Molecular Feedback Loop Research Articles

Identification of a novel circadian clock modulator controlling BMAL1 expression through a ROR/REV-ERB-response element-dependent mechanism

Circadian rhythms, biological oscillations with a period of about 24 h, are maintained by an innate genetically determined time-keeping system called the molecular circadian clockwork. Despite the physiological and clinical importance of the circadian clock, development of small molecule modulators targeting the core clock machinery has only recently been initiated. BMAL1, a core clock gene, is controlled by a ROR/REV-ERB-response element (RORE)-dependent mechanism, which plays an important role in stabilizing the period of the molecular circadian clock. Therefore, we aimed to identify a novel small molecule modulator that regulates Bmal1 gene expression in RORE-dependency, thereby influencing the molecular feedback loop of the circadian clock. For this purpose, we carried out a cell-based screen of more than 1000 drug-like compounds, using a luciferase reporter driven by the proximal region of the mouse Bmal1 promoter. One compound, designated KK-S6, repressed the RORE-dependent transcriptional activity of the mBmal1 promoter and reduced endogenous BMAL1 protein expression. More importantly, KK-S6 significantly altered the amplitude of circadian oscillations of Bmal1 and Per2 promoter activities in a dose-dependent manner, but barely affected the period length. KK-S6 effectively decreased mRNA expression of metabolic genes acting downstream of REV-ERBα, Pai-1 and Citrate synthase, that contain RORE cis-element in their promoter. KK-S6 likely acts in a RORE-dependent manner by reinforcing the REV-ERBα activity, though not by the same mechanism as known REV-ERB agonists. In conclusion, the present study demonstrates that KK-S6 functions as a novel modulator of the amplitude of molecular circadian rhythms by influencing RORE-mediated BMAL1 expression.

Neurobiology of Circadian Rhythm Regulation.

Over the past few decades, multilevel research has elucidated the basic neuroanatomy, neurochemistry, and molecular neurobiology of the master circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). The circadian timing system is composed of a large number of cellular oscillators located in the SCN, in non-SCN brain structures, and throughout the body. Cellular-level oscillations are generated by a molecular feedback loop in which circadian clock genes rhythmically regulate their own transcription, as well as that of hundreds of clock-controlled genes. The maintenance of proper coordination within this network of cellular- and tissue-level clocks is essential for health and well-being.

Drosophila TIM Binds Importin α1, and Acts as an Adapter to Transport PER to the Nucleus

Regulated nuclear entry of clock proteins is a conserved feature of eukaryotic circadian clocks and serves to separate the phase of mRNA activation from mRNA repression in the molecular feedback loop. In Drosophila, nuclear entry of the clock proteins, PERIOD (PER) and TIMELESS (TIM), is tightly controlled, and impairments of this process produce profound behavioral phenotypes. We report here that nuclear entry of PER-TIM in clock cells, and consequently behavioral rhythms, require a specific member of a classic nuclear import pathway, Importin α1 (IMPα1). In addition to IMPα1, rhythmic behavior and nuclear expression of PER-TIM require a specific nuclear pore protein, Nup153, and Ran-GTPase. IMPα1 can also drive rapid and efficient nuclear expression of TIM and PER in cultured cells, although the effect on PER is mediated by TIM. Mapping of interaction domains between IMPα1 and TIM/PER suggests that TIM is the primary cargo for the importin machinery. This is supported by attenuated interaction of IMPα1 with TIM carrying a mutation previously shown to prevent nuclear entry of TIM and PER. TIM is detected at the nuclear envelope, and computational modeling suggests that it contains HEAT-ARM repeats typically found in karyopherins, consistent with its role as a co-transporter for PER. These findings suggest that although PER is the major timekeeper of the clock, TIM is the primary target of nuclear import mechanisms. Thus, the circadian clock uses specific components of the importin pathway with a novel twist in that TIM serves a karyopherin-like role for PER.

PER1 Phosphorylation Specifies Feeding Rhythm in Mice

Organization of circadian behavior, physiology, and metabolism is important for human health. An S662G mutation in hPER2 has been linked to familial advanced sleep-phase syndrome (FASPS). Although the paralogous phosphorylation site S714 in PER1 is conserved in mice, its specific function in circadian organization remains unknown. Here, we find that the PER1S714G mutation accelerates the molecular feedback loop. Furthermore, hPER1S714G mice, but not hPER2S662G mice, exhibit peak time of food intake that is several hours before daily energy expenditure peaks. Both the advanced feeding behavior and the accelerated clock disrupt the phase of expression of several key metabolic regulators in the liver and adipose tissue. Consequently, hPER1S714G mice rapidly develop obesity on a high-fat diet. Our studies demonstrate that PER1 and PER2 are linked to different downstream pathways and that PER1 maintains coherence between the circadian clock and energy metabolism.

The regulation of clear cell renal cancer cells proliferation and tyrosine kinase inhibitors responsiveness by tumor micro-environmental factors.

488 Background: Tumor microenvironment is a complex network of multiple cell types, paracrine and endocrine signaling molecules, ECM and physical factors, which orchestrate the fate of tumor progression. In the field of ccRCC little attention has been paid to non-immunologic factors in tumor niche. Objectives of this study include the analysis of hypoxia, triiodothyronine (T3), insulin, insulin-like growth factor-1 and -2 impact on ccRCC cells. The effect of above mentioned tumor niche factors on TKI activity on ccRCC cells was to be quantified. The study was initiated as ccRCC cells are known to harbor disrupted hypoxia response and TRbeta (TRβ) - T3 receptor - functions as a tumor suppressor with tissue hypothyroidism involved in maintaining a proliferative advantage, whereas hypothyroidisms was defined as biomarker of sunitinib treatment efficacy. Moreover obesity and diabetes (DM2) are considered risk factors for ccRCC and high IGF-I expression is associated with poor long-term patient survival. This research aims to find molecular background for clinically relevant observations. Methods: ccRCC cells from primary and metastatic tumors (ATCC, MSK collections) along with cancer progenitor cells (CD133+) were cultured in normoxic or hypoxic conditions; in defined media supplemented with T3, IN, IGF, TRβ inhibitor and TKIs (sunitinib, axitinib). Cell proliferation was evaluated using AlamarBlue and MTT. Colony formation potential was evaluated. TRβ gene expression was measured with RealTime-RT PCR and sequenced under standard protocol. Normal kidney proximal tube cells were used as negative control. Results: ccRCC TKI responsiveness is limited by hypoxia, at the same time tumor cell proliferation may be promoted by T3, IN and IGF. Colony formation potential of ccRCC cells under TKI treatment in hypoxic conditions is downregulated. Conclusions: Multiple micro-environmental factors modify the proliferation and/or differentiation of ccRCC cell populations. Understanding the complex molecular feedback loops between cancer cells and the surrounding microenvironment should aid the identification of novel targets for improving TKIs clinical management.

Temporal orchestration of repressive chromatin modifiers by circadian clock Period complexes

The mammalian circadian clock is built on a molecular feedback loop in which the Period (PER) proteins, acting in a large, poorly understood complex, repress Clock-Bmal1, the transcription factor driving their expression. We found that mouse PER complexes include the histone methyltransferase HP1γ-Suv39h. PER proteins recruited HP1γ-Suv39h to the Per1 and Per2 promoters, and HP1γ-Suv39h proved important for circadian di- and trimethylation of histone H3 Lys9 (H3K9) at the Per1 promoter, feedback repression and clock function. HP1γ-Suv39h was recruited to the Per1 and Per2 promoters ~4 h after recruitment of HDAC1, a PER-associated protein previously implicated in clock function and H3K9 deacetylation at the Per1 promoter. PER complexes containing HDAC1 or HP1γ-Suv39h appeared to be physically separable. Circadian clock negative feedback by the PER complex thus involves dynamic, ordered recruitment of repressive chromatin modifiers to DNA-bound Clock-Bmal1.

Identification and Validation of Cryptochrome Inhibitors That Modulate the Molecular Circadian Clock

Circadian rhythms, biological oscillations with a period of about 24 h, are maintained by a genetically determined innate time-keeping system called the molecular circadian clockwork. Despite the physiological and clinical importance of the circadian clock, the development of small molecule modulators that directly target the core clock machinery has only been recently initiated. In the present study, we aimed to identify novel small molecule modulators influencing the molecular feedback loop of the circadian clock by applying our two-step cell-based screening strategy based on E-box-mediated transcriptional activity to test more than 1000 drug-like compounds. A derivative of 2-ethoxypropanoic acid designated as compound 15 was selected as the most promising candidate in terms of both efficacy and potency. We then performed pull-down assays with the biotinylated compound and find out that both cryptochrome (CRY)1 and 2 (CRY1/2), key negative components of the mammalian circadian clock, as molecular targets of compound 15. In accordance with the binding property, compound 15 enhanced E-box-mediated transcription in a CRY1/2-dependent manner, and more importantly, it attenuated the circadian oscillation of Per2-Luc and Bmal1-dLuc activities in cultured fibroblasts, indicating that compound 15 can functionally inhibit the effects of CRY1/2 in the molecular circadian clockwork. In conclusion, the present study describes the first novel chemical inhibitor of CRY1/2 that inhibits the repressive function of CRY1/2, thereby activating CLOCK-BMAL1-evoked E-box-mediated transcription. Further optimizations and subsequent functional studies of this compound may lead to development of efficient therapeutic strategies for a variety of physiological and metabolic disorders with circadian natures.

Root hairs.

Roots hairs are cylindrical extensions of root epidermal cells that are important for acquisition of nutrients, microbe interactions, and plant anchorage. The molecular mechanisms involved in the specification, differentiation, and physiology of root hairs in Arabidopsis are reviewed here. Root hair specification in Arabidopsis is determined by position-dependent signaling and molecular feedback loops causing differential accumulation of a WD-bHLH-Myb transcriptional complex. The initiation of root hairs is dependent on the RHD6 bHLH gene family and auxin to define the site of outgrowth. Root hair elongation relies on polarized cell expansion at the growing tip, which involves multiple integrated processes including cell secretion, endomembrane trafficking, cytoskeletal organization, and cell wall modifications. The study of root hair biology in Arabidopsis has provided a model cell type for insights into many aspects of plant development and cell biology.

Cyclin-dependent Kinase 5 (Cdk5) Regulates the Function of CLOCK Protein by Direct Phosphorylation

Circadian rhythm is a biological rhythm governing physiology and behavior with a period of ∼24 h. At the molecular level, circadian output is controlled by a molecular clock composed of positive and negative feedback loops in transcriptional and post-translational processes. CLOCK is a transcription factor known as a central component of the molecular clock feedback loops generating circadian oscillation. Although CLOCK is known to undergo multiple post-translational modifications, the knowledge of their entities remains limited. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine-threonine kinase that is involved in various neuronal processes. Here, we report that Cdk5 is a novel regulator of CLOCK protein. Cdk5 phosphorylates CLOCK at the Thr-451 and Thr-461 residues in association with transcriptional activation of CLOCK. The Cdk5-dependent regulation of CLOCK function is mediated by alterations of its stability and subcellular distribution. These results suggest that Cdk5 is a novel regulatory component of the core molecular clock machinery.

Role of miR-142-3p in the Post-Transcriptional Regulation of the Clock Gene Bmal1 in the Mouse SCN

MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional modulators by regulating stability or translation of target mRNAs. Recent studies have implicated miRNAs in the regulation of mammalian circadian rhythms. To explore the role of miRNAs in the post-transcriptional modulation of core clock genes in the master circadian pacemaker, we examined miR-142-3p for evidence of circadian expression in the suprachiasmatic nuclei (SCN), regulation of its putative clock gene target Bmal1 via specific binding sites in the 3′ UTR and overexpression-induced changes in the circadian rhythm of BMAL1 protein levels in SCN cells. In mice exposed to constant darkness (DD), miR-142-3p levels in the SCN were characterized by circadian rhythmicity with peak expression during early subjective day at CT 3. Mutagenesis studies indicate that two independent miRNA recognition elements located at nucleotides 1–7 and 335–357 contribute equally to miR-142-3p-induced repression of luciferase-reported Bmal1 3′ UTR activity. Importantly, overexpression of miR-142-3p in immortalized SCN cells abolished circadian variation in endogenous BMAL1 protein levels in vitro. Collectively, our results suggest that miR-142-3p may play a role in the post-transcriptional modulation of Bmal1 and its oscillatory regulation in molecular feedback loops mediating SCN circadian function.

Distinct and Separable Roles for Endogenous CRY1 and CRY2 within the Circadian Molecular Clockwork of the Suprachiasmatic Nucleus, as Revealed by the Fbxl3AfhMutation

The circadian clock of the suprachiasmatic nucleus (SCN) drives daily rhythms of behavior. Cryptochromes (CRYs) are powerful transcriptional repressors within the molecular negative feedback loops at the heart of the SCN clockwork, where they periodically suppress their own expression and that of clock-controlled genes. To determine the differential contributions of CRY1 and CRY2 within circadian timing in vivo, we exploited the N-ethyl-N-nitrosourea-induced afterhours mutant Fbxl3(Afh) to stabilize endogenous CRY. Importantly, this was conducted in CRY2- and CRY1-deficient mice to test each CRY in isolation. In both CRY-deficient backgrounds, circadian rhythms of wheel-running and SCN bioluminescence showed increased period length with increased Fbxl3(Afh) dosage. Although both CRY proteins slowed the clock, CRY1 was significantly more potent than CRY2, and in SCN slices, CRY1 but not CRY2 prolonged the interval of transcriptional suppression. Selective CRY-stabilization demonstrated that both CRYs are endogenous transcriptional repressors of clock-controlled genes, but again CRY1 was preeminent. Finally, although Cry1(-/-);Cry2(-/-) mice were behaviorally arrhythmic, their SCN expressed short period (~18 h) rhythms with variable stability. Fbxl3(Afh/Afh) had no effect on these CRY-independent rhythms, confirming its circadian action is mediated exclusively via CRYs. Thus, stabilization of both CRY1 and CRY2 are necessary and sufficient to explain circadian period lengthening by Fbxl3(Afh/Afh). Both CRY proteins dose-dependently lengthen the intrinsic, high-frequency SCN rhythm, and CRY2 also attenuates the more potent period-lengthening effects of CRY1. Incorporation of CRY-mediated transcriptional feedback thus confers stability to intrinsic SCN oscillations, establishing periods between 18 and 29 h, as determined by selective contributions of CRY1 and CRY2.

Mutual cytokine crosstalk between colon cancer cells and microenvironment initiates development of distant metastases

Tumor growth and cancer development are considered clear examples of Darwinian selection, whereby random mutational events in heterogeneous cancer cell populations that best fit the selective microenvironment are preferred.1 As a result, cancer cells evolve resistance to apoptosis, hide from immune surveillance and acquire the ability to invade other organs. Cancer cells, however, are not necessarily passive subjects of selection; they can actively subvert the host tissue to provide a favorable habitat for their growth. Recent findings by Calon et al. convincingly demonstrate that transforming growth factor-β-induced secretion of interleukin 11 by tumor stromal fibroblasts is a necessary prerequisite for the development of distant metastases in colorectal carcinoma. Thus, understanding the complex molecular feedback loops between cancer cells and the surrounding microenvironment (i.e., the tumor-associated stroma or invaded host tissue) should aid the identification of useful molecular targets for improving clinical management of advanced metastatic cancers.

Linking Morphogen and Chromatin in the Hair Follicle

In this issue of Developmental Cell, Xiong etal. (2013) identify a critical role for the chromatin remodeler Brg1 in hair follicle stem cell maintenance and epidermal repair. Brg1 interacts with the Shh signaling pathway to create a positive feedback loop that fuels hair follicle growth.

Transglutaminase 2 and NF-κB: an odd couple that shapes breast cancer phenotype

Owing to numerous pro-survival target genes, aberrant activation of the NF-κB transcription factor is associated with a drug-resistant phenotype and aggressive breast tumor behavior. Transglutaminase 2 (TG2), a ubiquitously expressed protein cross-linking enzyme, activates NF-κB through a non-conventional mechanism that disables the IκBα inhibitor. Our group has recently documented that the TG2 gene (termed TGM2) is a direct transcriptional target of NF-κB. These developments uncover a novel self-reinforcing molecular feedback loop where TG2 activates NF-κB and, in turn, NF-κB directly upregulates the transcription of TGM2. This manuscript reviews the literature that supports the existence of the TG2/NF-κB signaling loop, the nature of the signal transduction that activates this loop, and the phenotypic consequences stemming from the aberrant activation of this novel signaling mechanism in breast cancer.

Hepatocyte growth factor in the neighborhood reverses resistance to BRAF inhibitor in melanoma

Hepatocyte growth factor in the neighborhood reverses resistance to <scp>BRAF</scp> inhibitor in melanoma

Cloning, tissue expression pattern and daily rhythms of Period1, Period2, and Clock transcripts in the flatfish Senegalese sole, Solea senegalensis

An extensive network of endogenous oscillators governs vertebrate circadian rhythmicity. At the molecular level, they are composed of a set of clock genes that participate in transcriptional-translational feedback loops to control their own expression and that of downstream output genes. These clocks are synchronized with the environment, although entrainment by external periodic cues remains little explored in fish. In this work, partial cDNA sequences of clock genes representing both positive (Clock) and negative (Period1, Period2) elements of the molecular feedback loops were obtained from the nocturnal flatfish Senegalese sole, a relevant species for aquaculture and chronobiology. All of the above genes exhibited high identities with their respective teleost clock genes, and Per-Arnt-Sim or basic helix-loop-helix binding domains were recognized in their primary structure. They showed a widespread distribution through the animal body and some of them displayed daily mRNA rhythms in central (retina, optic tectum, diencephalon, and cerebellum) and peripheral (liver) tissues. These rhythms were most robust in retina and liver, exhibiting marked Period1 and Clock daily oscillations in transcript levels as revealed by ANOVA and cosinor analysis. Interestingly, expression profiles were inverted in retina and optic tectum compared to liver. Such differences suggest the existence of tissue-dependent zeitgebers for clock gene expression in this species (i.e., light for retina and optic tectum and feeding time for liver). This study provides novel insight into the location of the molecular clocks (central vs. peripheral) and their different phasing and synchronization pathways, which contributes to better understand the teleost circadian systems and its plasticity.

Time for Testosterone: The Suprachiasmatic Nucleus Gets Sexy

One mechanism that has evolved to support optimal survival and success is an endogenous daily timekeeping capability, or circadian system. This system coordinates the timing of physiology and behavior, including reproductive events such as hormone release, ovulation, mating, parturition, and offspring care (1, 2). Given the life history and physiologicaldifferencesbetweenmalesand females, it isnot surprising that these daily patterns exhibit sex differences and sensitivity to reproductive hormones (3). Natural life events, such as puberty, reproductive cycles, pregnancy, and menopause, as well as artificial hormone administration can alter the pacing (period) of endogenous rhythms or the alignment of these rhythms with time cues from the external environment, such as the solar day (3–7). Some of these changes are due to the activational (or immediate, transient) effects of reproductive hormones on circadian rhythms, but hormones can also give rise to organizational (long term, permanent) effects during development, producing sexual differentiation of circadian rhythm parameters and physiology (3). Until recently there was substantial debate over whether these effects of reproductive hormones on circadian rhythms could be the consequence of direct modulation of the central mammalian circadian regulator [the suprachiasmatic nuclei of the hypothalamus (SCN)]. This dispute arose because early reports were contradictory regarding the presence of hormone receptors in the region (4), suggesting that hormones might instead act downstream from the SCN by altering specific output pathways. This suspicion was further reinforced by the rapidity of some of the hormonal effects on circadian rhythms (8) because rhythms in the SCN were viewed as being slow to adjust after perturbation. The paper by Karatsoreos et al. (9) in the current issue sheds light on this debate because it provided a clear example of reproductive hormones (androgens) altering key components of SCN organization and function in male mice. The discovery that reproductive hormones could alter SCN function was facilitated by several large breakthroughs in our understanding of circadian physiology. Researchers determined that the daily cycle underlying circadian rhythms is generated by a molecular negative feedback loop within individual pacemaker cells in the SCN. Within this feedback loop, proteins produced by particular clock genes inhibit their own transcription, leading todaily cellularoscillations (10).Thesepacemaker cellsusereciprocalcommunicationtoamplifyoscillationand maintain a specific alignment between rhythmic events, a function referred to as oscillator coupling. It has been argued that the abundance of nonneuronal cells, called glia, in the SCN may play an important role in this coupling by modulating intercellular communication (11). Oscillator coupling is crucial because pacemaker cells can be divided into functional subgroups, characterized by different neurotransmitter content and connectivity. This provides a division of labor within the SCN. One functional subgroup (frequently referred to as the core of the SCN) is highly sensitive to environmental time cues. At particular times of day, time cues (typically light) cause an induction of components of the clock gene feedback loop, producing an overall phase shift (or realignment) of clock gene rhythms relative to the outside world. Another functional subgroup (often referred to as the shell of the SCN) sustains strong endogenous oscillation in the absence of rhythmic cues (12). Thus, the circadian system is entrained to maintain a particular alignment with environmental

Mammalian Molecular Clocks

As a consequence of the Earth's rotation, almost all organisms experience day and night cycles within a 24-hr period. To adapt and synchronize biological rhythms to external daily cycles, organisms have evolved an internal time-keeping system. In mammals, the master circadian pacemaker residing in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus generates circadian rhythmicity and orchestrates numerous subsidiary local clocks in other regions of the brain and peripheral tissues. Regardless of their locations, these circadian clocks are cell-autonomous and self-sustainable, implicating rhythmic oscillations in a variety of biochemical and metabolic processes. A group of core clock genes provides interlocking molecular feedback loops that drive the circadian rhythm even at the single-cell level. In addition to the core transcription/translation feedback loops, post-translational modifications also contribute to the fine regulation of molecular circadian clocks. In this article, we briefly review the molecular mechanisms and post-translational modifications of mammalian circadian clock regulation. We also discuss the organization of and communication between central and peripheral circadian oscillators of the mammalian circadian clock.

Differential Expression of the Circadian Clock in Maternal and Embryonic Tissues of Mice

BackgroundMolecular feedback loops involving transcription and translation and several key genes are at the core of circadian regulatory cycles affecting cellular pathways and metabolism. These cycles are active in most adult animal cells but little is known about their expression or influence during development.Methodology/Principal FindingsTo determine if circadian cycles are active during mammalian development we measured the expression of key circadian genes during embryogenesis in mice using quantitative real-time RT-PCR. All of the genes examined were expressed in whole embryos beginning at the earliest age examined, embryonic day 10. In contrast to adult tissues, circadian variation was absent for all genes at all of the embryonic ages examined in either whole embryos or individual tissues. Using a bioluminescent fusion protein that tracks translation of the circadian gene, per2, we also analyzed protein levels. Similar to mRNA, a protein rhythm was observed in adult tissue but not in embryonic tissues collected in-vivo. In contrast, when tissues were placed in culture for the continuous assay of bioluminescence, rhythms were observed in embryonic (E18) tissues. We found that placing embryonic tissues in culture set the timing (phase) of these rhythms, suggesting the importance of a synchronizing signal for the expression of circadian cycles in developing tissues.Conclusions/SignificanceThese results show that embryonic tissues express key circadian genes and have the capacity to express active circadian regulatory cycles. In vivo, circadian cycles are not expressed in embryonic tissues as they are in adult tissues. Individual cells might express oscillations, but are not synchronized until later in development.

Beyond the suprachiasmatic nucleus: Other circadian clocks in the brain

While the suprachiasmatic nucleus (SCN) is considered the “master pacemaker” governing mammalian circadian rhythms, there is substantial evidence for a methamphetamine-sensitive circadian oscillator (MASCO) capable of driving rhythms even in the absence of the SCN. We have recently demonstrated that MASCO does not require the canonical circadian molecular feedback loop to drive circadian rhythms of locomotor behavior. Here we argue that MASCO is an independent circadian pacemaker and remark upon its similarity to another extra-SCN oscillator—the food entrainable oscillator (FEO).