Histone deacetylase 9 promoter hypomethylation associated with adipocyte dysfunction is a statin-related metabolic effect.

BACKGROUND: Adipogenesis, the process whereby preadipocytes differentiate into mature adipocytes, is crucial for maintaining metabolic homeostasis. Cholesterol-lowering statins increase type 2 diabetes (T2D) risk possibly by affecting adipogenesis and insulin resistance but the (epi)genetic mechanisms involved are unknown. Here, we characterised the effects of statin treatment on adipocyte differentiation using in vitro human preadipocyte cell model to identify putative effective genes. RESULTS: Statin treatment during adipocyte differentiation caused a reduction in key genes involved in adipogenesis, such as ADIPOQ, GLUT4 and ABCG1. Using Illumina's Infinium '850K' Methylation EPIC array, we found a significant hypomethylation of cg14566882, located in the promoter of the histone deacetylase 9 (HDAC9) gene, in response to two types of statins (atorvastatin and mevastatin), which correlates with an increased HDAC9 mRNA expression. We confirmed that HDAC9 is a transcriptional repressor of the cholesterol efflux ABCG1 gene expression, which is epigenetically modified in obesity and prediabetic states. Thus, we assessed the putative impact of ABCG1 knockdown in mimicking the effect of statin in adipogenesis. ABCG1 KD reduced the expression of key genes involved in adipocyte differentiation and decreased insulin signalling and glucose uptake. In human blood cells from two cohorts, ABCG1 expression was impaired in response to statins, confirming that ABCG1 is targeted in vivo by these drugs. CONCLUSIONS: We identified an epigenetic link between adipogenesis and adipose tissue insulin resistance in the context of T2D risk associated with statin use, which has important implications as HDAC9 and ABCG1 are considered potential therapeutic targets for obesity and metabolic diseases.

The Circadian Clock Protein CRY1 Is a Negative Regulator of HIF-1α.

The circadian clock and the hypoxia-signaling pathway are regulated by an integrated interplay of positive and negative feedback limbs that incorporate energy homeostasis and carcinogenesis. We show that the negative circadian regulator CRY1 is also a negative regulator of hypoxia-inducible factor (HIF). Mechanistically, CRY1 interacts with the basic-helix-loop-helix domain of HIF-1α via its tail region. Subsequently, CRY1 reduces HIF-1α half-life and binding of HIFs to target gene promoters. This appeared to be CRY1 specific because genetic disruption of CRY1, but not CRY2, affected the hypoxia response. Furthermore, CRY1 deficiency could induce cellular HIF levels, proliferation, and migration, which could be reversed by CRISPR/Cas9- or short hairpin RNA-mediated HIF knockout. Altogether, our study provides a mechanistic explanation for genetic association studies linking a disruption of the circadian clock with hypoxia-associated processes such as carcinogenesis.

Loss-of-function mutations in ADCY3 cause monogenic severe obesity.

Study of monogenic forms of obesity has demonstrated the pivotal role of the central leptin-melanocortin pathway in controlling energy balance, appetite and body weight 1 . The majority of loss-of-function mutations (mostly recessive or co-dominant) have been identified in genes that are directly involved in leptin-melanocortin signaling. These genes, however, only explain obesity in <5% of cases, predominantly from outbred populations 2 . We previously showed that, in a consanguineous population in Pakistan, recessive mutations in known obesity-related genes explain ~30% of cases with severe obesity3-5. These data suggested that new monogenic forms of obesity could also be identified in this population. Here we identify and functionally characterize homozygous mutations in the ADCY3 gene encoding adenylate cyclase 3 in children with severe obesity from consanguineous Pakistani families, as well as compound heterozygous mutations in a severely obese child of European-American descent. These findings highlight ADCY3 as an important mediator of energy homeostasis and an attractive pharmacological target in the treatment of obesity.

Colorectal liver metastases with a disrupted circadian rhythm phase shift the peripheral clock in liver and kidney.

Circadian clock genes regulate 10-15% of the transcriptome, and might function as tumor suppressor genes. Relatively little is known about the circadian clock in tumors and its effect on surrounding healthy tissues. Therefore, we compared the 24-hr expression levels of key circadian clock genes in liver and kidney of healthy control mice with those of mice bearing C26 colorectal tumor metastases in the liver. Metastases were induced by injection of C26 colorectal carcinoma cells into the spleen. Subsequently, tumor, liver and kidney tissue was collected around the clock to compare circadian rhythmicity. Expression levels of five clock genes (Rev-Erbα, Per1, Per2, Bmal1 and Cry1) and three clock-controlled genes (CCGs; Dbp, p21 and Wee1) were determined by qRT-PCR. Liver and kidney tissue of healthy control mice showed normal 24-hr oscillations of all clock genes and CCGs, consistent with normal circadian rhythmicity. In colorectal liver metastases, however, 24-hr oscillations were completely absent for all clock genes and CCGs except Cry1. Liver and kidney tissue of tumor-bearing mice showed a shift in clock periodicity relative to control mice. In the liver we observed a phase advance, whereas in the kidney the phase was delayed. These data show that hepatic metastases of C26 colon carcinoma with a disrupted circadian rhythm phase shift liver and kidney tissue clocks, which strongly suggests a systemic effect on peripheral clocks. The possibility that tumors may modify peripheral clocks to escape from ordinary circadian rhythms and in this way contribute to fatigue and sleep disorders in cancer patients is discussed.

Phase locking and multiple oscillating attractors for the coupled mammalian clock and cell cycle.

Daily synchronous rhythms of cell division at the tissue or organism level are observed in many species and suggest that the circadian clock and cell cycle oscillators are coupled. For mammals, despite known mechanistic interactions, the effect of such coupling on clock and cell cycle progression, and hence its biological relevance, is not understood. In particular, we do not know how the temporal organization of cell division at the single-cell level produces this daily rhythm at the tissue level. Here we use multispectral imaging of single live cells, computational methods, and mathematical modeling to address this question in proliferating mouse fibroblasts. We show that in unsynchronized cells the cell cycle and circadian clock robustly phase lock each other in a 1:1 fashion so that in an expanding cell population the two oscillators oscillate in a synchronized way with a common frequency. Dexamethasone-induced synchronization reveals additional clock states. As well as the low-period phase-locked state there are distinct coexisting states with a significantly higher period clock. Cells transition to these states after dexamethasone synchronization. The temporal coordination of cell division by phase locking to the clock at a single-cell level has significant implications because disordered circadian function is increasingly being linked to the pathogenesis of many diseases, including cancer.

Mammalian TIMELESS is involved in period determination and DNA damage-dependent phase advancing of the circadian clock.

The transcription/translation feedback loop-based molecular oscillator underlying the generation of circadian gene expression is preserved in almost all organisms. Interestingly, the animal circadian clock proteins CRYPTOCHROME (CRY), PERIOD (PER) and TIMELESS (TIM) are strongly conserved at the amino acid level through evolution. Within this evolutionary frame, TIM represents a fascinating puzzle. While Drosophila contains two paralogs, dTIM and dTIM2, acting in clock/photoreception and chromosome integrity/photoreception respectively, mammals contain only one TIM homolog. Whereas TIM has been shown to regulate replication termination and cell cycle progression, its functional link to the circadian clock is under debate. Here we show that RNAi-mediated knockdown of TIM in NIH3T3 and U2OS cells shortens the period by 1 hour and diminishes DNA damage-dependent phase advancing. Furthermore, we reveal that the N-terminus of TIM is sufficient for interaction with CRY1 and CHK1 as well for homodimerization, and the C-terminus is necessary for nuclear localization. Interestingly, the long TIM isoform (l-TIM), but not the short (s-TIM), interacts with CRY1 and both proteins can reciprocally regulate their nuclear translocation in transiently transfected COS7 cells. Finally, we demonstrate that co-expression of PER2 abolishes the formation of the TIM/CRY1 complex through affinity binding competition to the C-terminal tail of CRY1. Notably, the presence of the latter protein region evolutionarily and structurally distinguishes mammalian from insect CRYs. We propose that the dynamic interaction between these three proteins could represent a post-translational aspect of the mammalian circadian clock that is important for its pace and adaption to external stimuli, such as DNA damage and/or light.

Histone methyltransferase MLL3 contributes to genome-scale circadian transcription.

Daily cyclical expression of thousands of genes in tissues such as the liver is orchestrated by the molecular circadian clock, the disruption of which is implicated in metabolic disorders and cancer. Although we understand much about the circadian transcription factors that can switch gene expression on and off, it is still unclear how global changes in rhythmic transcription are controlled at the genomic level. Here, we demonstrate circadian modification of an activating histone mark at a significant proportion of gene loci that undergo daily transcription, implicating widespread epigenetic modification as a key node regulated by the clockwork. Furthermore, we identify the histone-remodelling enzyme mixed lineage leukemia (MLL)3 as a clock-controlled factor that is able to directly and indirectly modulate over a hundred epigenetically targeted circadian "output" genes in the liver. Importantly, catalytic inactivation of the histone methyltransferase activity of MLL3 also severely compromises the oscillation of "core" clock gene promoters, including Bmal1, mCry1, mPer2, and Rev-erbα, suggesting that rhythmic histone methylation is vital for robust transcriptional oscillator function. This highlights a pathway by which the clockwork exerts genome-wide control over transcription, which is critical for sustaining temporal programming of tissue physiology.

Altered phase-relationship between peripheral oscillators and environmental time in Cry1 or Cry2 deficient mouse models for early and late chronotypes.

The mammalian circadian system is composed of a light-entrainable central clock in the suprachiasmatic nuclei (SCN) of the brain and peripheral clocks in virtually any other tissue. It allows the organism to optimally adjust metabolic, physiological and behavioral functions to the physiological needs it will have at specific time of the day. According to the resonance theory, such rhythms are only advantageous to an organism when in tune with the environment, which is illustrated by the adverse health effects originating from chronic circadian disruption by jetlag and shift work. Using short-period Cry1 and long-period Cry2 deficient mice as models for morningness and eveningness, respectively, we explored the effect of chronotype on the phase relationship between the central SCN clock and peripheral clocks in other organs. Whereas the behavioral activity patterns and circadian gene expression in the SCN of light-entrained Cry1(-/-) and Cry2(-/-) mice largely overlapped with that of wild type mice, expression of clock and clock controlled genes in liver, kidney, small intestine, and skin was shown to be markedly phase-advanced or phase-delayed, respectively. Likewise, circadian rhythms in urinary corticosterone were shown to display a significantly altered phase relationship similar to that of gene expression in peripheral tissues. We show that the daily dissonance between peripheral clocks and the environment did not affect the lifespan of Cry1(-/-) or Cry2(-/-) mice. Nonetheless, the phase-shifted peripheral clocks in light-entrained mice with morningness and eveningness-like phenotypes may have implications for personalized preventive and therapeutic (i.e. chronomodulation-based) health care for people with early and late chronotypes.

Flexible phase adjustment of circadian albumin D site-binding protein (DBP) gene expression by CRYPTOCHROME1.

The albumin D site-binding protein (DBP) governs circadian transcription of a number of hepatic detoxification and metabolic enzymes prior to the activity phase and subsequent food intake of mice. However, the behavior of mice is drastically affected by the photoperiod. Therefore, continuous adjustment of the phase of circadian Dbp expression is required in the liver. Here we describe a direct impact of CRYPTOCHROME1 (CRY1) on the phase of Dbp expression. Dbp and the nuclear receptor Rev-Erbalpha are circadian target genes of BMAL1 and CLOCK. Surprisingly, dynamic CRY1 binding to the Dbp promoter region delayed BMAL1 and CLOCK-mediated transcription of Dbp compared with Rev-Erbalpha. Extended presence of CRY1 in the nucleus enabled continuous uncoupling of the phase of Dbp from Rev-Erbalpha expression upon change from short to longer photoperiods. CRY1 thus maintained the peak of DBP accumulation close to the activity phase. In contrast, Rev-Erbalpha expression was phase-locked to the circadian oscillator and shaped by accumulation of its own gene product. Our data indicate that fine-tuning of circadian transcription in the liver is even more sophisticated than expected.

Impact of the circadian clock on in vitro genotoxic risk assessment assays.

Our society expects safety assessment for drugs, chemicals, cosmetics, and foods, which to date cannot be achieved without the use of laboratory animals. At the same time, society aims at refining, reducing, and (ultimately) replacing animal testing. As a consequence, much effort is taken to establish alternatives, such as toxicogenomics-based risk assessment assays on cultured cells and tissues. Evidently, the properties of cells in vitro will considerably differ from the in vivo situation. This review will discuss the impact of the circadian clock, an internal time keeping system that drives 24-h rhythms in metabolism, physiology and behavior, on in vitro genotoxic risk assessment. Our recent observation that DNA damaging agents can synchronize the circadian clock of individual cells in culture (and as a consequence the cyclic expression of clock-controlled genes, comprising up to 10% of the transcriptome) implies that the circadian clock should not be neglected when developing cell or tissue-based alternatives for chronic rodent toxicity assays.

Fragile X-related proteins regulate mammalian circadian behavioral rhythms.

Fragile X syndrome results from the absence of the fragile X mental retardation 1 (FMR1) gene product (FMRP). FMR1 has two paralogs in vertebrates: fragile X related gene 1 and 2 (FXR1 and FXR2). Here we show that Fmr1/Fxr2 double knockout (KO) and Fmr1 KO/Fxr2 heterozygous animals exhibit a loss of rhythmic activity in a light:dark (LD) cycle, and that Fmr1 or Fxr2 KO mice display a shorter free-running period of locomotor activity in total darkness (DD). Molecular analysis and in vitro electrophysiological studies suggest essentially normal function of cells in the suprachiasmatic nucleus (SCN) in Fmr1/Fxr2 double KO mice. However, the cyclical patterns of abundance of several core clock component messenger (m) RNAs are altered in the livers of double KO mice. Furthermore, FXR2P alone or FMRP and FXR2P together can increase PER1- or PER2-mediated BMAL1-Neuronal PAS2 (NPAS2) transcriptional activity in a dose-dependent manner. These data collectively demonstrate that FMR1 and FXR2 are required for the presence of rhythmic circadian behavior in mammals and suggest that this role may be relevant to sleep and other behavioral alterations observed in fragile X patients.

Phase resetting of the mammalian circadian clock by DNA damage.

To anticipate the momentum of the day, most organisms have developed an internal clock that drives circadian rhythms in metabolism, physiology, and behavior [1]. Recent studies indicate that cell-cycle progression and DNA-damage-response pathways are under circadian control [2-4]. Because circadian output processes can feed back into the clock, we investigated whether DNA damage affects the mammalian circadian clock. By using Rat-1 fibroblasts expressing an mPer2 promoter-driven luciferase reporter, we show that ionizing radiation exclusively phase advances circadian rhythms in a dose- and time-dependent manner. Notably, this in vitro finding translates to the living animal, because ionizing radiation also phase advanced behavioral rhythms in mice. The underlying mechanism involves ATM-mediated damage signaling as radiation-induced phase shifting was suppressed in fibroblasts from cancer-predisposed ataxia telangiectasia and Nijmegen breakage syndrome patients. Ionizing radiation-induced phase shifting depends on neither upregulation or downregulation of clock gene expression nor on de novo protein synthesis and, thus, differs mechanistically from dexamethasone- and forskolin-provoked clock resetting [5]. Interestingly, ultraviolet light and tert-butyl hydroperoxide also elicited a phase-advancing effect. Taken together, our data provide evidence that the mammalian circadian clock, like that of the lower eukaryote Neurospora[6], responds to DNA damage and suggest that clock resetting is a universal property of DNA damage.

Light entrainment of the mammalian circadian clock by a PRKCA-dependent posttranslational mechanism.

Light is the most potent stimulus for synchronizing endogenous circadian rhythms with external time. Photic clock resetting in mammals involves cAMP-responsive element binding protein (CREB)-mediated transcriptional activation of Period clock genes in the suprachiasmatic nuclei (SCN). Here we provide evidence for an additional photic input pathway to the mammalian circadian clock based on Protein Kinase C alpha (PRKCA). We found that Prkca-deficient mice show an impairment of light-mediated clock resetting. In the SCN of wild-type mice, light exposure evokes a transient interaction between PRKCA and PERIOD 2 (PER2) proteins that affects PER2 stability and nucleocytoplasmic distribution. These posttranslational events, together with CREB-mediated transcriptional regulation, are key factors in the molecular mechanism of photic clock resetting.

Manipulation of mammalian cell lines for circadian studies.

In mammals, the central circadian pacemaker resides in the hypothalamic suprachiasmatic nucleus (SCN), but circadian oscillators also exist in peripheral tissues. We have used wild-type and cryptochrome (mCry)-deficient mouse embryonic fibroblasts (MEFs) to demonstrate that the peripheral oscillator is mechanistically very similar to the oscillator in the SCN. Following serum shock activation, fibroblasts are able to sustain an SCN-like temporal expression profile of all known genes (i.e., antiphase oscillation of Bmal1 and Dbp genes), but are not able to produce oscillations in the absence of functional mCry genes. Remarkably, the analysis of mCry1-/- and mCry2-/- MEFs revealed the capacity to control period length in immortalized cell lines. Thus, the use of mammalian cells has become one of the most convenient methods for monitoring the molecular clock machinery and analyzing clock proteins at the functional/structural level. Here, we present the necessary protocols to (1) derive and culture a fibroblast cell line from wild-type and knockout mouse skin and (2) transfect cells at high efficiency to use in functional clock-protein studies.

Immunofluorescence analysis of circadian protein dynamics in cultured mammalian cells.

The timing of both entry and permanence of core-clock proteins in the nucleus is critical to maintain the correct pace of the clock mechanism. Several such proteins, namely CRYPTOCHROMEs (CRY), PERIODs (PER), and BMAL1, were recently shown to contain nuclear transport signals that facilitate their "shuttling" between the nucleus and the cytoplasm. This type of dynamic intracellular movement not only regulates protein localization, but also often affects functions by determining interactive partners and protein turnover. Because most clock genes have been identified by genetic screening in Drosophila and by gene knockdown in mammals, it is important to develop cellular techniques to study the structure-function and regulation of the corresponding proteins. Here we present working protocols for immunofluorescence studies of clock proteins in mammalian cultured cells. This technique allows the visualization in the cell of one or multiple proteins at the same time.

The BMAL1 C terminus regulates the circadian transcription feedback loop.

The circadian clock is driven by cell-autonomous transcription/translation feedback loops. The BMAL1 transcription factor is an indispensable component of the positive arm of this molecular oscillator in mammals. Here, we present a molecular genetic screening assay for mutant circadian clock proteins that is based on real-time circadian rhythm monitoring in cultured fibroblasts. By using this assay, we identified a domain in the extreme C terminus of BMAL1 that plays an essential role in the rhythmic control of E-box-mediated circadian transcription. Remarkably, the last 43 aa of BMAL1 are required for transcriptional activation, as well as for association with the circadian transcriptional repressor CRYPTOCHROME 1 (CRY1), depending on the coexistence of CLOCK protein. C-terminally truncated BMAL1 mutant proteins still associate with mPER2 (another protein of the negative feedback loop), suggesting that an additional repression mechanism may converge on the N terminus. Taken together, these results suggest that the C-terminal region of BMAL1 is involved in determining the balance between circadian transcriptional activation and suppression.

Functional evolution of the photolyase/cryptochrome protein family: importance of the C terminus of mammalian CRY1 for circadian core oscillator performance.

Cryptochromes (CRYs) are composed of a core domain with structural similarity to photolyase and a distinguishing C-terminal extension. While plant and fly CRYs act as circadian photoreceptors, using the C terminus for light signaling, mammalian CRY1 and CRY2 are integral components of the circadian oscillator. However, the function of their C terminus remains to be resolved. Here, we show that the C-terminal extension of mCRY1 harbors a nuclear localization signal and a putative coiled-coil domain that drive nuclear localization via two independent mechanisms and shift the equilibrium of shuttling mammalian CRY1 (mCRY1)/mammalian PER2 (mPER2) complexes towards the nucleus. Importantly, deletion of the complete C terminus prevents mCRY1 from repressing CLOCK/BMAL1-mediated transcription, whereas a plant photolyase gains this key clock function upon fusion to the last 100 amino acids of the mCRY1 core and its C terminus. Thus, the acquirement of different (species-specific) C termini during evolution not only functionally separated cryptochromes from photolyase but also caused diversity within the cryptochrome family.

Nucleocytoplasmic shuttling of clock proteins.

The mammalian circadian clock in the neurons of suprachiasmatic nuclei (SCN) in the brain and in cells of peripheral tissues is driven by a self-sustained molecular oscillator, which generates rhythmic gene expression with a periodicity of about 24?h (Reppert and Weaver, 2002). This molecular oscillator is composed of interacting positive and negative transcription/translation feedback loops in which the heterodimeric transcription activator CLOCK?BMAL1 promotes the transcription of E-box containing Cryptochrome (Cry1 and Cry2) and Period (Per1 and Per2) genes, as well as clock-controlled output genes. After being synthesized in the cytoplasm, CRY and PER proteins feedback in the nucleus to inhibit the transactivation mediated by positive regulators. The mPER2 protein acts at the interphase between positive and negative feedback loops by indirectly promoting the circadian transcription of the Bmal1 gene (through RevErbalpha) (Preitner et al., 2002; Shearman et al., 2000) and by interacting with mCRY proteins (Kume et al., 1999; Yagita et al., 2002) (for a detailed review, see Reppert and Weaver, 2002). In addition to cyclic transcription of clock genes, immunohistochemical studies on SCN neurons have revealed that mCRY1, mCRY2, mPER1, and mPER2 proteins undergo near synchronous circadian patterns of nuclear abundance (Field et al., 2000). The delay of approximately 6h between the peak in clock mRNA production and maximal levels of protein expression in the nucleus is believed to originate from posttranslational modification steps involving phosphorylation, ubiquitination, and proteosomal degradation. Thus, the timing of entry, as well as the residence time of core clock proteins into the nucleus, is a critical step in maintaining the correct pace of the circadian clock. Several clock proteins have been shown to contain nuclear export signal, sequences, on top of nuclear import signals, that facilitate their cellular trafficking (Chopin-Delannoy et al., 2003; Miyazaki et al., 2001; Yagita et al., 2002). This type of dynamic intracellular movement not only regulates protein localization, but also often affects functions by determining interactive partners and protein turnover. Because most of the clock genes have been identified by genetic screening in Drosophila and by gene knockdown in mammals, the development of innovative cellular techniques is essential in learning the structure-function and regulation of the corresponding proteins. This article discusses approaches, limitations, and applicable protocols to study the regulation of cellular localization of mammalian clock proteins, with a particular focus on mammalian CRY1 and PER2 proteins.

Nucleocytoplasmic shuttling and mCRY-dependent inhibition of ubiquitylation of the mPER2 clock protein.

The core oscillator generating circadian rhythms in eukaryotes is composed of transcription--translation-based autoregulatory feedback loops in which clock gene products negatively affect their own expression. A key step in this mechanism involves the periodic nuclear accumulation of clock proteins following their mRNA rhythms after approximately 6 h delay. Nuclear accumulation of mPER2 is promoted by mCRY proteins. Here, using COS7 cells and mCry1/mCry2 double mutant mouse embryonic fibroblasts transiently expressing GFP-tagged (mutant) mPER2, we show that the protein shuttles between nucleus and cytoplasm using functional nuclear localization and nuclear export sequences. Moreover, we provide evidence that mCRY proteins prevent ubiquitylation of mPER2 and subsequent degradation of the latter protein by the proteasome system. Interestingly, mPER2 in turn prevents ubiquitylation and degradation of mCRY proteins. On the basis of these data we propose a model in which shuttling mPER2 is ubiquitylated and degraded by the proteasome unless it is retained in the nucleus by mCRY proteins.