutive requirement of Src kinases for the association of HBx with these complexes. Notably, the HBx mutant that did not interact with cyclin E/A failed to destabilize p27(Kip1) or deregulate the cell cycle. Thus HBx appears to deregulate the cell cycle by interacting with the key cell cycle regulators independent of its well-established role in transactivation.

The molecular mechanisms underlying the events through which alterations in diurnal activities impinge on peripheral circadian clocks (PCCs), and reciprocally how the PCCs affect metabolism, thereby generating pathologies, are still poorly understood. Here, we deciphered how switching the diurnal feeding from the active to the rest phase, i.e., restricted feeding (RF), immediately creates a hypoinsulinemia during the active phase, which initiates a metabolic reprogramming by increasing FFA and glucagon levels. In turn, peroxisome proliferator-activated receptor alpha (PPARα) activation by free fatty acid (FFA), and cAMP response element-binding protein (CREB) activation by glucagon, lead to further metabolic alterations during the circadian active phase, as well as to aberrant activation of expression of the PCC components nuclear receptor subfamily 1, group D, member 1 (Nr1d1/RevErbα), Period (Per1 and Per2). Moreover, hypoinsulinemia leads to an increase in glycogen synthase kinase 3β (GSK3β) activity that, through phosphorylation, stabilizes and increases the level of the RevErbα protein during the active phase. This increase then leads to an untimely repression of expression of the genes containing a RORE DNA binding sequence (DBS), including the Bmal1 gene, thereby initiating in RF mice a 12-h PCC shift to which the CREB-mediated activation of Per1, Per2 by glucagon modestly contributes. We also show that the reported corticosterone extraproduction during the RF active phase reflects an adrenal aberrant activation of CREB signaling, which selectively delays the activation of the PPARα-RevErbα axis in muscle and heart and accounts for the retarded shift of their PCCs.shifted eating | shifted peripheral circadian clocks | metabolic alterations | RevErbα | PPARα P ioneering studies (1, 2) have established that switching the feeding time in mice from the "active" phase [dark period of the light-dark (L/D) cycle] to the "rest" phase (light period), i.e., restricted feeding (RF), overrides the suprachiasmatic nucleus (SCN) circadian clock (CC)-derived signals and acts as a "zeitgeber" for peripheral CCs (PCCs), leading to a 12-h shift in the time at which components of PCCs are expressed. Numerous studies have shown that under homeostatic conditions, the functions of PCCs and metabolism are tightly linked and that perturbations in their interactions leads to pathologies, e.g., obesity and metabolic syndrome (3-5).The identity of some of the molecular pathways that couple PCCs to metabolism are known (3-5), but it is largely unknown how environmental cues, e.g., altered feeding schedules, may directly perturb the expression of individual CC components (5-7), thereby leading to obesity and a metabolic syndrome-like pathology (5). Assuming that specific metabolic perturbations generated by switching the feeding time could selectively affect the time of expression of some of the CC core components, we looked for both metabolic and PCC alterations at early RF times. We report here a comprehensive temporal analysis, an...

ObjectiveHepatocellular carcinoma (HCC) is the fastest-growing cause of cancer-related mortality with chronic viral hepatitis and non-alcoholic steatohepatitis (NASH) as major aetiologies. Treatment options for HCC are unsatisfactory and chemopreventive approaches are absent. Chronic hepatitis C (CHC) results in epigenetic alterations driving HCC risk and persisting following cure. Here, we aimed to investigate epigenetic modifications as targets for liver cancer chemoprevention.DesignLiver tissues from patients with NASH and CHC were analysed by ChIP-Seq (H3K27ac) and RNA-Seq. The liver disease-specific epigenetic and transcriptional reprogramming in patients was modelled in a liver cell culture system. Perturbation studies combined with a targeted small molecule screen followed by in vivo and ex vivo validation were used to identify chromatin modifiers and readers for HCC chemoprevention.ResultsIn patients, CHC and NASH share similar epigenetic and transcriptomic modifications driving cancer risk. Using a cell-based system modelling epigenetic modifications in patients, we identified chromatin readers as targets to revert liver gene transcription driving clinical HCC risk. Proof-of-concept studies in a NASH-HCC mouse model showed that the pharmacological inhibition of chromatin reader bromodomain 4 inhibited liver disease progression and hepatocarcinogenesis by restoring transcriptional reprogramming of the genes that were epigenetically altered in patients.ConclusionOur results unravel the functional relevance of metabolic and virus-induced epigenetic alterations for pathogenesis of HCC development and identify chromatin readers as targets for chemoprevention in patients with chronic liver diseases.