AtGRP7, a nuclear RNA-binding protein as a component of a circadian-regulated negative feedback loop in Arabidopsis thaliana

The endogenous clock that drives circadian rhythms is thought to communicate temporal information within the cell via cycling downstream transcripts. A transcript encoding a glycine-rich RNA-binding protein, Atgrp7, in Arabidopsis thaliana undergoes circadian oscillations with peak levels in the evening. The AtGRP7 protein also cycles with a time delay so that Atgrp7 transcript levels decline when the AtGRP7 protein accumulates to high levels. After AtGRP7 protein concentration has fallen to trough levels, Atgrp7 transcript starts to reaccumulate. Overexpression of AtGRP7 in transgenic Arabidopsis plants severely depresses cycling of the endogenous Atgrp7 transcript. These data establish both transcript and protein as components of a negative feedback circuit capable of generating a stable oscillation. AtGRP7 overexpression also depresses the oscillation of the circadian-regulated transcript encoding the related RNA-binding protein AtGRP8 but does not affect the oscillation of transcripts such as cab or catalase mRNAs. We propose that the AtGRP7 autoregulatory loop represents a “slave” oscillator in Arabidopsis that receives temporal information from a central “master” oscillator, conserves the rhythmicity by negative feedback, and transduces it to the output pathway by regulating a subset of clock-controlled transcripts.

Translational coupling by modulation of feedback repression in the IF3 operon of Escherichia coli

A pseudoknot formed by a long-range interaction in the mRNA of the initiation factor 3 (IF3) operon is involved in the translational repression of the gene encoding ribosomal protein L35 by another ribosomal protein, L20. The nucleotides forming the 5′ strand of the key stem of the pseudoknot are located within the gene for IF3, whereas those forming the 3′ strand are located 280 nt downstream, immediately upstream of the Shine–Dalgarno sequence of the gene for L35. Here we show that premature termination of IF3 translation at a nonsense codon introduced upstream of the pseudoknot results in a substantial enhancement of L20-mediated repression of L35 expression. Conversely, an increase of IF3 translation decreases repression. These results, in addition to an analysis of the effect of mutations in sequences forming the pseudoknot, indicate that IF3 translation decreases L20-mediated repression of L35 expression. We propose that ribosomes translating IF3 disrupt the pseudoknot and thereby attenuate repression. The result is a novel type of translational coupling, where unfolding of the pseudoknot by ribosomes translating IF3 does not increase expression of L35 directly, but alleviates its repression by L20.

Feedback-inducible nuclear-receptor-driven reporter gene expression in transgenic mice

Understanding nuclear receptor signaling in vivo would be facilitated by an efficient methodology to determine where a nuclear receptor is active. Herein, we present a feedback-inducible expression system in transgenic mice to detect activated nuclear receptor effector proteins by using an inducible reporter gene. With this approach, reporter gene induction is not limited to a particular tissue, and, thus, this approach provides the opportunity for whole-animal screens. Furthermore, the effector and reporter genes are combined to generate a single strain of transgenic mice, which enables direct and rapid analysis of the offspring. The system was applied to localize sites where the retinoic acid receptor ligand-binding domain is activated in vivo. The results identify previously discovered sources of retinoids in the embryo and indicate the existence of previously undiscovered regions of retinoic acid receptor signaling in vivo. Notably, the feedback-inducible nuclear-receptor-driven assay, combined with an independent in vitro assay, provides evidence for a site of retinoid synthesis in the isthmic mesenchyme. These data illustrate the potential of feedback-inducible nuclear-receptor-driven analyses for assessing in vivo activation patterns of nuclear receptors and for analyzing pharmacological properties of natural and synthetic ligands of potential therapeutic value.

The negative feedback actions of progesterone on gonadotropinreleasing hormone secretion are transduced by the classical progesterone receptor

Progesterone (P) powerfully inhibits gonadotropin-releasing hormone (GnRH) secretion in ewes, as in other species, but the neural mechanisms underlying this effect remain poorly understood. Using an estrogen (E)-free ovine model, we investigated the immediate GnRH and luteinizing hormone (LH) response to acute manipulations of circulating P concentrations and whether this response was mediated by the nuclear P receptor. Simultaneous hypophyseal portal and jugular blood samples were collected over 36 hr: 0–12 hr, in the presence of exogenous P (P treatment begun 8 days earlier); 12–24 hr, P implant removed; 24–36 hr, P implant reinserted. P removal caused a significant rapid increase in the GnRH pulse frequency, which was detectable within two pulses (175 min). P insertion suppressed the GnRH pulse frequency even faster: the effect detectable within one pulse (49 min). LH pulsatility was modulated identically. The next two experiments demonstrated that these effects of P are mediated by the nuclear P receptor since intracerebroventricularly infused P suppressed LH release but 3α-hydroxy-5α-pregnan-20-one, which operates through the type A γ-aminobutyric acid receptor, was without effect and pretreatment with the P-receptor antagonist RU486 blocked the ability of P to inhibit LH. Our final study showed that P exerts its acute suppression of GnRH through an E-dependent system because the effects of P on LH secretion, lost after long-term E deprivation, are restored after 2 weeks of E treatment. Thus we demonstrate that P acutely inhibits GnRH through an E-dependent nuclear P-receptor system.

A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control

The p53 tumor suppressor protein and the MDM2 oncoprotein form a feedback-control loop that up-regulates cellular MDM2 production, blocks p53 activity, and promotes p53 decay. tsg101 was discovered as a gene whose deficiency results in neoplastic transformation of NIH 3T3 cells and the ability to generate metastatic tumors in nude mice. Its protein product contains a domain, Ubc, characteristic of the catalytic domain of ubiquitin conjugase (E2) enzymes but lacking an active-site cysteine crucial for ubiquitin conjugase activity. Here we report that TSG101 participates with MDM2 in an autoregulatory loop that modulates the cellular levels of both proteins, and also of p53, by affecting protein decay. We show that the Ubc domain of TSG101 interferes with ubiquitination of MDM2, that TSG101 inhibits MDM2 decay and elevates its steady-state level, and that these events are associated with down-regulation of p53 protein. Conversely, pulse–chase and Western blot experiments in wild-type and mutant fibroblasts indicate that elevation of MDM2 by overexpression of wild-type p53, by amplification of the endogenous MDM2 gene, or by transfection of MDM2-expressing constructs promotes TSG101 loss, which we show occurs by 26S proteasome-dependent decay. Our results identify TSG101 as both a regulator of, and target of, MDM2/p53 circuitry.

RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: A negative feedback circuit

The RPN4 (SON1, UFD5) protein of the yeast Saccharomyces cerevisiae is required for normal levels of intracellular proteolysis. RPN4 is a transcriptional activator of genes encoding proteasomal subunits. Here we show that RPN4 is required for normal levels of these subunits. Further, we demonstrate that RPN4 is extremely short-lived (t1/2 ≈2 min), that it directly interacts with RPN2, a subunit of the 26S proteasome, and that rpn4Δ cells are perturbed in their cell cycle. The degradation signal of RPN4 was mapped to its N-terminal region, outside the transcription–activation domains of RPN4. The ability of RPN4 to augment the synthesis of proteasomal subunits while being metabolically unstable yields a negative feedback circuit in which the same protein up-regulates the proteasome production and is destroyed by the assembled active proteasome.

A feedback-controlled ensemble model of the stress-responsive hypothalamo-pituitary-adrenal axis

The present work develops and implements a biomathematical statement of how reciprocal connectivity drives stress-adaptive homeostasis in the corticotropic (hypothalamo-pituitary-adrenal) axis. In initial analyses with this interactive construct, we test six specific a priori hypotheses of mechanisms linking circadian (24-h) rhythmicity to pulsatile secretory output. This formulation offers a dynamic framework for later statistical estimation of unobserved in vivo neurohormone secretion and within-axis, dose-responsive interfaces in health and disease. Explication of the core dynamics of the stress-responsive corticotropic axis based on secure physiological precepts should help to unveil new biomedical hypotheses of stressor-specific system failure.

Feedback Control and Diurnal Regulation of Gibberellin 20-Oxidase Transcript Levels in Potato1

Tuber formation in potato (Solanum tuberosum) is promoted by short photoperiods and is inhibited by gibberellins (GAs). Endogenous levels of GA1 were shown to decrease in stolons and leaves of potato plants induced to tuberize, which suggests that photoperiodic regulation of GA biosynthesis may play a role in tuber induction. We report the isolation of three potato cDNA clones (StGA20ox1–3) encoding GA 20-oxidase, a key regulatory enzyme in the GA-biosynthetic pathway. Using northern analysis, we detected a differential pattern of tissue-specific expression of the mRNAs corresponding to these clones. StGA20ox mRNAs were also very abundant in leaves of the potato ga1 mutant, which is blocked in the 13-hydroxylation step, and were strongly down-regulated by gibberellic acid, suggesting a feedback regulation of these genes. In plants grown in short-day (inductive) conditions, levels of the StGA20ox transcripts in leaves fluctuated during a 24-h period, with a peak of accumulation observed about 4 h after the lights were turned off. Interruption of the night with a 30-min “night break” of light (noninductive conditions) did not have a marked effect on the levels of accumulation of the three GA 20-oxidase mRNAs during the day, but it induced a second peak of expression of StGA20ox1 and StGA20ox3 transcripts late in the night. This observation, together with the finding that StGA20ox1 mRNA is expressed at high levels in leaves, suggests that night-break induction of this gene might play a role in the control of tuberization by regulating endogenous levels of GAs in response to daylength conditions.

Feedback Inhibition of Chlorophyll Synthesis in the Phytochrome Chromophore-Deficient aurea and yellow-green-2 Mutants of Tomato

The aurea (au) and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum L.) are unable to synthesize the linear tetrapyrrole chromophore of phytochrome, resulting in plants with a yellow-green phenotype. To understand the basis of this phenotype, we investigated the consequences of the au and yg-2 mutations on tetrapyrrole metabolism. Dark-grown seedlings of both mutants have reduced levels of protochlorophyllide (Pchlide) due to an inhibition of Pchlide synthesis. Feeding experiments with the tetrapyrrole precursor 5-aminolevulinic acid (ALA) demonstrate that the pathway between ALA and Pchlide is intact in au and yg-2 and suggest that the reduction in Pchlide is a result of the inhibition of ALA synthesis. This inhibition was independent of any deficiency in seed phytochrome, and experiments using an iron chelator to block heme synthesis demonstrated that both mutations inhibited the degradation of the physiologically active heme pool, suggesting that the reduction in Pchlide synthesis is a consequence of feedback inhibition by heme. We discuss the significance of these results in understanding the chlorophyll-deficient phenotype of the au and yg-2 mutants.

Desert dust suppressing precipitation: A possible desertification feedback loop

The effect of desert dust on cloud properties and precipitation has so far been studied solely by using theoretical models, which predict that rainfall would be enhanced. Here we present observations showing the contrary; the effect of dust on cloud properties is to inhibit precipitation. Using satellite and aircraft observations we show that clouds forming within desert dust contain small droplets and produce little precipitation by drop coalescence. Measurement of the size distribution and the chemical analysis of individual Saharan dust particles collected in such a dust storm suggest a possible mechanism for the diminished rainfall. The detrimental impact of dust on rainfall is smaller than that caused by smoke from biomass burning or anthropogenic air pollution, but the large abundance of desert dust in the atmosphere renders it important. The reduction of precipitation from clouds affected by desert dust can cause drier soil, which in turn raises more dust, thus providing a possible feedback loop to further decrease precipitation. Furthermore, anthropogenic changes of land use exposing the topsoil can initiate such a desertification feedback process.

Timing of metamorphosis and the onset of the negative feedback loop between the thyroid gland and the pituitary is controlled by type II iodothyronine deiodinase in Xenopus laevis

Two important features of amphibian metamorphosis are the sequential response of tissues to different concentrations of thyroid hormone (TH) and the development of the negative feedback loop between the pituitary and the thyroid gland that regulates TH synthesis by the thyroid gland. At the climax of metamorphosis in Xenopus laevis (when the TH level is highest), the ratio of the circulating precursor thyroxine (T4) to the active form 3,5,3′-triiodothyronine (T3) in the blood is many times higher than it is in tissues. This difference is because of the conversion of T4 to T3 in target cells of the tadpole catalyzed by the enzyme type II iodothyronine deiodinase (D2) and the local effect (cell autonomy) of this activity. Limb buds and tails express D2 early and late in metamorphosis, respectively, correlating with the time that these organs undergo TH-induced change. T3 is required to complete metamorphosis because the peak concentration of T4 that is reached at metamorphic climax cannot induce the final morphological changes. At the climax of metamorphosis, D2 expression is activated specifically in the anterior pituitary cells that express the genes for thyroid-stimulating hormone but not in the cells that express proopiomelanocortin. Physiological concentrations of T3 but not T4 can suppress thyrotropin subunit β gene expression. The timing and the remarkable specificity of D2 expression in the thyrotrophs of the anterior pituitary coupled with the requirement for locally synthesized T3 strongly support a role for D2 in the onset of the negative feedback loop at the climax of metamorphosis.

Interlocked feedback loops contribute to the robustness of the Neurospora circadian clock

Interlocked feedback loops may represent a common feature among the regulatory systems controlling circadian rhythms. The Neurospora circadian feedback loops involve white collar-1 (wc-1), wc-2, and frequency (frq) genes. We show that WC-1 and WC-2 proteins activate the transcription of frq gene, whereas FRQ protein plays dual roles: repressing its own transcription, probably by interacting with the WC-1/WC-2 complex, and activating the expression of both WC proteins. Thus, they form two interlocked feedback loops: one negative and one positive. We establish the physiological significance of the interlocked positive feedback loops by showing that the levels of WC-1 and WC-2 determine the robustness and stability of the clock. Our data demonstrate that with WC-1 being the limiting factor in the WC-1/WC-2 complex, the greater the levels of WC-1 and WC-2, the higher the level of the FRQ oscillation and the more robust the overt rhythms. Our data also show that, despite considerable changes in the levels of WC-1, WC-2, and FRQ, the period of the clock has been limited to a small range, suggesting that the interlocked circadian feedback loops are also important for determining the circadian period length of the clock.