Autoregulatory Feedback Research Articles

The PAS Protein VIVID Defines a Clock-Associated Feedback Loop that Represses Light Input, Modulates Gating, and Regulates Clock Resetting

vvd, a gene regulating light responses in Neurospora, encodes a novel member of the PAS/LOV protein superfamily. VVD defines a circadian clock–associated autoregulatory feedback loop that influences light resetting, modulates circadian gating of input by connecting output and input, and regulates light adaptation. Rapidly light induced, vvd is an early repressor of light-regulated processes. Further, vvd is clock controlled; the clock gates light induction of vvd and the clock gene frq so identical signals yield greater induction in the morning. Mutation of vvd severely dampens gating, especially of frq, consistent with VVD modulating gating and phasing light-resetting responses. vvd null strains display distinct alterations in the phase–response curve to light. Thus VVD, although not part of the clock, contributes significantly to regulation within the Neurospora circadian system.

View of a mouse clock gene ticking

Circadian clocks consist of an ingenious autoregulatory feedback loop whereby the cyclically expressed products of the clock gene are able to inhibit their own expression<1. Here we follow the rhythmic expression of the clock gene mPer1 in the brain of a living mouse. This model system enables real-time gene expression to be monitored in the intact brain under physiological conditions.

The human PER1 gene is transcriptionally regulated by multiple signaling pathways

The mammalian period ( Per) genes are components of the circadian clock and appear to be regulated via an autoregulatory feedback loop. Here we show that the human PER1 ( hPER1) gene is synergistically activated by protein kinases A and C (PKA, PKC) and cAMP responsive element binding protein. Activators and inhibitors of PKA as well as PKC modulate endogenous hPER1 expression and hPER1 promoter-driven reporter gene activity in a dose-dependent manner. Our results suggest that the hPER1 promoter acts as a sensor for multiple signaling molecules thereby integrating different physiological parameters. This regulation of hPER1 appears to be significant for rapid adaptation to changing environmental conditions.

GENETICS OF THE MAMMALIAN CIRCADIAN SYSTEM: Photic Entrainment, Circadian Pacemaker Mechanisms, and Posttranslational Regulation

During the past four years, significant progress has been made in identifying the molecular components of the mammalian circadian clock system. An autoregulatory transcriptional feedback loop similar to that described in Drosophila appears to form the core circadian rhythm generating mechanism in mammals. Two basic helix-loop-helix (bHLH) PAS (PER-ARNT-SIM) transcription factors, CLOCK and BMAL1, form the positive elements of the system and drive transcription of three Period and two Cryptochrome genes. The protein products of these genes are components of a negative feedback complex that inhibits CLOCK and BMAL1 to close the circadian loop. In this review, we focus on three aspects of the circadian story in mammals: the genetics of the photic entrainment pathway; the molecular components of the circadian pacemaker in the hypothalamic suprachiasmatic nucleus; and the role of posttranslational regulation of circadian elements. A molecular description of the mammalian circadian system has revealed that circadian oscillations may be a fundamental property of many cells in the body and that a circadian hierarchy underlies the temporal organization of animals.

Circadian clockwork: two loops are better than one.

The spectacularly successful race over the past three years to place our understanding of the circadian clockwork of mammals into a molecular framework is beginning to yield the cardinal example of the molecular-genetic control of behaviour. This perspective describes recent evidence for the conservation of a double-loop, autoregulatory feedback mechanism across the best understood eukaryotic circadian systems, and discusses how these findings may illuminate some long-standing puzzles concerning our subliminal sense of circadian time.

An autoregulatory function of Dfos during Drosophila endoderm induction.

The endoderm of Drosophila is patterned during embryogenesis by an inductive cascade emanating from the adhering mesoderm. An immediate-early endodermal target gene of this induction is Dfos whose expression is upregulated in the middle midgut by Dpp signalling. Previous evidence based on a dominant-negative Dfos construct indicated that Dfos may cooperate with Dpp signalling to induce the HOX gene labial, the ultimate target gene of the inductive cascade. Here, we examine kayak mutants that lack Dfos to establish that Dfos is indeed required for labial induction. We provide evidence that Dfos acts through a CRE-like sequence, previously identified to be a target for signalling by Dpp and by the Epidermal growth factor receptor (Egfr) in the embryonic midgut. We show that Dfos expression is stimulated by Egfr signalling. Finally, we find that Dfos function is required for its own upregulation. Thus, endoderm induction is based on at least four tiers of positive autoregulatory feedback loops.

Signaling angiogenesis via p42/p44 MAP kinase and hypoxia

Angiogenesis is associated with a number of pathological situations. In this study, we have focused our attention on the role of p42/p44 MAP (mitogen-activated protein) kinases and hypoxia in the control of angiogenesis. We demonstrate that p42/p44 MAP kinases play a pivotal role in angiogenesis by exerting a determinant action at three levels: i) persistent activation of p42/p44 MAP kinases abrogates apoptosis; ii) p42/p44 MAP kinase activity is critical for controlling proliferation and growth arrest of confluent endothelial cells; and iii) p42/p44 MAP kinases promote VEGF (vascular endothelial growth factor) expression by activating its transcription via recruitment of the AP-2/Sp1 (activator protein-2) complex on the proximal region (−88/−66) of the VEGF promoter and by direct phosphorylation of hypoxia-inducible factor 1α (HIF-1α). HIF-1α plays a crucial role in the control of HIF-1 activity, which mediates hypoxia-induced VEGF expression. We show that oxygen-regulated HIF-1α protein levels are not affected by intracellular localization (nucleus versus cytoplasm). Finally, we propose a model which suggests an autoregulatory feedback mechanism controlling HIF-1α and therefore HIF-1-dependent gene expression.

Relative roles of the fla/che P(A), P(D-3), and P(sigD) promoters in regulating motility and sigD expression in Bacillus subtilis.

Three promoters have been identified as having potentially important regulatory roles in governing expression of the fla/che operon and of sigD, a gene that lies near the 3' end of the operon. Two of these promoters, fla/che P(A) and P(D-3), lie upstream of the >26-kb fla/che operon. The third promoter, P(sigD), lies within the operon, immediately upstream of sigD. fla/che P(A), transcribed by E sigma(A), lies >/=24 kb upstream of sigD and appears to be largely responsible for sigD expression. P(D-3), transcribed by E sigma(D), has been proposed to participate in an autoregulatory positive feedback loop. P(sigD), a minor sigma(A)-dependent promoter, has been implicated as essential for normal expression of the fla/che operon. We tested the proposed functions of these promoters in experiments that utilized strains that bear chromosomal deletions of fla/che P(A), P(D-3), or P(sigD). Our analysis of these strains indicates that fla/che P(A) is absolutely essential for motility, that P(D-3) does not function in positive feedback regulation of sigD expression, and that P(sigD) is not essential for normal fla/che expression. Further, our results suggest that an additional promoter(s) contributes to sigD expression.

Local feedback mechanisms in human breast cancer.

Breast function and development are controlled by a variety of both local and systemic signals. Many of these signals are exerted by hormones and cytokines which are believed to be effectors in autoregulatory feedback loops. Recent studies have also suggested the involvement of such mechanisms in human breast cancer. For example, the disruption of a negative feedback system by malignant transformation can result in the loss of growth control or in increased malignant behavior of tumor cells. Conversely, pathological positive feedback loops can develop that enhance tumor growth and invasion by excessive release of stimulatory factors. These loops are often located at the site of tumor invasion and involve stromal-epithelial interactions. They can be composed of mutually stimulating or inhibiting cytokines and may include locally expressed sex steroids. Although most studies have concentrated on cell-cell interactions at the site of the primary tumor, a number of observations indicate their importance in metastases as well. A thorough analysis of the regulatory mechanisms within a malignant tumor is essential for the understanding of its unique behavior and for the investigation of more specific breast cancer therapies.

Circadian clock-protein expression in cyanobacteria: rhythms and phase setting.

The cyanobacterial gene cluster kaiABC encodes three essential circadian clock proteins: KaiA, KaiB and KaiC. The KaiB and KaiC protein levels are robustly rhythmical, whereas the KaiA protein abundance undergoes little if any circadian oscillation in constant light. The level of the KaiC protein is crucial for correct functioning of the clock because induction of the protein at phases when the protein level is normally low elicits phase resetting. Titration of the effects of the inducer upon phase resetting versus KaiC level shows a direct correlation between induction of the KaiC protein within the physiological range and significant phase shifting. The protein synthesis inhibitor chloramphenicol prevents the induction of KaiC and blocks phase shifting. When the metabolism is repressed by either translational inhibition or constant darkness, the rhythm of KaiC abundance persists; therefore, clock protein expression has a preferred status under a variety of conditions. These data indicate that rhythmic expression of KaiC appears to be a crucial component of clock precession in cyanobacteria.

Ca2+-sensitive Inactivation and Facilitation of L-type Ca2+ Channels Both Depend on Specific Amino Acid Residues in a Consensus Calmodulin-binding Motif in theα1C subunit

L-type Ca(2+) channels are unusual in displaying two opposing forms of autoregulatory feedback, Ca(2+)-dependent inactivation and facilitation. Previous studies suggest that both involve direct interactions between calmodulin (CaM) and a consensus CaM-binding sequence (IQ motif) in the C terminus of the channel's alpha(1C) subunit. Here we report the functional effects of an extensive series of modifications of the IQ motif aimed at dissecting the structural determinants of the different forms of modulation. Although the combined substitution by alanine at five key positions (Ile(1624), Gln(1625), Phe(1628), Arg(1629), and Lys(1630)) abolished all Ca(2+) dependence, corresponding single alanine replacements behaved similarly to the wild-type channel (77wt) in four of five cases. The mutant I1624A stood out in displaying little or no Ca(2+)-dependent inactivation, but clear Ca(2+)- and frequency-dependent facilitation. An even more pronounced tilt in favor of facilitation was seen with the double mutant I1624A/Q1625A: overt facilitation was observed even during a single depolarizing pulse, as confirmed by two-pulse experiments. Replacement of Ile(1624) by 13 other amino acids produced graded and distinct patterns of change in the two forms of modulation. The extent of Ca(2+)-dependent facilitation was monotonically correlated with the affinity of CaM for the mutant IQ motif, determined in peptide binding experiments in vitro. Ca(2+)-dependent inactivation also depended on strong CaM binding to the IQ motif, but showed an additional requirement for a bulky, hydrophobic side chain at position 1624. Abolition of Ca(2+)-dependent modulation by IQ motif modifications mimicked and occluded the effects of overexpressing a dominant-negative CaM mutant.

Gene Regulatory Networks Generating the Phenomena of Additivity, Dominance and Epistasis

We show how the phenomena of genetic dominance, overdominance, additivity, and epistasis are generic features of simple diploid gene regulatory networks. These regulatory network models are together sufficiently complex to catch most of the suggested molecular mechanisms responsible for generating dominant mutations. These include reduced gene dosage, expression or protein activity (haploinsufficiency), increased gene dosage, ectopic or temporarily altered mRNA expression, increased or constitutive protein activity, and dominant negative effects. As classical genetics regards the phenomenon of dominance to be generated by intralocus interactions, we have studied two one-locus models, one with a negative autoregulatory feedback loop, and one with a positive autoregulatory feedback loop. To include the phenomena of epistasis and downstream regulatory effects, a model of a three-locus signal transduction network is also analyzed. It is found that genetic dominance as well as overdominance may be an intra- as well as interlocus interaction phenomenon. In the latter case the dominance phenomenon is intimately connected to either feedback-mediated epistasis or downstream-mediated epistasis. It appears that in the intra- as well as the interlocus case there is considerable room for additive gene action, which may explain to some degree the predictive power of quantitative genetic theory, with its emphasis on this type of gene action. Furthermore, the results illuminate and reconcile the prevailing explanations of heterosis, and they support the old conjecture that the phenomenon of dominance may have an evolutionary explanation related to life history strategy.

Nitric Oxide Inhibits Inducible Nitric Oxide Synthase mRNA Expression in RAW 264.7 Macrophages

Using cultured murine RAW 264.7 macrophages, the present study investigates the influence of nitric oxide (NO) on the expression of the inducible NO synthase (iNOS) enzyme at the transcriptional level. Incubation of cells with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) led to a marked increase in iNOS mRNA levels. Inhibition of LPS/IFN-γ-induced NO synthesis with the L-arginine analogue NG-monomethyl-L-arginine (L-NMMA) was accompanied by a significant up-regulation of iNOS mRNA that was reversed in the presence of the NO donor sodium nitroprusside (SNP). Treatment of cells with SNP alone decreased LPS/IFN-γ-induced iNOS mRNA levels in a concentration-dependent manner. The inhibitory effect of SNP on iNOS mRNA expression was not prevented by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of the soluble guanylyl cyclase. In agreement with this finding, incubation of cells with the membrane-permeable cyclic GMP analogue 8-bromo cyclic GMP left LPS/IFN-γ-induced iNOS mRNA expression virtually unaltered. Together, our results demonstrate that both iNOS-derived and exogenous NO exert an inhibitory effect on the expression of iNOS by a mechanism independent of the soluble guanylyl cyclase/cyclic GMP pathway. In conclusion, NO may control the extent of iNOS mRNA expression by a negative autoregulatory feedback.

Conservation of polyamine regulation by translational frameshifting from yeast to mammals.

Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. Here we show that a similar mechanism exists in the fission yeast Schizosaccharomyces pombe. The expression of mammalian antizyme genes requires a specific +1 translational frameshift. The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop. As shown here, the yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression. Twelve nucleotides around the frameshift site are identical between S.pombe and the mammalian counterparts. The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals.

Timekeeping in worms

During the past few years, circadian clock genes have been discovered in various model systems, from Drosophila and Neurospora to cyanobacteria. Many clock components share conserved features and encode transcription factors of the PAS domain family, which function within transcriptional autoregulatory feedback loops. Surprisingly, given the wealth of information concerning its genetics and development, one animal model, Caenorhabditis elegans, has been conspicuously absent from these studies because a circadian rhythm, as yet, had not been demonstrated in it. In this paper1xSimilarity of the C. elegans developmental timing protein LIN-42 to circadian rhythm proteins. Jeon, M. et al. Science. 1999; 286: 1141–1146Crossref | PubMed | Scopus (110)See all References1, Jeon et al. show that worms do have clock gene homologues. By studying heterochronic mutants in which the timing and sequence of various developmental events is abnormal, they found that one gene, lin-42, which affects hypodermal differentiation, encodes a PAS domain protein that closely resembles the Drosophila clock gene per product, period. However, unlike the circadian rhythm of per expression in Drosophila, lin-42 oscillates with a 6-hour rhythm that correlates with the molting cycle. Also unlike per, lin-42 expression continues to oscillate in lin-42 mutant worms – thus, the protein is not required to generate the transcript rhythm. Drosophila period heterodimerizes with another clock component, timeless (encoded by tim), to translocate into the nucleus. Expression of a C. elegans tim homologue, which Jeon et al. identified by computer analysis of the complete genomic sequence, does not oscillate, unlike Drosophila tim expression. Furthermore, its expression pattern does not overlap with that of lin-42. Nevertheless, by linking a clock gene homologue with a developmental timing function, this work opens up the exciting possibility that different biological timing systems might have a similar molecular organization and might even share common components.

Molecular clocks (joint Juan March/EMBO workshop). Madrid, May 10-12, 1999.

Circadian rhythms in the physiology of plants and animals constitute key adaptations to survive day–night changes in the environment. In all species, an endogenous circadian clock directs these rhythms. A distinctive property of the clock is that it continues to function even under constant environmental conditions to generate an ∼24 h (circadian) rhythm. The rhythm is synchronized (entrained) with the external day–night cycle by environmental signals, the most important of which is light. The clock seems to consist of three functional components: (i) an autonomous circadian pacemaker that generates the oscillation; (ii) an input pathway that synchronizes the pacemaker with the day–night cycle; and (iii) an output pathway whereby the pacemaker generates physiological rhythms. During the past decade considerable effort has been made to identify clock components. The most successful approach has been based on mutant screens using Arabidopsis , cyanobacteria, Drosophila , Neurospora and, more recently, mice. A number of mutants have now been isolated and characterized in which clock outputs show altered timing. Genetic analysis has ultimately led to the cloning of genes involved in the function of the central pacemaker. It has become increasingly clear that the basic properties and organization of clock components are highly conserved even between these divergent species. Most of the clock genes identified encode transcription factors, which positively or negatively regulate their own transcription and thus constitute autoregulatory feedback loops. Additional clock components have been shown to regulate the intracellular localization, stability and transactivation potential of these transcriptional regulators. The last 3 years have seen an avalanche of publications revealing new features of the circadian clock in the different model systems. For this reason, the ‘Molecular clocks’ meeting organized by the Juan March Foundation in Madrid, Spain, was particularly timely. It brought together many of the major players in the field and …

MDM2 oncogene as a novel target for human cancer therapy.

The MDM2 oncogene was first cloned as an amplified gene on a murine double-minute chromosome in the 3T3DM cell line, a spontaneously transformed derivative of BALB/c 3T3 cells. The MDM2 oncogene has now been shown to be amplified or overexpressed in many human cancers. It also has been suggested that MDM2 levels are associated with poor prognosis of several human cancers. The most exciting finding is the MDM2-p53 autoregulatory feedback loop that regulates the function of the p53 tumor suppressor gene. The MDM2 gene is a target for direct transcriptional activation by p53, and the MDM2 protein is a negative regulator of p53. The MDM2 oncoprotein binds to the p53 protein, inhibiting p53 functions as a transcription factor and inducing p53 degradation. The p53 tumor suppressor has an important role in cancer therapy, with p53-mediated cell growth arrest and/or apoptosis being major mechanisms of action for many clinically used cancer chemotherapeutic agents and radiation therapy. Therefore, the MDM2-p53 interaction may be a target for cancer therapy. In addition, the negative regulation of p53 by MDM2 may limit the magnitude of p53 activation by DNA damaging agents, thereby limiting their therapeutic effectiveness. If the MDM2 feed-back inhibition of p53 is interrupted, a significant increase in functional p53 levels will increase p53-mediated therapeutic effectiveness. Several approaches have now been tested using this strategy, including polypeptides targeted to MDM2-p53 binding domain and antisense oligonucleotides that specifically inhibit MDM2 expression. In addition to the interaction with p53, the MDM2 protein has been found to have interactions with other cellular proteins such as pRb and E2F-1. Although the exact function and significance of these interactions are not fully understood, the p53-independent functions of MDM2 may have a role in cancer etiology and progression, indicating that the MDM2 oncogene is a potential molecular target for cancer therapy.

A regulatory network for the efficient control of transgene expression.

Expression of heterologous genes in mammalian cells or organisms for therapeutic or experimental purposes often requires tight control of transgene expression. Specifically, the following criteria should be met: no background gene activity in the off-state, high gene expression in the on-state, regulated expression over an extended period, and multiple switching between on- and off-states. Here, we describe a genetic switch system for controlled transgene transcription using chimeric repressor and activator proteins functioning in a novel regulatory network. In the off-state, the target transgene is actively silenced by a chimeric protein consisting of multimerized eukaryotic transcriptional repression domains fused to the DNA-binding tetracycline repressor. In the on-state, the inducer drug doxycycline affects both the derepression of the target gene promoter and activation by the GAL4-VP16 transactivator, which in turn is under the control of an autoregulatory feedback loop. The hallmark of this new system is the efficient transgene silencing in the off-state, as demonstrated by the tightly controlled expression of the highly cytotoxic diphtheria toxin A gene. Addition of the inducer drug allows robust activation of transgene expression. In stably transfected cells, this control is still observed after months of repeated cycling between the repressed and activated states of the target genes. This system permits tight long-term regulation when stably introduced into cell lines. The underlying principles of this network system should have general applications in biotechnology and gene therapy.

Molecular genetics of circadian rhythms in mammals.

Recent gene discovery approaches have led to a new era in our understanding of the molecular basis of circadian oscillators in animals. A conserved set of genes in Drosophila and mammals (Clock, Bmal1, Period, and Timeless) provide a molecular framework for the circadian mechanism. These genes define a transcription-translation-based negative autoregulatory feedback loop that comprises the core elements generating circadian rhythmicity. This circadian core provides a focal point for understanding how circadian rhythms arise, how environmental inputs entrain the oscillatory system, and how the circadian system regulates its outputs. The addition of molecular genetic approaches to the existing physiological understanding of the mammalian circadian system provides new opportunities for understanding this basic life process.

The clock gene period of the housefly, Musca domestica, rescues behavioral rhythmicity in Drosophila melanogaster. Evidence for intermolecular coevolution?

In Drosophila, the clock gene period (per), is an integral component of the circadian clock and acts via a negative autoregulatory feedback loop. Comparative analyses of per genes in insects and mammals have revealed that they may function in similar ways. However in the giant silkmoth, Antheraea pernyi, per expression and that of the partner gene, tim, is not consistent with the negative feedback role. As an initial step in developing an alternative dipteran model to Drosophila, we have identified the per orthologue in the housefly, Musca domestica. The Musca per sequence highlights a pattern of conservation and divergence similar to other insect per genes. The PAS dimerization domain shows an unexpected phylogenetic relationship in comparison with the corresponding region of other Drosophila species, and this appears to correlate with a functional assay of the Musca per transgene in Drosophila melanogaster per-mutant hosts. A simple hypothesis based on the coevolution of the PERIOD and TIMELESS proteins with respect to the PER PAS domain can explain the behavioral data gathered from transformants.