Ruprecht-Karls-Universität Heidelberg
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Circadian Clock Biology

Prof. Dr. Nick Foulkes

 
Cuesta IH(1), Lahiri K, Lopez-Olmeda JF, Loosli F, Foulkes NS, Vallone D. (2014). Differential maturation of rhythmic clock gene expression during early development in medaka (Oryzias latipes). Chronobiol Int. 31(4):468-78.
Abstract
One key challenge for the field of chronobiology is to identify how circadian clock function emerges during early embryonic development. Teleosts such as the zebrafish are ideal models for studying circadian clock ontogeny since the entire process of development occurs ex utero in an optically transparent chorion. Medaka (Oryzias latipes) represents another powerful fish model for exploring early clock function with, like the zebrafish, many tools available for detailed genetic analysis. However, to date there have been no reports documenting circadian clock gene expression during medaka development. Here we have characterized the expression of key clock genes in various developmental stages and in adult tissues of medaka. As previously reported for other fish, light dark cycles are required for the emergence of clock gene expression rhythms in this species. While rhythmic expression of per and cry genes is detected very early during development and seems to be light driven, rhythmic clock and bmal expression appears much later around hatching time. Furthermore, the maturation of clock function seems to correlate with the appearance of rhythmic expression of these positive elements of the clock feedback loop. By accelerating development through elevated temperatures or by artificially removing the chorion, we show an earlier onset of rhythmicity in clock and bmal expression. Thus, differential maturation of key elements of the medaka clock mechanism depends on the developmental stage and the presence of the chorion.
Ben-Moshe Z(1), Alon S, Mracek P, Faigenbloom L, Tovin A, Vatine GD, Eisenberg E, Foulkes NS, Gothilf Y. (2014). The light-induced transcriptome of the zebrafish pineal gland reveals complex regulation of the circadian clockwork by light. Nucleic Acids Res. 42(6):3750-67.
Abstract
Light constitutes a primary signal whereby endogenous circadian clocks are synchronized ('entrained') with the day/night cycle. The molecular mechanisms underlying this vital process are known to require gene activation, yet are incompletely understood. Here, the light-induced transcriptome in the zebrafish central clock organ, the pineal gland, was characterized by messenger RNA (mRNA) sequencing (mRNA-seq) and microarray analyses, resulting in the identification of multiple light-induced mRNAs. Interestingly, a considerable portion of the molecular clock (14 genes) is light-induced in the pineal gland. Four of these genes, encoding the transcription factors dec1, reverbb1, e4bp4-5 and e4bp4-6, differentially affected clock- and light-regulated promoter activation, suggesting that light-input is conveyed to the core clock machinery via diverse mechanisms. Moreover, we show that dec1, as well as the core clock gene per2, is essential for light-entrainment of rhythmic locomotor activity in zebrafish larvae. Additionally, we used microRNA (miRNA) sequencing (miR-seq) and identified pineal-enhanced and light-induced miRNAs. One such miRNA, miR-183, is shown to downregulate e4bp4-6 mRNA through a 3'UTR target site, and importantly, to regulate the rhythmic mRNA levels of aanat2, the key enzyme in melatonin synthesis. Together, this genome-wide approach and functional characterization of light-induced factors indicate a multi-level regulation of the circadian clockwork by light.
Villamizar N(1), Vera LM, Foulkes NS, Sánchez-Vázquez FJ. (2013). Effect of lighting conditions on zebrafish growth and development. Zebrafish. 11(2):173-81.
Abstract
Abstract In the underwater environment, the properties of light (intensity and spectrum) change rapidly with depth and water quality. In this article, we have described how and to what extent lighting conditions can influence the development, growth, and survival of zebrafish. Fertilized eggs and the corresponding larvae were exposed to different visible light wavelengths (violet, blue, green, yellow, red, and white) in a 12-h light-12-h dark (LD) cycle until 30 days posthatching (dph), when the expression of morphometric parameters and growth (igf1a, igf2a)- and stress-related (crh and pomca) genes were examined. Another group of larvae was raised under constant darkness (DD) until 5 or 10 dph, after which they were transferred to a LD of white light. A third group remained under DD to investigate the effects of light deprivation upon zebrafish development. The results revealed that the hatching rate was highest under blue and violet light, while total length at 30 dph was greatest under blue, white, and violet light. Red light led to reduced feeding activity and poor survival (100% mortality). Larvae raised under constant white light (LL) showed a higher proportion of malformations, as did larvae raised under LD violet light. The expression of growth and stress factors was upregulated in the violet (igf1a, igf2a, pomca, and chr) and blue (igf2a) groups, which is consistent with the higher growth recorded and the higher proportion of malformations detected under the violet light. All larvae kept under DD died before 18 dph, but the survival rates improved in larvae transferred to LD at 5 dph and at 10 dph. In summary, these findings revealed that lighting conditions are crucial factors influencing zebrafish larval development and growth.
Mracek P(1), Pagano C, Fröhlich N, Idda ML, Cuesta IH, Lopez-Olmeda JF, Sánchez-Vázquez FJ, Vallone D, Foulkes NS. (2013). ERK Signaling Regulates Light-Induced Gene Expression via D-Box Enhancers in a Differential, Wavelength-Dependent Manner. PLoS One. 8(6):e67858. Print 2013.
Abstract
The day-night and seasonal cycles are dominated by regular changes in the intensity as well as spectral composition of sunlight. In aquatic environments the spectrum of sunlight is also strongly affected by the depth and quality of water. During evolution, organisms have adopted various key strategies in order to adapt to these changes, including the development of clocks and photoreceptor mechanisms. These mechanisms enable the detection and anticipation of regular changes in lighting conditions and thereby direct an appropriate physiological response. In teleosts, a growing body of evidence points to most cell types possessing complex photoreceptive systems. However, our understanding of precisely how these systems are regulated and in turn dictate changes in gene expression remains incomplete. In this manuscript we attempt to unravel this complexity by comparing the effects of two specific wavelengths of light upon signal transduction and gene expression regulatory mechanisms in zebrafish cells. We reveal a significant difference in the kinetics of light-induced gene expression upon blue and red light exposure. Importantly, both red and blue light-induced gene expression relies upon D-box enhancer promoter elements. Using pharmacological and genetic approaches we demonstrate that the ERK/MAPK pathway acts as a negative regulator of blue but not red light activated transcription. Thus, we reveal that D-box-driven gene expression is regulated via ERK/MAPK signaling in a strongly wavelength-dependent manner.
Cavodeassi F(1), Del Bene F, Fürthauer M, Grabher C, Herzog W, Lehtonen S, Linker C, Mercader N, Mikut R, Norton W, Strähle U, Tiso N, Foulkes NS. (2013). Report of the Second European Zebrafish Principal Investigator Meeting in Karlsruhe, Germany, March 21-24, 2012. Zebrafish. 10(1):119-23.
Abstract
The second European Zebrafish Principal Investigator (PI) Meeting was held in March, 2012, in Karlsruhe, Germany. It brought together PIs from all over Europe who work with fish models such as zebrafish and medaka to discuss their latest results, as well as to resolve strategic issues faced by this research community. Scientific discussion ranged from the development of new technologies for working with fish models to progress in various fields of research such as injury and repair, disease models, and cell polarity and dynamics. This meeting also marked the establishment of the European Zebrafish Resource Centre (EZRC) at Karlsruhe that in the future will serve as an important focus and community resource for zebrafish- and medaka-based research.
Elbaz I(1), Foulkes NS, Gothilf Y, Appelbaum L. (2013). Circadian clocks, rhythmic synaptic plasticity and the sleep-wake cycle in zebrafish. Front Neural Circuits. 7:9.
Abstract
The circadian clock and homeostatic processes are fundamental mechanisms that regulate sleep. Surprisingly, despite decades of research, we still do not know why we sleep. Intriguing hypotheses suggest that sleep regulates synaptic plasticity and consequently has a beneficial role in learning and memory. However, direct evidence is still limited and the molecular regulatory mechanisms remain unclear. The zebrafish provides a powerful vertebrate model system that enables simple genetic manipulation, imaging of neuronal circuits and synapses in living animals, and the monitoring of behavioral performance during day and night. Thus, the zebrafish has become an attractive model to study circadian and homeostatic processes that regulate sleep. Zebrafish clock- and sleep-related genes have been cloned, neuronal circuits that exhibit circadian rhythms of activity and synaptic plasticity have been studied, and rhythmic behavioral outputs have been characterized. Integration of this data could lead to a better understanding of sleep regulation. Here, we review the progress of circadian clock and sleep studies in zebrafish with special emphasis on the genetic and neuroendocrine mechanisms that regulate rhythms of melatonin secretion, structural synaptic plasticity, locomotor activity and sleep.
Smadja Storz S(1), Tovin A, Mracek P, Alon S, Foulkes NS, Gothilf Y. (2013). Casein kinase 1δ activity: a key element in the zebrafish circadian timing system. 7. PLoS One. 2013;8(1):e54189.
Abstract
Zebrafish have become a popular model for studies of the circadian timing mechanism. Taking advantage of its rapid development of a functional circadian clock and the availability of light-entrainable clock-containing cell lines, much knowledge has been gained about the circadian clock system in this species. However, the post-translational modifications of clock proteins, and in particular the phosphorylation of PER proteins by Casein kinase I delta and epsilon (CK1δ and CK1ε), have so far not been examined in the zebrafish. Using pharmacological inhibitors for CK1δ and CK1ε, a pan-CK1δ/ε inhibitor PF-670462, and a CK1ε -selective inhibitor PF-4800567, we show that CK1δ activity is crucial for the functioning of the circadian timing mechanism of zebrafish, while CK1ε plays a minor role. The CK1δ/ε inhibitor disrupted circadian rhythms of promoter activity in the circadian clock-containing zebrafish cell line, PAC-2, while the CK1ε inhibitor had no effect. Zebrafish larvae that were exposed to the CK1δ/ε inhibitor showed no rhythms of locomotor activity while the CK1ε inhibitor had only a minor effect on locomotor activity. Moreover, the addition of the CK1δ/ε inhibitor disrupted rhythms of aanat2 mRNA expression in the pineal gland. The pineal gland is considered to act as a central clock organ in fish, delivering a rhythmic hormonal signal, melatonin, which is regulated by AANAT2 enzymatic activity. Therefore, CK1δ plays a key role in the circadian timing system of the zebrafish. Furthermore, the effect of CK1δ inhibition on rhythmic locomotor activity may reflect its effect on the function of the central clock in the pineal gland as well as its regulation of peripheral clocks.
Tovin A(1), Alon S, Ben-Moshe Z, Mracek P, Vatine G, Foulkes NS, Jacob-Hirsch J, Rechavi G, Toyama R, Coon SL, Klein DC, Eisenberg E, Gothilf Y. (2012). Systematic identification of rhythmic genes reveals camk1gb as a new element in the circadian clockwork. 8. PLoS Genet. 2012;8(12):e1003116.
Abstract
A wide variety of biochemical, physiological, and molecular processes are known to have daily rhythms driven by an endogenous circadian clock. While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock, the links between this clock and dependent processes have remained elusive. To address this gap in our knowledge, we have used RNA sequencing (RNA-seq) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland. In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue, this approach has revealed novel potential elements of the circadian timing system. We have implicated one rhythmically expressed gene, camk1gb, in connecting the clock with downstream physiology of the pineal gland. Remarkably, knockdown of camk1gb disrupts locomotor activity in the whole larva, even though it is predominantly expressed within the pineal gland. Therefore, it appears that camk1gb plays a role in linking the pineal master clock with the periphery.
Mracek P(1), Santoriello C, Idda ML, Pagano C, Ben-Moshe Z, Gothilf Y, Vallone D, Foulkes NS. (2012). Regulation of per and cry genes reveals a central role for the D-box enhancer in light-dependent gene expression. 9. PLoS One. 2012;7(12):e51278.
Abstract
Light serves as a key environmental signal for synchronizing the circadian clock with the day night cycle. The zebrafish represents an attractive model for exploring how light influences the vertebrate clock mechanism. Direct illumination of most fish tissues and cell lines induces expression of a broad range of genes including DNA repair, stress response and key clock genes. We have previously identified D- and E-box elements within the promoter of the zebrafish per2 gene that together direct light-induced gene expression. However, is the combined regulation by E- and D-boxes a general feature for all light-induced gene expression? We have tackled this question by examining the regulation of additional light-inducible genes. Our results demonstrate that with the exception of per2, all other genes tested are not induced by light upon blocking of de novo protein synthesis. We reveal that a single D-box serves as the principal light responsive element within the cry1a promoter. Furthermore, upon inhibition of protein synthesis D-box mediated gene expression is abolished while the E-box confers light driven activation as observed in the per2 gene. Given the existence of different photoreceptors in fish cells, our results implicate the D-box enhancer as a general convergence point for light driven signaling.
Idda ML(1), Bertolucci C, Vallone D, Gothilf Y, Sánchez-Vázquez FJ, Foulkes NS. ( 10. Prog Brain Res. 2012;199:41-57. doi: 10.1016/B978-0-444-59427-3.00003-4. ). Circadian clocks: lessons from fish. 10. Prog Brain Res. 2012;199:41-57.
Abstract
Our understanding of the molecular and cellular organization of the circadian timing system in vertebrates has increased enormously over the past decade. In large part, progress has been based on genetic studies in the mouse as well as on fundamental similarities between vertebrate and Drosophila clocks. The zebrafish was initially considered as a potentially attractive genetic model for identifying vertebrate clock genes. However, instead, fish have ultimately proven to be valuable complementary models for studying various aspects of clock biology. For example, many fish can shift from diurnal to nocturnal activity implying specific flexibility in their clock function. We have learned much about the function of light input pathways, and the ontogeny and function of the pineal organ, the fish central pacemaker. Finally, blind cavefish have also provided new insight into the evolution of the circadian clock under extreme environmental conditions.
Pubmed 
Tarttelin EE(1), Frigato E, Bellingham J, Di Rosa V, Berti R, Foulkes NS, Lucas RJ, Bertolucci C. (2012). Encephalic photoreception and phototactic response in the troglobiont Somalian blind cavefish Phreatichthys andruzzii. J Exp Biol. 215(Pt 16):2898-903.
Abstract
Many physiological and behavioural responses to changes in environmental lighting conditions are mediated by extraocular photoreceptors. Here we investigate encephalic photoreception in Phreatichthys andruzzii, a typical cave-dwelling fish showing an extreme phenotype with complete anophthalmy and a reduction in size of associated brain structures. We firstly identified two P. andruzzii photopigments, orthologues of rod opsin and exo-rod opsin. In vitro, both opsins serve as light-absorbing photopigments with λ(max) around 500 nm when reconstituted with an A(1) chromophore. When corrected for the summed absorption from the skin and skull, the spectral sensitivity profiles shifted to longer wavelengths (rod opsin: 521 nm; exo-rod opsin: 520 nm). We next explored the involvement of both opsins in the negative phototaxis reported for this species. A comparison of the spectral sensitivity of the photophobic response with the putative A(2) absorbance spectra corrected for skin/skull absorbance indicates that the A(2) versions of either or both of these pigments could explain the observed behavioural spectral sensitivity.
Idda ML(1), Kage E, Lopez-Olmeda JF, Mracek P, Foulkes NS, Vallone D. (2012). Circadian timing of injury-induced cell proliferation in zebrafish. 12. PLoS One. 2012;7(3):e34203.
Abstract
In certain vertebrates such as the zebrafish, most tissues and organs including the heart and central nervous system possess the remarkable ability to regenerate following severe injury. Both spatial and temporal control of cell proliferation and differentiation is essential for the successful repair and re-growth of damaged tissues. Here, using the regenerating adult zebrafish caudal fin as a model, we have demonstrated an involvement of the circadian clock in timing cell proliferation following injury. Using a BrdU incorporation assay with a short labeling period, we reveal high amplitude daily rhythms in S-phase in the epidermal cell layer of the fin under normal conditions. Peak numbers of S-phase cells occur at the end of the light period while lowest levels are observed at the end of the dark period. Remarkably, immediately following amputation the basal level of epidermal cell proliferation increases significantly with kinetics, depending upon the time of day when the amputation is performed. In sharp contrast, we failed to detect circadian rhythms of S-phase in the highly proliferative mesenchymal cells of the blastema. Subsequently, during the entire period of outgrowth of the new fin, elevated, cycling levels of epidermal cell proliferation persist. Thus, our results point to a preferential role for the circadian clock in the timing of epidermal cell proliferation in response to injury.
Cavallari N(1), Frigato E, Vallone D, Fröhlich N, Lopez-Olmeda JF, Foà A, Berti R, Sánchez-Vázquez FJ, Bertolucci C, Foulkes NS. (2011). A blind circadian clock in cavefish reveals that opsins mediate peripheral clock photoreception. PLoS Biol. 9(9):e1001142.
Abstract
Comment in PLoS Biol. 2011 Sep;9(9):e1001141.
Pubmed 
Vatine G(1), Vallone D, Gothilf Y, Foulkes NS. (2011). It's time to swim! Zebrafish and the circadian clock. FEBS Lett. 585(10):1485-94.
Abstract
The zebrafish represents a fascinating model for studying key aspects of the vertebrate circadian timing system. Easy access to early embryonic development has made this species ideal for investigating how the clock is first established during embryogenesis. In particular, the molecular basis for the functional development of the zebrafish pineal gland has received much attention. In addition to this dedicated clock and photoreceptor organ, and unlike the situation in mammals, the clocks in zebrafish peripheral tissues and even cell lines are entrainable by direct exposure to light thus providing unique insight into the function and evolution of the light input pathway. Finally, the small size, low maintenance costs and high fecundity of this fish together with the availability of genetic tools make this an attractive model for forward genetic analysis of the circadian clock. Here, we review the work that has established the zebrafish as a valuable clock model organism and highlight the key questions that will shape the future direction of research.
Pubmed 
Weger BD(1), Sahinbas M, Otto GW, Mracek P, Armant O, Dolle D, Lahiri K, Vallone D, Ettwiller L, Geisler R, Foulkes NS, Dickmeis T. (2011). The light responsive transcriptome of the zebrafish: function and regulation. PLoS One. 6(2):e17080.
Abstract
Most organisms possess circadian clocks that are able to anticipate the day/night cycle and are reset or "entrained" by the ambient light. In the zebrafish, many organs and even cultured cell lines are directly light responsive, allowing for direct entrainment of the clock by light. Here, we have characterized light induced gene transcription in the zebrafish at several organizational levels. Larvae, heart organ cultures and cell cultures were exposed to 1- or 3-hour light pulses, and changes in gene expression were compared with controls kept in the dark. We identified 117 light regulated genes, with the majority being induced and some repressed by light. Cluster analysis groups the genes into five major classes that show regulation at all levels of organization or in different subset combinations. The regulated genes cover a variety of functions, and the analysis of gene ontology categories reveals an enrichment of genes involved in circadian rhythms, stress response and DNA repair, consistent with the exposure to visible wavelengths of light priming cells for UV-induced damage repair. Promoter analysis of the induced genes shows an enrichment of various short sequence motifs, including E- and D-box enhancers that have previously been implicated in light regulation of the zebrafish period2 gene. Heterologous reporter constructs with sequences matching these motifs reveal light regulation of D-box elements in both cells and larvae. Morpholino-mediated knock-down studies of two homologues of the D-box binding factor Tef indicate that these are differentially involved in the cell autonomous light induction in a gene-specific manner. These findings suggest that the mechanisms involved in period2 regulation might represent a more general pathway leading to light induced gene expression.
Ben-Moshe Z(1), Vatine G, Alon S, Tovin A, Mracek P, Foulkes NS, Gothilf Y. (2010). Multiple PAR and E4BP4 bZIP transcription factors in zebrafish: diverse spatial and temporal expression patterns. Chronobiol Int. 27(8):1509-31.
Abstract
Erratum in Chronobiol Int. 2010 Sep;27(8):1672.
Pubmed 
Dickmeis T(1), Foulkes NS. (2010). Glucocorticoids and circadian clock control of cell proliferation: at the interface between three dynamic systems. Mol Cell Endocrinol. 331(1):11-22.
Abstract
The circadian clock, an endogenous timekeeper that regulates daily rhythms of physiology, also influences the dynamic release of glucocorticoids. The release of glucocorticoids is characteristically pulsatile and is further modulated in a circadian fashion. A circadian pacemaker in the brain regulates daily rhythms of hypothalamic-pituitary-adrenal axis and autonomic nervous system activity that both influence glucocorticoid release from the adrenal gland. This systemic regulation interacts with rhythms in the adrenal gland itself that are driven by its own circadian clock. One function of glucocorticoids is the regulation of cell proliferation. Depending on the tissue, this can involve both negative and positive regulation of a variety of processes, including cell differentiation and cell death. Cell proliferation is also under circadian control, and recent evidence suggests that this regulation may involve glucocorticoid signalling. Here, we review the dynamic processes participating in the interplay between the circadian clock, glucocorticoids and cell proliferation, and we discuss the potential implications for therapy.
Pubmed 
Vatine G(1), Vallone D, Appelbaum L, Mracek P, Ben-Moshe Z, Lahiri K, Gothilf Y, Foulkes NS. (2009). Light directs zebrafish period2 expression via conserved D and E boxes. PLoS Biol. 7(10):e1000223.
Abstract
For most species, light represents the principal environmental signal for entraining the endogenous circadian clock. The zebrafish is a fascinating vertebrate model for studying this process since unlike mammals, direct exposure of most of its tissues to light leads to local clock entrainment. Importantly, light induces the expression of a set of genes including certain clock genes in most zebrafish cell types in vivo and in vitro. However, the mechanism linking light to gene expression remains poorly understood. To elucidate this key mechanism, here we focus on how light regulates transcription of the zebrafish period2 (per2) gene. Using transgenic fish and stably transfected cell line-based assays, we define a Light Responsive Module (LRM) within the per2 promoter. The LRM lies proximal to the transcription start site and is both necessary and sufficient for light-driven gene expression and also for a light-dependent circadian clock regulation. Curiously, the LRM sequence is strongly conserved in other vertebrate per2 genes, even in species lacking directly light-sensitive peripheral clocks. Furthermore, we reveal that the human LRM can substitute for the zebrafish LRM to confer light-regulated transcription in zebrafish cells. The LRM contains E- and D-box elements that are critical for its function. While the E-box directs circadian clock regulation by mediating BMAL/CLOCK activity, the D-box confers light-driven expression. The zebrafish homolog of the thyrotroph embryonic factor binds efficiently to the LRM D-box and transactivates expression. We demonstrate that tef mRNA levels are light inducible and that knock-down of tef expression attenuates light-driven transcription from the per2 promoter in vivo. Together, our results support a model where a light-dependent crosstalk between E- and D-box binding factors is a central determinant of per2 expression. These findings extend the general understanding of the mechanism whereby the clock is entrained by light and how the regulation of clock gene expression by light has evolved in vertebrates.
Cossins AR(1), Williams DR, Foulkes NS, Berenbrink M, Kipar A. (2009). Diverse cell-specific expression of myoglobin isoforms in brain, kidney, gill and liver of the hypoxia-tolerant carp and zebrafish. J Exp Biol. 212(Pt 5):627-38.
Abstract
Myoglobin (Mb) is famous as a muscle-specific protein--yet the common carp expresses the gene (cMb1) encoding this protein in a range of non-muscle tissues and also expresses a novel isoform (cMb2) in the brain. Using a homologous antibody and riboprobes, we have established the relative amounts and cellular sites of non-muscle Mb expression in different tissues. The amounts of carp myoglobin (cMb) in supernatants of different tissues were just 0.4-0.7% relative to that of heart supernatants and were upregulated by two-to-four fold in liver, gill and brain following 5 days of hypoxic treatment. Brain exhibited both cMb proteins in western analysis, whereas all other tissues had only cMb1. We have also identified cells expressing cMb protein and cMb mRNA using immunohistology and RNA in situ hybridisation (RNA-ISH), respectively. Mb was strongly expressed throughout all cardiac myocytes and a subset of skeletal muscle fibres, whereas it was restricted to a small range of specific cell types in each of the non-muscle tissues. These include pillar and epithelial cells in secondary gill lamellae, hepatocytes, some neurones, and tubular epithelial cells in the kidney. Capillaries and small blood vessels in all tissues exhibited Mb expression within vascular endothelial cells. The cMb2 riboprobe located expression to a subset of neurones but not to endothelial cells. In zebrafish, which possesses only one Mb gene, a similar expression pattern of Mb protein and mRNA was observed. This establishes a surprisingly cell-specific distribution of Mb within non-muscle tissues in both carp and zebrafish, where it probably plays an important role in the regulation of microvascular, renal and brain function.
Vallone D(1), Lahiri K, Dickmeis T, Foulkes NS. ( 20. Zebrafish. 2005;2(3):171-87. doi: 10.1089/zeb.2005.2.171. ). Zebrafish cell clocks feel the heat and see the light! 20. Zebrafish. 2005;2(3):171-87.
Abstract
The zebrafish has rapidly become established as one of the most valuable vertebrate models for studying circadian clock function. A major initial attraction was its utility in large-scale genetic screens. It subsequently emerged that most zebrafish cells possess circadian clocks that can be entrained directly by exposure to temperature or light dark cycles, a property shared by several zebrafish cell lines. This is not the case for mammals, where the retina is the primary source of light input to the clock. Furthermore, mammalian cell culture clocks can only be entrained by acute culture treatments such as serum shocks. Thus, the zebrafish is proving invaluable to study light and temperature input to the vertebrate clock. In addition, the accessibility of its early developmental stages has placed the zebrafish at the forefront of studies aimed at understanding how the circadian clock is established during embryogenesis.
Vallone D(1), Santoriello C, Gondi SB, Foulkes NS. ( 21. Methods Mol Biol. 2007;362:429-41. ). Basic protocols for zebrafish cell lines: maintenance and transfection. 21. Methods Mol Biol. 2007;362:429-41.
Abstract
Cell lines derived from zebrafish embryos show great potential as cell culture tools to study the regulation and function of the vertebrate circadian clock. They exhibit directly light-entrainable rhythms of clock gene expression that can be established by simply exposing cultures to light-dark cycles. Mammalian cell lines require treatments with serum or activators of signaling pathways to initiate transient, rapidly dampening clock rhythms. Furthermore, zebrafish cells grow at room temperature, are viable for long periods at confluence, and do not require a CO2-enriched atmosphere, greatly simplifying culture conditions. Here we describe detailed methods for establishing zebrafish cell cultures as well as optimizing transient and stable transfections. These protocols have been successfully used to introduce luciferase reporter constructs into the cells and thereby monitor clock gene expression in vivo. The bioluminescence assay described here lends itself particularly well to high-throughput analysis.
Rembold M(1), Lahiri K, Foulkes NS, Wittbrodt J. ( 22. Nat Protoc. 2006;1(3):1133-9. ). Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct. 22. Nat Protoc. 2006;1(3):1133-9.
Abstract
Small fish are a popular laboratory model for studying gene expression and function by transgenesis. If, however, the transgenes are not readily detectable by visual inspection, a large number of embryos must be injected, raised and screened to identify positive founder fish. Here, we describe a strategy to efficiently generate and preselect transgenic lines harbouring any transgene of interest. Co-injection of a selectable reporter construct (e.g., GFP), together with the transgene of interest on a separate plasmid using the I-SceI meganuclease approach, results in co-distribution of the two plasmids. The quality of GFP expression within the F0 generation therefore reflects the quality of injection and allows efficient and reliable selection of founder fish that are also positive for the second transgene of interest. In our experience, a large fraction (up to 50%) of GFP-positive fish will also be transgenic for the second transgene, thus providing a rapid (within 3-4 months) and efficient way to establish transgenic lines for any gene of interest in medaka and zebrafish.
Dickmeis T(1), Lahiri K, Nica G, Vallone D, Santoriello C, Neumann CJ, Hammerschmidt M, Foulkes NS. (2007). Glucocorticoids play a key role in circadian cell cycle rhythms. PLoS Biol. 5(4):e78.
Abstract
Clock output pathways play a pivotal role by relaying timing information from the circadian clock to a diversity of physiological systems. Both cell-autonomous and systemic mechanisms have been implicated as clock outputs; however, the relative importance and interplay between these mechanisms are poorly understood. The cell cycle represents a highly conserved regulatory target of the circadian timing system. Previously, we have demonstrated that in zebrafish, the circadian clock has the capacity to generate daily rhythms of S phase by a cell-autonomous mechanism in vitro. Here, by studying a panel of zebrafish mutants, we reveal that the pituitary-adrenal axis also plays an essential role in establishing these rhythms in the whole animal. Mutants with a reduction or a complete absence of corticotrope pituitary cells show attenuated cell-proliferation rhythms, whereas expression of circadian clock genes is not affected. We show that the corticotrope deficiency is associated with reduced cortisol levels, implicating glucocorticoids as a component of a systemic signaling pathway required for circadian cell cycle rhythmicity. Strikingly, high-amplitude rhythms can be rescued by exposing mutant larvae to a tonic concentration of a glucocorticoid agonist. Our work suggests that cell-autonomous clock mechanisms are not sufficient to establish circadian cell cycle rhythms at the whole-animal level. Instead, they act in concert with a systemic signaling environment of which glucocorticoids are an essential part.
Zilberman-Peled B(1), Appelbaum L, Vallone D, Foulkes NS, Anava S, Anzulovich A, Coon SL, Klein DC, Falcón J, Ron B, Gothilf Y. (2007). Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream. J Neuroendocrinol. 19(1):46-53.
Abstract
Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.
Vallone D(1), Lahiri K, Dickmeis T, Foulkes NS. (2007). Start the clock! Circadian rhythms and development. Dev Dyn. 236(1):142-55.
Abstract
The contribution of timing cues from the environment to the coordination of early developmental processes is poorly understood. The day-night cycle represents one of the most important, regular environmental changes that animals are exposed to. A key adaptation that allows animals to anticipate daily environmental changes is the circadian clock. In this review, we aim to address when a light-regulated circadian clock first emerges during development and what its functions are at this early stage. In particular, do circadian clocks regulate early developmental processes? We will focus on results obtained with Drosophila and vertebrates, where both circadian clock and developmental control mechanisms have been intensively studied.
Vallone D(1), Frigato E, Vernesi C, Foà A, Foulkes NS, Bertolucci C. (2006). Hypothermia modulates circadian clock gene expression in lizard peripheral tissues. Am J Physiol Regul Integr Comp Physiol. 292(1):R160-6.
Abstract
The molecular mechanisms whereby the circadian clock responds to temperature changes are poorly understood. The ruin lizard Podarcis sicula has historically proven to be a valuable vertebrate model for exploring the influence of temperature on circadian physiology. It is an ectotherm that naturally experiences an impressive range of temperatures during the course of the year. However, no tools have been available to dissect the molecular basis of the clock in this organism. Here, we report the cloning of three lizard clock gene homologs (Period2, Cryptochrome1, and Clock) that have a close phylogenetic relationship with avian clock genes. These genes are expressed in many tissues and show a rhythmic expression profile at 29 degrees C in light-dark and constant darkness lighting conditions, with phases comparable to their mammalian and avian counterparts. Interestingly, we show that at low temperatures (6 degrees C), cycling clock gene expression is attenuated in peripheral clocks with a characteristic increase in basal expression levels. We speculate that this represents a conserved vertebrate clock gene response to low temperatures. Furthermore, these results bring new insight into the issue of whether circadian clock function is compatible with hypothermia.
Frigato E(1), Vallone D, Bertolucci C, Foulkes NS. (2006). Isolation and characterization of melanopsin and pinopsin expression within photoreceptive sites of reptiles. Naturwissenschaften. 93(8):379-85.
Abstract
Non-mammalian vertebrates have multiple extraocular photoreceptors, mainly localised in the pineal complex and the brain, to mediate irradiance detection. In this study, we report the full-length cDNA cloning of ruin lizard melanopsin and pinopsin. The high level of identity with opsins in both the transmembrane regions, where the chromophore binding site is located, and the intracellular loops, where the G-proteins interact, suggests that both melanopsin and pinopsin should be able to generate a stable photopigment, capable of triggering a transduction cascade mediated by G-proteins. Phylogenetic analysis showed that both opsins are located on the expected branches of the corresponding sequences of ortholog proteins. Subsequently, using RT-PCR and RPA analysis, we verified the expression of ruin lizard melanopsin and pinopsin in directly photosensitive organs, such as the lateral eye, brain, pineal gland and parietal eye. Melanopsin expression was detected in the lateral eye and all major regions of the brain. However, different from the situation in Xenopus and chicken, melanopsin is not expressed in the ruin lizard pineal. Pinopsin mRNA expression was only detected in the pineal complex. As a result of their phylogenetic position and ecology, reptiles provide the circadian field with some of the most interesting models for understanding the evolution of the vertebrate circadian timing system and its response to light. This characterization of melanopsin and pinopsin expression in the ruin lizard will be important for future studies aimed at understanding the molecular basis of circadian light detection in reptiles.
Helfer G(1), Fidler AE, Vallone D, Foulkes NS, Brandstaetter R. ( 28. Chronobiol Int. 2006;23(1-2):113-27. ). Molecular analysis of clock gene expression in the avian brain. 28. Chronobiol Int. 2006;23(1-2):113-27.
Abstract
Birds are equipped with a complex circadian pacemaking system that regulates the rhythmicity of physiology and behavior. As with all organisms, transcriptional and translational feedback loops of clock genes represent the basic molecular mechanism of rhythm generation in birds. To investigate avian clock gene expression, partial cDNA sequences of six mammalian clock gene homologs (Bmal1, Clock, Per2, Per3, Cry1, and Cry2) and a novel avian cryptochrome gene (Cry4) were cloned from the house sparrow, a model system in circadian research. Expression patterns were analyzed by semi-quantitative RT-PCR and RNase protection assays using total RNA extracted from adult male house sparrow brains. With the exception of Cry4, pronounced rhythmic mRNA expression of all the clock genes analyzed was encountered, with mRNA levels varying considerably between the various genes. Although some basic features of the molecular circadian feedback loop appear to be similar between mammals and birds, the precise phase relationships of the clock gene mRNA rhythms relative to each other and to the light zeitgeber differ significantly between the house sparrow and mammals. Our results point to the existence of differences in the organization of avian and mammalian circadian clock mechanisms.
Appelbaum L(1), Vallone D, Anzulovich A, Ziv L, Tom M, Foulkes NS, Gothilf Y. (2006). Zebrafish arylalkylamine-N-acetyltransferase genes - targets for regulation of the circadian clock. J Mol Endocrinol. 36(2):337-47.
Abstract
Daily rhythms of melatonin production are controlled by changes in the activity of arylalkylamine-N-acetyltransferase (AANAT). Zebrafish possess two aanats, aanat1 and aanat2; the former is expressed only in the retina and the latter is expressed in both the retina and the pineal gland. Here, their differential expression and regulation were studied using transcript quantification and transient and stable in vivo and in vitro transfection assays. In the pineal gland, the aanat2 promoter exhibited circadian clock-controlled activity, as indicated by circadian rhythms of Enhanced green fluorescent protein (EGFP) mRNA in AANAT2:EGFP transgenic fish. In vivo transient expression analyses of the aanat2 promoter indicated that E-box and photoreceptor conserved elements (PCE) are required for expression in the pineal gland. In the retina, the expression of both genes was characterized by a robust circadian rhythm of their transcript levels. In constant darkness, the rhythmic expression of retinal aanat2 persisted while the aanat1 rhythm disappeared; indicating that the former is controlled by a circadian clock and the latter is also light driven. In the light-entrainable clock-containing PAC-2 zebrafish cell line, both stably transfected aanat1 and aanat2 promoters exhibited a clock-controlled circadian rhythm, characteristic for an E-box-driven expression. Transient co-transfection experiments in NIH-3T3 cells revealed that the two, E-box- and PCE-containing, promoters are driven by the synergistic action of BMAL/CLOCK and orthehodenticle homeobox 5. This study has revealed a shared mechanism for the regulation of two related genes, yet describes their differential phases and photic responses which may be driven by other gene-specific regulatory mechanisms and tissue-specific transcription factor profiles.
Lahiri K(1), Vallone D, Gondi SB, Santoriello C, Dickmeis T, Foulkes NS. (2005). Temperature regulates transcription in the zebrafish circadian clock. PLoS Biol. 3(11):e351.
Abstract
It has been well-documented that temperature influences key aspects of the circadian clock. Temperature cycles entrain the clock, while the period length of the circadian cycle is adjusted so that it remains relatively constant over a wide range of temperatures (temperature compensation). In vertebrates, the molecular basis of these properties is poorly understood. Here, using the zebrafish as an ectothermic model, we demonstrate first that in the absence of light, exposure of embryos and primary cell lines to temperature cycles entrains circadian rhythms of clock gene expression. Temperature steps drive changes in the basal expression of certain clock genes in a gene-specific manner, a mechanism potentially contributing to entrainment. In the case of the per4 gene, while E-box promoter elements mediate circadian clock regulation, they do not direct the temperature-driven changes in transcription. Second, by studying E-box-regulated transcription as a reporter of the core clock mechanism, we reveal that the zebrafish clock is temperature-compensated. In addition, temperature strongly influences the amplitude of circadian transcriptional rhythms during and following entrainment by light-dark cycles, a property that could confer temperature compensation. Finally, we show temperature-dependent changes in the expression levels, phosphorylation, and function of the clock protein, CLK. This suggests a mechanism that could account for changes in the amplitude of the E-box-directed rhythm. Together, our results imply that several key transcriptional regulatory elements at the core of the zebrafish clock respond to temperature.
Vallone D(1), Gondi SB, Whitmore D, Foulkes NS. (2004). E-box function in a period gene repressed by light. Proc Natl Acad Sci U S A. 101(12):4106-11.
Abstract
In most organisms, light plays a key role in the synchronization of the circadian timing system with the environmental day-night cycle. Light pulses that phase-shift the circadian clock also induce the expression of period (per) genes in vertebrates. Here, we report the cloning of a zebrafish per gene, zfper4, which is remarkable in being repressed by light. We have developed an in vivo luciferase reporter assay for this gene in cells that contain a light-entrainable clock. High-definition bioluminescence traces have enabled us to accurately measure phase-shifting of the clock by light. We have also exploited this model to study how four E-box elements in the zfper4 promoter regulate expression. Mutagenesis reveals that the integrity of these four E-boxes is crucial for maintaining low basal expression together with robust rhythmicity and repression by light. Importantly, in the context of a minimal heterologous promoter, the E-box elements also direct a robust circadian rhythm of expression that is significantly phase-advanced compared with the original zfper4 promoter and lacks the light-repression property. Thus, these results reveal flexibility in the phase and light responsiveness of E-box-directed rhythmic expression, depending on the promoter context.
Tamai TK(1), Vardhanabhuti V, Foulkes NS, Whitmore D. (2004). Early embryonic light detection improves survival. Curr Biol. 14(3):R104-5.
Abstract
Erratum in Curr Biol. 2004 Mar 9;14(5):446.
Dekens MP(1), Santoriello C, Vallone D, Grassi G, Whitmore D, Foulkes NS. (2003). Light regulates the cell cycle in zebrafish. Curr Biol. 13(23):2051-7.
Abstract
The timing of cell proliferation is a key factor contributing to the regulation of normal growth. Daily rhythms of cell cycle progression have been documented in a wide range of organisms. However, little is known about how environmental, humoral, and cell-autonomous factors contribute to these rhythms. Here, we demonstrate that light plays a key role in cell cycle regulation in the zebrafish. Exposure of larvae to light-dark (LD) cycles causes a range of different cell types to enter S phase predominantly at the end of the day. When larvae are raised in constant darkness (DD), a low level of arrhythmic S phase is observed. In addition, light-entrained cell cycle rhythms persist for several days after transfer to DD, both observations pointing to the involvement of the circadian clock. We show that the number of LD cycles experienced is essential for establishing this rhythm during larval development. Furthermore, we reveal that the same phenomenon exists in a zebrafish cell line. This represents the first example of a vertebrate cell culture system where circadian rhythms of the cell cycle are observed. Thus, we implicate the cell-autonomous circadian clock in the regulation of the vertebrate cell cycle by light.
Tamai TK(1), Vardhanabhuti V, Arthur S, Foulkes NS, Whitmore D. (2003). Flies and fish: birds of a feather. J Neuroendocrinol. 15(4):344-9.
Abstract
The identification of specific clock-containing structures has been a major endeavour of the circadian field for many years. This has lead to the identification of many key components of the circadian system, including the suprachiasmatic nucleus in mammals, and the eyes and pineal glands in lower vertebrates. However, the idea that these structures represent the only clocks in animals has been challenged by the discovery of peripheral pacemakers in most organs and tissues, and even a number of cell lines. In Drosophila, and vertebrates such as the zebrafish, these peripheral clocks appear to be highly autonomous, being set directly by the environmental light/dark cycle. However, a hierarchy of clocks may still exist in mammals. In this review, we examine some of the current views regarding peripheral clocks, their organization and how they are entrained.
Vyas S(1), Biguet NF, Michel PP, Monaco L, Foulkes NS, Evan GI, Sassone-Corsi P, Agid Y. (2002). Molecular mechanisms of neuronal cell death: implications for nuclear factors responding to cAMP and phorbol esters. Mol Cell Neurosci. 21(1):1-14.
Abstract
Chronic treatment with calcium ionophore A23187 in NGF-differentiated cells results in cell death that is time- and concentration-dependent. Additionally, PC12 cells codifferentiated with NGF and dBcAMP become dependent on these factors for survival and undergo apoptosis when both factors are withdrawn. We show that in both cases there is a prolonged induction of c-Fos which correlates with cell death. Its continual activation in PC12 cells overexpressing c-FosER results in caspase-3 cleavage and rapid cell death. Specific phosphorylation of CREB/CREM(tau) transactivators or their binding to CRE of c-fos was observed. Our results indicate that prolonged c-Fos induction activates p53. There is increased nuclear localization of p53, p21 and Bax levels are induced in NGF/dBcAMP-deprived c-FosER cells, and dominant negative p53 inhibits cell death induced either by serum deprivation or by c-Fos. Overall these data implicate AP-1 as a nuclear target of signal transduction pathways which plays a role in the activation of apoptosis.
Whitmore D(1), Cermakian N, Crosio C, Foulkes NS, Pando MP, Travnickova Z, Sassone-Corsi P. (2000). A clockwork organ. Biol Chem. 381(9-10):793-800.
Abstract
The vertebrate circadian clock was thought to be highly localized to specific anatomical structures: the mammalian suprachiasmatic nucleus (SCN), and the retina and pineal gland in lower vertebrates. However, recent findings in the zebrafish, rat and in cultured cells have suggested that the vertebrate circadian timing system may in fact be highly distributed, with most if not all cells containing a clock. Our understanding of the clock mechanism has progressed extensively through the use of mutant screening and forward genetic approaches. The first vertebrate clock gene was identified only a few years ago in the mouse by such an approach. More recently, using a syntenic comparative genetic approach, the molecular basis of the the tau mutation in the hamster was determined. The tau gene in the hamster appears to encode casein kinase 1 epsilon, a protein previously shown to be important for PER protein turnover in the Drosophila circadian system. A number of additional clock genes have now been described. These proteins appear to play central roles in the transcription-translation negative feedback loop responsible for clock function. Post-translational modification, protein dimerization and nuclear transport all appear to be essential features of how clocks are thought to tick.
Cermakian N(1), Whitmore D, Foulkes NS, Sassone-Corsi P. (2000). Asynchronous oscillations of two zebrafish CLOCK partners reveal differential clock control and function. Proc Natl Acad Sci U S A. 97(8):4339-44.
Abstract
Most clock genes encode transcription factors that interact to elicit cooperative control of clock function. Using a two-hybrid system approach, we have isolated two different partners of zebrafish (zf) CLOCK, which are similar to the mammalian BMAL1 (brain and muscle arylhydrocarbon receptor nuclear translocator-like protein 1). The two homologs, zfBMAL1 and zfBMAL2, contain conserved basic helix-loop-helix-PAS (Period-Arylhydrocarbon receptor-Singleminded) domains but diverge in the carboxyl termini, thus bearing different transcriptional activation potential. As for zfClock, the expression of both zfBmals oscillates in most tissues in the animal. However, in many tissues, the peak, levels, and kinetics of expression are different between the two genes and for the same gene from tissue to tissue. These results support the existence of independent peripheral oscillators and suggest that zfBMAL1 and zfBMAL2 may exert distinct circadian functions, interacting differentially with zfCLOCK at various times in different tissues. Our findings also indicate that multiple controls may be exerted by the central clock and/or that peripheral oscillators can differentially interpret central clock signals.
Foulkes NS(1), Cermakian N, Whitmore D, Sassone-Corsi P. ( 38. Novartis Found Symp. 2000;227:5-14; discussion 15-8. ). Rhythmic transcription: the molecular basis of oscillatory melatonin synthesis. 38. Novartis Found Symp. 2000;227:5-14; discussion 15-8.
Abstract
Pulsatile hormone synthesis and secretion are characteristic features of various oscillatory biological systems. Circadian rhythms are critical in the regulation of most physiological functions, and much interest has been centred on the understanding of the molecular mechanisms governing them. Adaptation to a changing environment is an essential feature of physiological regulation. The day-night rhythm is translated into hormonal oscillations governing the metabolism of all living organisms. In mammals the pineal gland is responsible for the circadian synthesis of the hormone melatonin in response to signals originating from the endogenous clock located in the hypothalamic suprachiasmatic nucleus (SCN). The molecular mechanisms involved in rhythmic synthesis of melatonin involve the CREM gene, which encodes transcription factors responsive to activation of the cAMP signalling pathway. The CREM product, ICER, is rhythmically expressed and participates in a transcriptional autoregulatory loop which also controls the amplitude of oscillations of serotonin N-acetyl transferase, the rate-limiting enzyme of melatonin synthesis. Thus, a transcription factor modulates the oscillatory levels of a hormone.
Whitmore D(1), Foulkes NS, Sassone-Corsi P. (2000). Light acts directly on organs and cells in culture to set the vertebrate circadian clock. Nature. 404(6773):87-91.
Abstract
Comment in Nature. 2000 Mar 2;404(6773):25, 27-8.
Pubmed 
Foulkes NS(1), Sassone-Corsi P. (2000). Molecular clocks (joint Juan March/EMBO workshop). Madrid, May 10-12, 1999. EMBO J. 19(5):789-91.
Abstract
PMCID: PMC379300 PMID: 10698920 [PubMed - indexed for MEDLINE]
Foulkes NS(1), Naranjo JR, Sassone-Corsi P. (1999). Setting the clock in Madrid. Trends Cell Biol. 9(9):371-2.
Abstract
PMID: 10532822 [PubMed - indexed for MEDLINE]
Whitmore D(1), Foulkes NS, Strähle U, Sassone-Corsi P. (1998). Zebrafish Clock rhythmic expression reveals independent peripheral circadian oscillators. Nat Neurosci. 1(8):701-7.
Abstract
The only vertebrate clock gene identified by mutagenesis is mouse Clock, which encodes a bHLH-PAS transcription factor. We have cloned Clock in zebrafish and show that, in contrast to its mouse homologue, it is expressed with a pronounced circadian rhythm in the brain and in two defined pacemaker structures, the eye and the pineal gland. Clock oscillation was also found in other tissues, including kidney and heart. In these tissues, expression of Clock continues to oscillate in vitro. This demonstrates that self-sustaining circadian oscillators exist in several vertebrate organs, as was previously reported for invertebrates.
Whitmore D(1), Sassone-Corsi P, Foulkes NS. (1998). PASting together the mammalian clock. Curr Opin Neurobiol. 8(5):635-41.
Abstract
Over the past year, the first components of the mammalian clock have been identified; Clock, bmal1 and three homologs of Drosophila period have been cloned, all of which encode PAS proteins. Expression of the mammalian period gene oscillates in many tissues in vivo and in immortalized cell cultures in vitro. Now, can we say that every cell has a circadian clock?
Della Fazia MA(1), Servillo G, Foulkes NS, Sassone-Corsi P. (1998). Stress-induced expression of transcriptional repressor ICER in the adrenal gland. FEBS Lett. 434(1-2):33-6.
Abstract
Second messenger cyclic AMP plays a central role in signalling within the hypothalamo-pituitary-adrenal (HPA) axis. Changes in gene expression are central to long-term adaptations made in response to stress in the adrenal gland. Here we demonstrate that expression of the cAMP inducible transcriptional repressor, ICER (Inducible cAMP Early Repressor), is rapidly and powerfully induced in response to surgical stress in the rat adrenal gland. Hypophysectomisation blocks stress-induced ICER expression. Finally we demonstrate that injection of the pituitary hormone ACTH (Adrenocorticotropin Hormone) induces robust ICER expression in the adrenal cortex. Thus, induction of the transcriptional repressor ICER is coupled to the HPA axis response to stress.
Foulkes NS(1), Whitmore D, Sassone-Corsi P. (1997). Rhythmic transcription: the molecular basis of circadian melatonin synthesis. Biol Cell. 89(8):487-94.
Abstract
Adaptation to a changing environment is an essential feature of physiological regulation. The day/night rhythm is translated into hormonal oscillations governing the physiology of all living organisms. In mammals the pineal gland is responsible for the synthesis of the hormone melatonin in response to signals originating from the endogenous clock located in the hypothalamic suprachiasmatic nucleus (SCN). The molecular mechanisms involved in rhythmic synthesis of melatonin involve the CREM gene, which encodes transcription factors responsive to activation of the cAMP signalling pathway. The CREM product, ICER, is rhythmically expressed and participates in a transcriptional autoregulatory loop which also controls the amplitude of oscillations of serotonin N-acetyl transferase (AANAT), the rate-limiting enzyme of melatonin synthesis. In contrast, chick pinealocytes possess an endogenous circadian pacemaker which directs AANAT rhythmic expression. cAMP-responsive activator transcription factors CREB and ATF1 and the repressor ICER are highly conserved in the chick with the notable exception of ATF1 that possesses two glutamine-rich domains in contrast to the single domain encountered to date in mammalian systems. ICER is cAMP inducible and undergoes a characteristic day-night oscillation in expression reminiscent of AA-NAT, but with a peak towards the end of the night. Interestingly CREB appears to be phosphorylated constitutively with a transient fall occurring at the beginning of the night. Thus, a transcription factor modulates the oscillatory levels of a hormone.
Naranjo JR(1), Mellström B, Carrión AM, Lucas JJ, Foulkes NS, Sassone-Corsi P. (1997). Peripheral noxious stimulation induces CREM expression in dorsal horn: involvement of glutamate. Eur J Neurosci. 9(12):2778-83.
Abstract
Peripheral noxious stimulation is known to trigger signalling cascades in neurons of the spinal cord. The response to pain and stress at the level of gene expression involves transcriptional activation of several cyclic AMP responsive genes. Here, we show induction of the CREM (cyclic-AMP responsive element modulator) gene in distinct subpopulations of spinal cord neurons upon thermal noxious stimulation. The addition of forskolin or glutamate to cultured spinal cord neurons results in the induction of the CREM isoform, ICER (Inducible cyclic-AMP Early Repressor), a powerful repressor of cAMP-induced transcription. Overexpression of ICER in cultured spinal cord neurons results in the repression of the c-fos and c-jun promoters induced by forskolin and glutamate. On this basis, we postulate that early activation of ICER in spinal cord participates in the attenuation of early gene induction following noxious stimulation.
Foulkes NS(1), Borjigin J, Snyder SH, Sassone-Corsi P. (1997). Rhythmic transcription: the molecular basis of circadian melatonin synthesis. Trends Neurosci. 20(10):487-92.
Abstract
Adaptation to a changing environment is an essential feature of physiological regulation. The day-night rhythm is translated into hormonal oscillations governing the metabolism of all living organisms. In mammals the pineal gland is responsible for the synthesis of the hormone melatonin in response to signals originating from the endogenous clock located in the hypothalamic suprachiasmatic nucleus (SCN). The molecular mechanisms involved in rhythmic synthesis of melatonin involve the cAMP response element modulator (crem) gene, which encodes transcription factors responsive to activation of the cAMP signalling pathway. The CREM product, inducible cAMP early repressor (ICER), is rhythmically expressed and participates in a transcriptional autoregulatory loop that also controls the amplitude of oscillations of 5-HT N-acetyl transferase, the rate-limiting enzyme of melatonin synthesis. Thus, a transcription factor modulates the oscillatory levels of a hormone.
Lamas M(1), Molina C, Foulkes NS, Jansen E, Sassone-Corsi P. (1997). Ectopic ICER expression in pituitary corticotroph AtT20 cells: effects on morphology, cell cycle, and hormonal production. Mol Endocrinol. 11(10):1425-34.
Abstract
The products of the cAMP response element modulator (CREM) gene play an important role in the transcriptional response to cAMP in endocrine cells. By virtue of an alternative, intronic promoter within the gene, the inducible cAMP early repressor (ICER) isoform is generated. ICER was shown to act as a dominant negative regulator and to be cAMP-inducible in various neuroendocrine cells and tissues. ICER negatively autoregulates its own expression and has been postulated to participate in the molecular events governing oscillatory hormonal regulations. To elucidate ICER function in pituitary physiology, we have generated AtT20 corticotroph cell lines expressing the sense or antisense ICER transcript under the control of the cadmium-inducible human methallothionein IIA promoter. Here we demonstrate that changes in the regulated levels of ICER have drastic consequences on the physiology of the corticotrophs. Ectopic ICER expression induces remarkable modifications in AtT20 morphology. Cells with persistent, nonregulated high levels of ICER are blocked in the G2/M phase of the cell cycle, while the opposite effect is obtained in cells expressing an antisense ICER transcript. We show that the effect of ICER on the AtT20 cell cycle is correlated to a direct down-regulation of the cyclin A gene promoter by ICER. Finally, we show that ACTH hormonal secretion from the corticotrophs is completely blocked by ICER ectopic expression. Interestingly, this effect is not due to a direct regulation of the POMC gene, but is mediated by a transcriptional control of the prohormone convertase 1 gene. These results point to a key regulatory function of CREM in pituitary physiology.
Ruchaud S(1), Seité P, Foulkes NS, Sassone-Corsi P, Lanotte M. (1997). The transcriptional repressor ICER and cAMP-induced programmed cell death. Oncogene. 15(7):827-36.
Abstract
The cAMP pathway plays a central role in the response to hormonal signals for cell proliferation, differentiation and apoptosis. In IPC-81 leukaemia cells, activation of the cAMP pathway by prostaglandin E1 treatment, or other cAMP-elevating agents, induces apoptosis within 4-6 h. Inhibition of mRNA or protein synthesis during the first 2 h of cAMP induction protects cells from apoptosis, suggesting a requirement for early gene expression. cAMP-dependent protein kinase phosphorylates a class of nuclear factors and thereby regulates the transcription of a specific set of genes. Here we show that CREM (cAMP Responsive Element Modulator) expression is induced rapidly upon prostaglandin E1 treatment of IPC-81 cells. The induced transcripts correspond to the early product ICER (Inducible cAMP Early Repressor). ICER expression remains elevated until the burst of cell death. Protein synthesis inhibitors which prevent cAMP-induced apoptosis also block de novo ICER synthesis. Transfected IPC-81 cell lines, constitutively expressing high level of ICER are resistant to cAMP-induced cell death. In these transfected cells, cAMP fails to upregulate the ICER transcripts demonstrating that ICER exerts strongly its repressor function on CRE-containing genes. That an early expression of ICER blocks apoptosis, suggests that gene repression by endogenous ICER in IPC-81 is insufficient or occurs too late to protect cells against death. ICER transfected cells rescued from cAMP-induced apoptosis are growth arrested. It shows for the first time that CREM activation directly participates to the decision of the cell to die. ICER, by sequentially repressing distinct sets of CRE-containing genes could modulate cell fate.
Servillo G(1), Penna L, Foulkes NS, Magni MV, Della Fazia MA, Sassone-Corsi P. (1997). Cyclic AMP signalling pathway and cellular proliferation: induction of CREM during liver regeneration. Oncogene. 14(13):1601-6.
Abstract
The CREM gene encodes both activators and repressors of cAMP-induced gene expression. An isoform of CREM encodes the powerful transcriptional repressor ICER (Inducible cAMP Early Repressor), which has been shown to be inducible by virtue of an alternative, intronic promoter. The CREM gene belongs to the early response class and displays a characteristic neuroendocrine cell- and tissue-specific expression. To date ICER inducibility has been described in non-replicating, terminally differentiated tissues. In this paper we document a robust induction of CREM expression in the regenerating rat liver after partial hepatectomy. This represents the first link of inducible CREM expression to the phenomenon of cellular proliferation. Furthermore, it represents the first example of transcriptional activation of a cAMP-responsive factor in the regenerating liver. This has significant physiological relevance since the adenylate cyclase signalling pathway is strongly implicated in liver regeneration. Finally, we show that the repressor ICER is inducible in the hepatoma cell line H35 upon activation of the adenylate cyclase and phosphorylation of the activator CREB.
Monaco L(1), Lamas M, Tamai K, Lalli E, Zazopoulos E, Penna L, Nantel F, Foulkes NS, Mazzucchelli C, Sassone-Corsi P. ( 51. Adv Second Messenger Phosphoprotein Res. 1997;31:63-74. ). Coupling transcription to signaling pathways: cAMP and nuclear factor cAMP-responsive element modulator. 51. Adv Second Messenger Phosphoprotein Res. 1997;31:63-74.
Abstract
PMID: 9344242 [PubMed - indexed for MEDLINE]
Foulkes NS(1), Sassone-Corsi P. (1996). Transcription factors coupled to the cAMP-signalling pathway. Biochim Biophys Acta. 1288(3):F101-21.
Abstract
PMID: 9011175 [PubMed - indexed for MEDLINE]
Foulkes NS(1), Borjigin J, Snyder SH, Sassone-Corsi P. (1996). Transcriptional control of circadian hormone synthesis via the CREM feedback loop. Proc Natl Acad Sci U S A. 93(24):14140-5.
Abstract
Transcription factor cAMP-responsive element modulator (CREM) plays a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. The use of an alternative, intronic promoter within the CREM gene is responsible for the production of a cAMP-inducible repressor, inducible cAMP early repressor (ICER). ICER negatively autoregulates the ICER promoter, thus generating a feedback loop. We have previously documented a striking, clock-driven circadian fluctuation of CREM expression in the pineal gland. Oscillating ICER levels tightly correlate with fluctuations in the synthesis of the pineal hormone melatonin, whose production is also driven by the endogenous clock. Melatonin in turn regulates the hypothalamic-pituitary axis. The enzyme serotonin N-acetyltransferase (NAT) catalyzes the rate limiting step in melatonin synthesis. Thus, oscillations in NAT levels determine the circadian synthesis of melatonin. Here we demonstrate that NAT expression is dramatically increased in CREM-deficient mice that we have generated by homologous recombination. Characterization of the NAT promoter shows the presence of a ICER binding site. In addition, transfection studies show that ICER powerfully represses NAT transcription. Our results implicate CREM as a central regulator of output functions of the clock. Indeed, CREM acts as a key regulator of oscillatory hormonal synthesis.
Foulkes NS(1), Duval G, Sassone-Corsi P. (1996). Adaptive inducibility of CREM as transcriptional memory of circadian rhythms. Nature. 381(6577):83-5.
Abstract
The CREM gene encodes the transcriptional repressor ICER, which has been implicated in the molecular mechanisms controlling circadian rhythms in mammals. ICER is rhythmically expressed in the pineal gland, with peak levels occurring at night. ICER levels are regulated by light by means of the suprachiasmatic nucleus (SCN); transcription is induced during darkness by adrenergic input to the pineal gland from the SCN, which activates the ICER promoter using cyclic AMP and the transcriptional activator CREB. This induction is transient because ICER represses its own transcription. Here we show that the response of the CREM gene to adrenergic stimulation is determined by night length. Depending on the photoperiod of the prior entraining cycles, the CREM gene is either subsensitive or supersensitive to induction. This differential responsiveness is controlled by the changing balance between positive (CREB) and negative (ICER) transcriptional regulators. Thus, the transcriptional response of the CREM gene is determined by the memory of past photoperiods.
Lamas M(1), Monaco L, Zazopoulos E, Lalli E, Tamai K, Penna L, Mazzucchelli C, Nantel F, Foulkes NS, Sassone-Corsi P. (1996). CREM: a master-switch in the transcriptional response to cAMP. Philos Trans R Soc Lond B Biol Sci. 351(1339):561-7.
Abstract
The CREM gene encodes both repressors and activators of cAMP-dependent transcription in a tissue and developmentally regulated manner. In addition, multiple and cooperative phosphorylation events regulate the function of the CREM proteins. CREM plays a key physiological and developmental role within the hypothalamic-pituitary axis. There is a functional switch in CREM expression during the development of male germ cells which is directed by the pituitary hormone FSH. The CREM protein in germ cells is a powerful activator which appears to function as a master-switch in the regulation of postmeiotic genes. CREM is inducible by activation of the cAMP signalling pathway with the kinetics of an early response gene. The induction is transient, cell-specific, does not involve increased transcript stability and does not require protein synthesis. The subsequent decline in CREM expression requires de novo protein synthesis. The induced transcript encodes ICER and is generated from an alternative, intronic promoter. ICER functions as a powerful repressor of cAMP-induced transcription, and represses the activity of its own promoter, thus constituting a negative autoregulatory loop.
Nantel F(1), Monaco L, Foulkes NS, Masquilier D, LeMeur M, Henriksén K, Dierich A, Parvinen M, Sassone-Corsi P. (1996). Spermiogenesis deficiency and germ-cell apoptosis in CREM-mutant mice. Nature. 380(6570):159-62.
Abstract
Spermiogenesis is a complex process by which postmeiotic male germ cells differentiate into mature spermatozoa. This process involves remarkable structural and biochemical changes including nuclear DNA compaction and acrosome formation. Transcription activator CREM (cyclic AMP-responsive element modulator) is highly expressed in postmeiotic cells, and CREM may be responsible for the activation of several haploid germ cell-specific genes involved in the structuring of the spermatozoon. The specific role of CREM in spermiogenesis was addressed using CREM-mutant mice generated by homologous recombination. Analysis of the seminiferous epithelium in mutant male mice reveals postmeiotic arrest at the first step of spermiogenesis. Late spermatids are completely absent, and there is a significant increase in apoptotic germ cells. We show that CREM deficiency results in the lack of postmeiotic cell-specific gene expression. The complete lack of spermatozoa in the mutant mice is reminiscent of cases of human infertility.
Lalli E(1), Lee JS, Lamas M, Tamai K, Zazopoulos E, Nantel F, Penna L, Foulkes NS, Sassone-Corsi P. (1996). The nuclear response to cAMP: role of transcription factor CREM. Philos Trans R Soc Lond B Biol Sci. 351(1336):201-9.
Abstract
In eukaryotes, transcriptional regulation upon stimulation of the adenylate cyclase signalling pathway is mediated by a family of cAMP-responsive nuclear factors. This family consists of a large number of members which may act as activators or repressors. These factors contain the basic domain/leucine zipper motifs and bind as dimers to cAMP-response elements (CRE). The function of CRE-binding proteins is modulated by phosphorylation by several kinases. The ICER (inducible cAMP early repressor) protein is the only inducible member of this family. The induction of this powerful repressor is likely to be important for the transient nature of cAMP-induced gene expression. CRE-binding proteins have been found to play an important role in the physiology of the pituitary gland, in regulating spermatogenesis, in the response to circadian rhythms and in the molecular basis of memory.
Lamas M(1), Lalli E, Foulkes NS, Sassone-Corsi P. ( 58. Cold Spring Harb Symp Quant Biol. 1996;61:285-94. ). Rhythmic transcription and autoregulatory loops: nuclear pacemaker CREM. 58. Cold Spring Harb Symp Quant Biol. 1996;61:285-94.
Abstract
PMID: 9246457 [PubMed - indexed for MEDLINE]
Monaco L(1), Foulkes NS, Sassone-Corsi P. (1995). Pituitary follicle-stimulating hormone (FSH) induces CREM gene expression in Sertoli cells: involvement in long-term desensitization of the FSH receptor. Proc Natl Acad Sci U S A. 92(23):10673-7.
Abstract
Transcription factor CREM (cAMP-responsive element modulator) plays a pivotal role in the nuclear response to cAMP in neuroendocrine cells. We have previously shown that follicle-stimulating hormone (FSH) directs CREM expression in male germ cells. The physiological importance of FSH in Sertoli cell function prompted us to analyze its effect on CREM expression in these cells. We observed a dramatic and specific increase in the CREM isoform ICER (inducible cAMP early repressor) expression, with a peak 4 h after FSH treatment of primary Sertoli cells. Interestingly, induced levels of ICER protein persist for a considerably longer time. Induction of the repressor ICER accompanies early down-regulation of the FSH receptor transcript, which leads to long-term desensitization. Here we show that ICER represses FSH receptor expression by binding to a CRE-like sequence in the regulatory region of the gene. Our results confirm the crucial role played by CREM in hormonal control and suggest its role in the long-term desensitization phenomenon of peptide membrane receptors.
Stehle JH(1), Foulkes NS, Pévet P, Sassone-Corsi P. (1995). Developmental maturation of pineal gland function: synchronized CREM inducibility and adrenergic stimulation. Mol Endocrinol. 9(6):706-16.
Abstract
The cAMP response element modulator (CREM) gene encodes multiple activators and repressors of cAMP-responsive transcription. Differential splicing generates a developmental switch in CREM function during spermatogenesis, while the use of an alternative promoter is responsible for the production of a cAMP-inducible transcriptional repressor, ICER (inducible cAMP early repressor). The ICER promoter is strongly inducible by cAMP because of the presence of four tandemly repeated cAMP response elements. Furthermore, ICER negatively autoregulates the ICER promoter activity, thus generating a feedback loop. CREM constitutes an early response gene of the cAMP pathway in several neuroendocrine cells. We have previously shown that CREM is highly expressed in the adult rat pineal gland at nighttime. Here, we show that the only additional site of rhythmic ICER expression within the photoneuroendocrine system is the lamina intercalaris. Ontogenetically, the ICER day-night switch and cAMP inducibility mature in the pineal gland at the end of the first postnatal week. Importantly, this correlates with the onset of melatonin synthesis and the establishment of functional adrenergic innervation. At this developmental phase we document a significant increase in protein kinase A levels, thus suggesting that ICER inducibility reflects a complete maturation of the cAMP-dependent signaling pathway at the nuclear level.
Desdouets C(1), Matesic G, Molina CA, Foulkes NS, Sassone-Corsi P, Brechot C, Sobczak-Thepot J. (1995). Cell cycle regulation of cyclin A gene expression by the cyclic AMP-responsive transcription factors CREB and CREM. Mol Cell Biol. 15(6):3301-9.
Abstract
Cyclin A is a pivotal regulatory protein which, in mammalian cells, is involved in the S phase of the cell cycle. Transcription of the human cyclin A gene is cell cycle regulated. We have investigated the role of the cyclic AMP (cAMP)-dependent signalling pathway in this cell cycle-dependent control. In human diploid fibroblasts (Hs 27), induction of cyclin A gene expression at G1/S is stimulated by 8-bromo-cAMP and suppressed by the protein kinase A inhibitor H89, which was found to delay S phase entry. Transfection experiments showed that the cyclin A promoter is inducible by activation of the adenylyl cyclase signalling pathway. Stimulation is mediated predominantly via a cAMP response element (CRE) located at positions -80 to -73 with respect to the transcription initiation site and is able to bind CRE-binding proteins and CRE modulators. Moreover, activation by phosphorylation of the activators CRE-binding proteins and CRE modulator tau and levels of the inducible cAMP early repressor are cell cycle regulated, which is consistent with the pattern of cyclin A inducibility by cAMP during the cell cycle. These results suggest that the CRE is, at least partly, implicated in stimulation of cyclin A transcription at G1/S.
Delmas V(1), Molina CA, Lalli E, de Groot R, Foulkes NS, Masquilier D, Sassone-Corsi P. ( 62. Rev Physiol Biochem Pharmacol. 1994;124:1-28. ). Complexity and versatility of the transcriptional response to cAMP. 62. Rev Physiol Biochem Pharmacol. 1994;124:1-28.
Abstract
PMID: 8209138 [PubMed - indexed for MEDLINE]
Molina CA(1), Foulkes NS, Lalli E, Sassone-Corsi P. (1993). Inducibility and negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early response repressor. Cell. 75(5):875-86.
Abstract
cAMP-responsive element modulator (CREM) expression is tissue specific and developmentally regulated. Here we report that CREM is unique within the family of cAMP-responsive promoter element (CRE)-binding factors since it is inducible by activation of the cAMP signaling pathway. The kinetic of expression is characteristic of an early response gene. The induction is transient and cell specific, does not involve increased transcript stability, and does not require protein synthesis. Significantly, the subsequent decline in CREM expression requires de novo protein synthesis. The induced transcript encodes a novel repressor, inducible cAMP early repressor (ICER), and is generated from an alternative intronic promoter. A cluster of four CREs in this promoter directs cAMP inducibility. ICER binds to these elements and thereby represses the activity of its own promoter, thus constituting a negative autoregulatory loop.
Lalli E(1), Lee JS, Masquilier D, Schlotter F, Foulkes NS, Molina CA, Sassone-Corsi P. (1993). Nuclear response to cyclic AMP: central role of transcription factor CREM (cyclic-AMP-responsive-element modulator). Biochem Soc Trans. 21(4):912-7.
Abstract
PMID: 8132092 [PubMed - indexed for MEDLINE]
Masquilier D(1), Foulkes NS, Mattei MG, Sassone-Corsi P. (1993). Human CREM gene: evolutionary conservation, chromosomal localization, and inducibility of the transcript. Cell Growth Differ. 4(11):931-7.
Abstract
The CREM (cyclic AMP-responsive element modulator) gene encodes multiple regulators of the cyclic AMP transcriptional response. CREM expression has been linked with several key physiological aspects of neuroendocrine pathways. We investigated the conservation of CREM during evolution. Here, we show conservation of CREM sequences in the pig, humans, the chicken, the lemur, and Xenopus. We have also determined the chromosomal localization of the CREM and CREB genes both in the mouse and in humans. We cloned the full human CREM complementary DNA sequence and demonstrate that it has a high degree of sequence identity with the mouse gene. Finally, we show the conservation of CREM cyclic AMP transcriptional inducibility in humans and establish that the induced transcripts correspond to the mouse ICER products.
Stehle JH(1), Foulkes NS, Molina CA, Simonneaux V, Pévet P, Sassone-Corsi P. (1993). Adrenergic signals direct rhythmic expression of transcriptional repressor CREM in the pineal gland. Nature. 365(6444):314-20.
Abstract
Comment in Nature. 1993 Sep 23;365(6444):299-300.
Pubmed 
Mellström B(1), Naranjo JR, Foulkes NS, Lafarga M, Sassone-Corsi P. (1993). Transcriptional response to cAMP in brain: specific distribution and induction of CREM antagonists. Neuron. 10(4):655-65.
Abstract
Changes in cAMP levels are often associated with the modulation of neuronal function. The CREM gene encodes both antagonists and activators of the cAMP-dependent transcriptional response by alternative splicing. CREM transcripts in rat brain show a characteristic pattern of expression, being specific for the inner layer of the cerebral cortex, anterior thalamus, hippocampus, and hypothalamus. Strikingly, the CREM transcripts correspond to the antagonist isoforms in these areas, suggesting a down-regulatory role for CREM in brain; in contrast, the expression of CREM tau and CREB activators is more diffuse and generalized. In the supraoptic nucleus, CREM expression is induced after osmotic stimulus. Importantly, this demonstrates physiological inducibility of CREM, which is novel within the CRE/ATF family.
Foulkes NS(1), Schlotter F, Pévet P, Sassone-Corsi P. (1993). Pituitary hormone FSH directs the CREM functional switch during spermatogenesis. Nature. 362(6417):264-7.
Abstract
The CREM (cyclic AMP-responsive element modulator) gene encodes multiple regulators of the cAMP-transcriptional response by alternative splicing. A developmental switch in CREM expression occurs during spermatogenesis, whereby CREM function is converted from an antagonist to an activator (CREM tau; ref. 2) which accumulates to extremely high levels from the premeiotic spermatocyte stage onwards. To define the physiological mechanisms controlling the CREM developmental switch, we have hypophysectomized rats and observed the extinction of CREM tau expression in testis, thereby demonstrating a central role of the pituitary-hypothalamic axis. We then used the seasonal-dependent modulation of spermatogenesis in hamsters to dissect the hormonal programme controlling this developmental process. By this approach, combined with direct administration of pituitary-derived hormones, we have established that follicle-stimulating hormone (FSH) is responsible for the CREM switch. FSH appears to regulate CREM expression by alternative polyadenylation, which results in a dramatic enhancement of transcript stability.
Laoide BM(1), Foulkes NS, Schlotter F, Sassone-Corsi P. (1993). The functional versatility of CREM is determined by its modular structure. EMBO J. 12(3):1179-91.
Abstract
The CREM gene (cAMP-responsive element modulator) generates both activators and repressors of cAMP-induced transcription by alternative splicing. We determined the exon structure of the CREM gene and have identified new isoforms. We show that CREM isoforms with different structural characteristics are generated by the shuffling of exons to produce proteins with various combinations of functional domains. CREM proteins bind efficiently to CREs and here we demonstrate that the various isoforms heterodimerize in vivo with each other and with CREB. The two alternative DNA binding domains of CREM, which are differentially spliced in the various isoforms, show distinct binding efficiencies, while CREM alpha/CREB heterodimers exhibit stronger binding than CREM beta/CREB heterodimers to a consensus CRE in vitro. We identify the protein domains involved in activation function and find that the phosphorylation domain and a single glutamine-rich domain are sufficient for activation. A minimal CREM repressor, containing only the b-Zip motif, efficiently antagonizes cAMP-induced transcription. In addition, phosphorylation may reduce repressor function, as a CREM beta mutant carrying a mutation of the serine phosphoacceptor site (CREM beta 68) represses more efficiently than the wild-type CREM beta.
Delmas V(1), Laoide BM, Masquilier D, de Groot RP, Foulkes NS, Sassone-Corsi P. (1992). Alternative usage of initiation codons in mRNA encoding the cAMP-responsive-element modulator generates regulators with opposite functions. Proc Natl Acad Sci U S A. 89(10):4226-30.
Abstract
The cAMP-responsive-element modulator (CREM) gene encodes both antagonists (CREM alpha/beta/gamma) and an activator (CREM tau) of cAMP-responsive transcription by alternative splicing. In adult mouse brain a predominant 21-kDa protein, not corresponding to any previously characterized transcript, is detected with specific CREM antibodies. A developmental switch occurs in brain as expression changes at birth from CREM alpha/beta to the 21-kDa protein. We show that the 21-kDa protein corresponds to S-CREM (short CREM), a protein produced by the use of an internal AUG initiation codon in the CREM tau transcript. S-CREM shares with the other CREM proteins the basic DNA-binding and leucine-zipper dimerization domain. S-CREM functions as a transcriptional repressor of cAMP-induced transcription. Thus, two proteins with opposite functions are generated by alternative translation using two AUG codons within the same reading frame.
Foulkes NS(1), Sassone-Corsi P. (1992). More is better: activators and repressors from the same gene. Cell. 68(3):411-4.
Abstract
PMID: 1739963 [PubMed - indexed for MEDLINE]
Foulkes NS(1), Mellström B, Benusiglio E, Sassone-Corsi P. (1992). Developmental switch of CREM function during spermatogenesis: from antagonist to activator. Nature. 355(6355):80-4.
Abstract
Mammalian spermatogenesis consists of a series of complex developmental processes controlled by the pituitary-hypothalamic axis. This flow of biochemical information is directly regulated by the adenylate cyclase signal transduction pathway. We have previously described the CREM (cyclic AMP-responsive element modulator) gene which generates, by cell-specific splicing, alternative antagonists of the cAMP transcriptional response. Here we report the expression of a novel CREM isoform (CREM tau) in adult testis. CREM tau differs from the previously characterized CREM antagonists by the coordinate insertion of two glutamine-rich domains that confer transcriptional activation function. During spermatogenesis there was an abrupt switch in CREM expression. In premeiotic germ cells CREM is expressed at low amounts in the antagonist form. Subsequently, from the pachytene spermatocyte stage onwards, a splicing event generates exclusively the CREM tau activator, which accumulates in extremely high amounts. This splicing-dependent reversal in CREM function represents an important example of developmental modulation in gene expression.
Borrelli E(1), Montmayeur JP, Foulkes NS, Sassone-Corsi P. ( 73. Crit Rev Oncog. 1992;3(4):321-38. ). Signal transduction and gene control: the cAMP pathway. 73. Crit Rev Oncog. 1992;3(4):321-38.
Abstract
The transcriptional activity of a gene can be regulated by a multitude of trans-acting factors that interact with specific cis-acting elements, mostly located in the promoter regions. The function of transcription factors is modulated by intracellular signal transduction pathways, which are activated by specific ligands binding to the appropriate membrane receptors. Here we discuss the links between the activation of the adenylyl cyclase pathway and the transcriptional response of cAMP-inducible genes that is achieved by the interplay of a multitude of nuclear transcription factors such as CREB and CREM.
Foulkes NS(1), Laoide BM, Schlotter F, Sassone-Corsi P. (1991). Transcriptional antagonist cAMP-responsive element modulator (CREM) down-regulates c-fos cAMP-induced expression. Proc Natl Acad Sci U S A. 88(12):5448-52.
Abstract
Protooncogene c-fos is induced by activation of adenylate cyclase through the major cAMP-responsive element (CRE) centered at position -60 of the promoter. cAMP induction is followed by a rapid decrease in transcriptional rate, reminiscent of down-regulation after serum stimulation. Fos protein is known to negatively autoregulate serum-induced transcription of c-fos promoter, but whether Fos is responsible for down-regulation of cAMP-induced transcription is unclear. Here we show that Fos is unable to down-regulate CRE-mediated activation. We present evidence that the transcriptional antagonist CRE modulator (CREM) can bind to c-fos CRE and heterodimerize with activator CRE-binding protein, thereby blocking cAMP induction. Furthermore, expression of antisense CREM enhances c-fos basal and cAMP-induced transcription. CREM does not antagonize serum-induced transcription; therefore, we conclude that down-regulation of c-fos is exerted by different effectors, depending upon which signal transduction pathway is activated. We speculate that, by its c-fos down-regulatory function, CREM may act as an antioncogene.
Foulkes NS(1), Borrelli E, Sassone-Corsi P. (1991). CREM gene: use of alternative DNA-binding domains generates multiple antagonists of cAMP-induced transcription. Cell. 64(4):739-49.
Abstract
We isolated a gene from a mouse pituitary cDNA library that encodes a protein highly homologous to nuclear factor CREB, an activator of cAMP-responsive promoter elements (CREs). We demonstrate that while CREB is expressed uniformly in several cell types, this gene, termed CREM, shows cell-specific expression. CREM has a remarkable organization, since down-stream of the stop codon there is a second, out-of-frame DNA-binding domain. Using PCR and RNAase protection analysis, we have identified three mRNA isoforms that appear to be obtained by differential cell-specific splicing. Sequencing of the isoforms demonstrated alternative usage of the two DNA-binding domains. CREM proteins reveal the same efficiency and specificity of binding to CRE sequences as CREB, but in contrast to CREB, CREM acts as a down-regulator of cAMP-induced transcription.
Poggi V(1), Town M, Foulkes NS, Luzzatto L. (1990). Identification of a single base change in a new human mutant glucose-6-phosphate dehydrogenase gene by polymerase-chain-reaction amplification of the entire coding region from genomic DNA. Biochem J. 271(1):157-60.
Abstract
We report the characterization at the molecular level of a mutant glucose-6-phosphate dehydrogenase (G6PD) gene in a Greek boy who presented with a chronic non-spherocytic haemolytic anaemia. In order to identify the mutation from a small amount of patient material, we adopted an approach which by-passes the need to construct a library by using the polymerase chain reaction. The entire coding region was amplified in eight sections, with genomic DNA as template. The DNA fragments were then cloned in an M13 vector and sequenced. The only difference from the sequence of normal G6PD was a T----G substitution at nucleotide position 648 in exon 7, which predicts a substitution of leucine for phenylalanine at amino acid position 216. This mutation creates a new recognition site for the restriction nuclease BalI. We confirmed the presence of the mutation in the DNA of the patient's mother, who was found to be heterozygous for the new BalI site. This is the first transversion among the point mutations thus far reported in the human G6PD gene.
Jones MD(1), Foulkes NS. (1989). Reverse transcription of mRNA by Thermus aquaticus DNA polymerase. Nucleic Acids Res. 17(20):8387-8.
Abstract
PMCID: PMC334993 PMID: 2478963 [PubMed - indexed for MEDLINE]
Mason PJ(1), Vulliamy TJ, Foulkes NS, Town M, Haidar B, Luzzatto L. (1988). The production of normal and variant human glucose-6-phosphate dehydrogenase in cos cells. Eur J Biochem. 178(1):109-13.
Abstract
Full-length cDNA coding for human glucose-6-phosphate dehydrogenase (G6PD) was inserted into a eukaryotic expression vector containing the immediate early promoter of cytomegalovirus. When this plasmid was introduced into cos cells by transfection it led to the production of high levels of human G6PD. cDNAs containing mutations found in G6PD-deficient individuals were constructed by in vitro mutagenesis and expressed in the same system. Characterization of the G6PD proteins obtained in this way confirmed the primary structure inferred for the variant enzymes. An enzyme in which lysine-205 had been mutated to threonine was produced and found to have no G6PD activity, proving that this lysine residue is essential for enzyme activity in human G6PD.
de Franchis R(1), Cross NC, Foulkes NS, Cox TM. (1988). A potent inhibitor of Taq polymerase copurifies with human genomic DNA. Nucleic Acids Res. 16(21):10355.
Abstract
PMCID: PMC338857 PMID: 3194201 [PubMed - indexed for MEDLINE]
Vulliamy TJ(1), D'Urso M, Battistuzzi G, Estrada M, Foulkes NS, Martini G, Calabro V, Poggi V, Giordano R, Town M, et al. (1988). Diverse point mutations in the human glucose-6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia. Proc Natl Acad Sci U S A. 85(14):5171-5.
Abstract
Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is a common genetic abnormality affecting an estimated 400 million people worldwide. Clinical and biochemical analyses have identified many variants exhibiting a range of phenotypes, which have been well characterized from the hematological point of view. However, until now, their precise molecular basis has remained unknown. We have cloned and sequenced seven mutant G6PD alleles. In the nondeficient polymorphic African variant G6PD A we have found a single point mutation. The other six mutants investigated were all associated with enzyme deficiency. In one of the commonest, G6PD Mediterranean, which is associated with favism among other clinical manifestations, a single amino acid replacement was found (serine----phenylalanine): it must be responsible for the decreased stability and the reduced catalytic efficiency of this enzyme. Single point mutations were also found in G6PD Metaponto (Southern Italy) and in G6PD Ilesha (Nigeria), which are asymptomatic, and in G6PD Chatham, which was observed in an Indian boy with neonatal jaundice. In G6PD "Matera," which is now known to be the same as G6PD A-, two separate point mutations were found, one of which is the same as in G6PD A. In G6PD Santiago, a de novo mutation (glycine----arginine) is associated with severe chronic hemolytic anemia. The mutations observed show a striking predominance of C----T transitions, with CG doublets involved in four of seven cases. Thus, diverse point mutations may account largely for the phenotypic heterogeneity of G6PD deficiency.
Foulkes NS(1), Pandolfi de Rinaldis PP, Macdonnell J, Cross NC, Luzzatto L. (1988). Polymerase chain reaction automated at low cost. Nucleic Acids Res. 16(12):5687-8.
Abstract
PMCID: PMC336795 PMID: 2838825 [PubMed - indexed for MEDLINE]

/var/www/cos/ / http://www.cos.uni-heidelberg.de/ Prof. Dr. Nick Foulkes _e