Franck Delaunay

Estrogen receptor β acts as a dominant regulator of estrogen signaling

The physiological effects of estrogens are mediated by two intracellular transcription factors, the estrogen receptors (ERs), that regulate transcription of target genes through binding to specific DNA target sequences. Here we describe alterations in cellular responses to different ER agonists and to the anti-estrogenic compound tamoxifen resulting from co-expression of the two ERs in transient co-transfection experiments. Our results demonstrate that ERβ can act as a negative or positive dominant regulator of ER activity. This is manifested through reduced transcriptional activity at low concentrations of estradiol (E2); increased antagonistic effects of tamoxifen on E2 stimulated activity; and enhanced agonistic action of the phytoestrogenic compound genistein. Furthermore, using chimeric proteins lacking the N-terminal activation function 1 (AF-1), we show that the differential responses of ERα and ERβ to different agonists and antagonists are primarily dictated by inherent differences in the C-terminal ligand-binding domains of the receptors, whereas the magnitude of transcriptional activity is influenced by ERα AF-1, but not ERβ AF-1. The ERα AF-1 activity appears to be modulated upon co-expression of both ERs. The alterations in transcriptional activity resulting from co-expression of ERα and ERβ are probably due to the formation of α/β heterodimeric complexes. This study demonstrates that co-localization and subsequent heterodimerization of ERα and ERβ may result in receptor activity distinct from that of ER homodimers.

Effects of Chronic Jet Lag on Tumor Progression in Mice

Frequent transmeridian flights or predominant work at night can increase cancer risk. Altered circadian rhythms also predict for poor survival in cancer patients, whereas physical destruction of the suprachiasmatic nuclei (SCN), the hypothalamic circadian pacemaker, accelerates tumor growth in mice. Here we tested the effect of functional disruption of circadian system on tumor progression in a novel experimental model of chronic jet lag. B6D2F1 mice were synchronized with 12 hours of light and 12 hours of darkness or underwent repeat 8-hour advances of the light/dark cycle every 2 days before inoculation of Glasgow osteosarcoma. The 24-hour changes were assessed for plasma corticosterone, clock protein mPER1 expression in the SCN, and mRNA expression of clock genes mPer2 and mRev-erbα in liver and tumor. Time series were analyzed by spectral analysis and/or Cosinor. Differences were compared with analysis of variance (ANOVA). The 24-hour rest/activity cycle was ablated, and the rhythms of body temperature, serum corticosterone, and mPER1 protein expression in the SCN were markedly altered in jet-lagged mice as compared with controls (ANOVA, P < 0.001 for corticosterone and P = 0.01 for mPER1). Tumor grew faster in the jet-lagged animals as compared with controls (ANOVA, P < 0.001), whereas exposure to constant light or darkness had no effect (ANOVA, P = 0.66 and P = 0.8, respectively). The expression of mPer2 and mRev-erbα mRNAs in controls showed significant circadian rhythms in the liver (P = 0.006 and P = 0.003, respectively, Cosinor) and in the tumor (P = 0.04 and P < 0.001). Both rhythms were suppressed in the liver (P = 0.2 and P = 0.1, respectively, Cosinor) and in the tumor (P = 0.5) of jet-lagged mice. Altered environmental conditions can disrupt circadian clock molecular coordination in peripheral organs including tumors and play a significant role in malignant progression.

The circadian clock component BMAL1 is a critical regulator of p21WAF1/CIP1 expression and hepatocyte proliferation.

Most living organisms show circadian (∼24 h) rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. Although circadian gating of mitosis has been reported for many years in some peripheral tissues, the underlying molecular mechanisms have remained poorly understood. Here we show that the cell cycle inhibitor p21WAF1/CIP1 is rhythmically expressed in mouse peripheral organs. This rhythmic pattern of mRNA and protein expression was recapitulated in vitro in serum-shocked differentiated skeletal muscle cells. p21WAF1/CIP1 circadian expression is dramatically increased and no longer rhythmic in clock-deficient Bmal1–/– knock-out mice. Biochemical and genetic data show that oscillation of p21WAF1/CIP1 gene transcription is regulated by the antagonistic activities of the orphan nuclear receptors REV-ERBα/β and RORα4/γ, which are core clock regulators. Importantly, p21WAF1/CIP1 overexpressing Bmal1–/– primary hepatocytes exhibit a decreased proliferation rate. This phenotype could be reversed using small interfering RNA-mediated knockdown of p21WAF1/CIP1. These data establish a novel molecular link between clock and cell cycle genes and suggest that the G1 progression phase is a target of the circadian clock during liver cell proliferation.

The nuclear receptor REV-ERBα is required for the daily balance of carbohydrate and lipid metabolism

Mutations of clock genes can lead to diabetes and obesity. REV‐ERBα, a nuclear receptor involved in the circadian clockwork, has been shown to control lipid metabolism. To gain insight into the role of REV‐ERBα in energy homeostasis in vivo, we explored daily metabolism of carbohydrates and lipids in chow‐fed, unfed, or high‐fat‐fed Rev‐erbα−/− mice and their wild‐type littermates. Chow‐fed Rev‐erbα−/− mice displayed increased adiposity (2.5‐fold) and mild hyperglycemia (∼10%) without insulin resistance. Indirect calorimetry indicates that chow‐fed Rev‐erbα−/− mice utilize more fatty acids during daytime. A 24‐h nonfeeding period in Rev‐erbα−/− animals favors further fatty acid mobilization at the expense of glycogen utilization and gluconeogenesis, without triggering hypoglycemia and hypothermia. High‐fat feeding in Rev‐erbα−/− mice amplified metabolic disturbances, including expression of lipogenic factors. Lipoprotein lipase (Lpl) gene, critical in lipid utilization/storage, is triggered in liver at night and constitutively up‐regulated (∼ 2‐fold) in muscle and adipose tissue of Rev‐erbα−/− mice. We show that CLOCK, up‐regulated (2‐fold) at night in Rev‐erbα−/− mice, can transactivate Lpl. Thus, overexpression of Lpl facilitates muscle fatty acid utilization and contributes to fat overload. This study demonstrates the importance of clock‐driven Lpl expression in energy balance and highlights circadian disruption as a potential cause for the metabolic syndrome.—Delezie, J., Dumont, S., Dardente, H., Oudart, H., Gréchez‐Cassiau, A., Klosen, P., Teboul, M., Delaunay, F., Pévet, P., Challet, E. The nuclear receptor REV‐ERBα is required for the daily balance of carbohydrate and lipid metabolism. FASEB J. 26, 3321–3335 (2012). www.fasebj.org

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

Significance In tissues such as bone marrow, intestinal mucosa, or regenerating liver, the daily rhythm of cell division is controlled by the cell’s circadian clock. Determining how this clock organizes important processes such as cell division, apoptosis, and DNA damage repair is key to understanding the links between circadian dysfunction and malignant cell proliferation. We show that in proliferating mouse fibroblasts there is more than one way in which the clock and cell cycle synchronize their oscillations and that one of them is the biological equivalent of the phase locking first discovered by Huygens in the 17th century when he coupled two clocks together. When phase-locked two coupled oscillators have a fixed relative phase and oscillate with a common frequency. 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.

Cancer Inhibition through Circadian Reprogramming of Tumor Transcriptome with Meal Timing

Circadian disruption accelerates cancer progression, whereas circadian reinforcement could halt it. Mice with P03 pancreatic adenocarcinoma (n = 77) were synchronized and fed ad libitum (AL) or with meal timing (MT) from Zeitgeber time (ZT) 2 to ZT6 with normal or fat diet. Tumor gene expression profiling was determined with DNA microarrays at endogenous circadian time (CT) 4 and CT16. Circadian mRNA expression patterns were determined for clock genes Rev-erbalpha, Per2, and Bmal1, cellular stress genes Hspa8 and Cirbp, and cyclin A2 gene Ccna2 in liver and tumor. The 24-hour patterns in telemetered rest-activity and body temperature and plasma corticosterone and insulin-like growth factor-I (IGF-I) were assessed. We showed that MT inhibited cancer growth by approximately 40% as compared with AL (P = 0.011) irrespective of calorie intake. Clock gene transcription remained arrhythmic in tumors irrespective of feeding schedule or diet. Yet, MT upregulated or downregulated the expression of 423 tumor genes, according to CT. Moreover, 36 genes involved in cellular stress, cell cycle, and metabolism were upregulated at one CT and downregulated 12 h apart. MT induced >10-fold circadian expression of Hspa8, Cirbp, and Ccna2 in tumors. Corticosterone or IGF-I patterns played no role in tumor growth inhibition. In contrast, MT consistently doubled the circadian amplitude of body temperature. Peak and trough respectively corresponded to peak expressions of Hspa8 and Cirbp in tumors. The reinforcement of the host circadian timing system with MT induced 24-hour rhythmic expression of critical genes in clock-deficient tumors, which translated into cancer growth inhibition. Targeting circadian clocks represents a novel potential challenge for cancer therapeutics.

Expression Levels of Estrogen Receptor β Are Modulated by Components of the Molecular Clock

ABSTRACT Circadian regulation of gene expression plays a major role in health and disease. The precise role of the circadian system remains to be clarified, but it is known that circadian proteins generate physiological rhythms in organisms by regulating clock-controlled target genes. The estrogen receptor beta (ERβ) is, together with ERα, a member of the nuclear receptor superfamily and a key mediator of estrogen action. Interestingly, recent studies show that disturbed circadian rhythmicity in humans can increase the risk of reproductive malfunctions, suggesting a link between the circadian system and ER-mediated transcription pathways. Here, we identify a novel level of regulation of estrogen signaling where ERβ, but not ERα, is controlled by circadian clock proteins. We show that ERβ mRNA levels fluctuate in different peripheral tissues following a robust circadian pattern, with a peak at the light-dark transition, which is maintained under free-running conditions. Interestingly, this oscillation is abolished in clock-deficient BMAL1 knockout mice. Circadian control of ERβ expression is exerted through a conserved E-box element in the ERβ promoter region that recruits circadian regulatory factors. Furthermore, using small interfering RNA-mediated knockdown assays, we show that the expression levels of the circadian regulatory factors directly influence estrogen signaling by regulating the intracellular levels of endogenous ERβ.

Kruppel-like factor KLF10 is a link between the circadian clock and metabolism in liver.

ABSTRACT The circadian timing system coordinates many aspects of mammalian physiology and behavior in synchrony with the external light/dark cycle. These rhythms are driven by endogenous molecular clocks present in most body cells. Many clock outputs are transcriptional regulators, suggesting that clock genes primarily control physiology through indirect pathways. Here, we show that Krüppel-like factor 10 (KLF10) displays a robust circadian expression pattern in wild-type mouse liver but not in clock-deficient Bmal1 knockout mice. Consistently, the Klf10 promoter recruited the BMAL1 core clock protein and was transactivated by the CLOCK-BMAL1 heterodimer through a conserved E-box response element. Profiling the liver transcriptome from Klf10−/− mice identified 158 regulated genes with significant enrichment for transcripts involved in lipid and carbohydrate metabolism. Importantly, approximately 56% of these metabolic genes are clock controlled. Male Klf10−/− mice displayed postprandial and fasting hyperglycemia, a phenotype accompanied by a significant time-of-day-dependent upregulation of the gluconeogenic gene Pepck and increased hepatic glucose production. Consistently, functional data showed that the proximal Pepck promoter is repressed directly by KLF10. Klf10−/− females were normoglycemic but displayed higher plasma triglycerides. Correspondingly, rhythmic gene expression of components of the lipogenic pathway, including Srebp1c, Fas, and Elovl6, was altered in females. Collectively, these data establish KLF10 as a required circadian transcriptional regulator that links the molecular clock to energy metabolism in the liver.

Coupling between the Circadian Clock and Cell Cycle Oscillators: Implication for Healthy Cells and Malignant Growth

Uncontrolled cell proliferation is one of the key features leading to cancer. Seminal works in chronobiology have revealed that disruption of the circadian timing system in mice, either by surgical, genetic, or environmental manipulation, increased tumor development. In humans, shift work is a risk factor for cancer. Based on these observations, the link between the circadian clock and cell cycle has become intuitive. But despite identification of molecular connections between the two processes, the influence of the clock on the dynamics of the cell cycle has never been formally observed. Recently, two studies combining single live cell imaging with computational methods have shed light on robust coupling between clock and cell cycle oscillators. We recapitulate here these novel findings and integrate them with earlier results in both healthy and cancerous cells. Moreover, we propose that the cell cycle may be synchronized or slowed down through coupling with the circadian clock, which results in reduced tumor growth. More than ever, systems biology has become instrumental to understand the dynamic interaction between the circadian clock and cell cycle, which is critical in cellular coordination and for diseases such as cancer.

Differential regulation of Period 2 and Period 3 expression during development of the zebrafish circadian clock.

Circadian ( approximately 24h) clocks are endogenous time-keeping systems that drive the daily biological rhythms observed in most living organisms. The oscillation is generated by a transcriptional/translational autoregulatory feedback loop that is reset by external time cues such as the light/dark cycle and which in turn controls rhythms in physiology and behavior through downstream clock-controlled genes (Nature 417 (2002) 329). Genetic and biochemical analysis of Drosophila and mammalian clock genes has provided a comprehensive model for the molecular oscillator that generates these rhythms, but the ontogeny of this oscillator remains poorly understood. A circadian oscillator involving the clock genes Per3 and Rev-erb alpha was identified during early development in zebrafish (Science 289 (2000) 297). Here, we report the isolation of zebrafish Per2 and show the presence of a Per2 maternal mRNA in early embryos as for Per3. However, Per2 rhythmic expression occurs late during embryogenesis as compared to that of Per3. Furthermore, our data indicate that Per2 is not required during embryogenesis for the rhythmicity of physiological outputs such as melatonin synthesis. In addition, Per2 but not Per3 is constitutively expressed in the developing olfactory bulb and pituitary. This differential spatio-temporal expression patterns suggest specific roles for Per2 and Per3 in the establishment of the embryonic circadian system.