Regulatory Gate Research Articles

Reinvestigating the regulatory gate hypothesis for a better understanding of microbial redundancy in soil

Reinvestigating the regulatory gate hypothesis for a better understanding of microbial redundancy in soil

Single-cell multi-omics analysis identifies context-specific gene regulatory gates and mechanisms.

There is a growing interest in inferring context specific gene regulatory networks from single-cell RNA sequencing (scRNA-seq) data. This involves identifying the regulatory relationships between transcription factors (TFs) and genes in individual cells, and then characterizing these relationships at the level of specific cell types or cell states. In this study, we introduce scGATE (single-cell gene regulatory gate) as a novel computational tool for inferring TF-gene interaction networks and reconstructing Boolean logic gates involving regulatory TFs using scRNA-seq data. In contrast to current Boolean models, scGATE eliminates the need for individual formulations and likelihood calculations for each Boolean rule (e.g. AND, OR, XOR). By employing a Bayesian framework, scGATE infers the Boolean rule after fitting the model to the data, resulting in significant reductions in time-complexities for logic-based studies. We have applied assay for transposase-accessible chromatin with sequencing (scATAC-seq) data and TF DNA binding motifs to filter out non-relevant TFs in gene regulations. By integrating single-cell clustering with these external cues, scGATE is able to infer context specific networks. The performance of scGATE is evaluated using synthetic and real single-cell multi-omics data from mouse tissues and human blood, demonstrating its superiority over existing tools for reconstructing TF-gene networks. Additionally, scGATE provides a flexible framework for understanding the complex combinatorial and cooperative relationships among TFs regulating target genes by inferring Boolean logic gates among them.

LeishIF4E2 is a cap-binding protein that plays a role in Leishmania cell cycle progression.

Leishmania encode six paralogs of the cap-binding protein eIF4E and five eIF4G candidates, forming unique complexes. Two cap-binding proteins, LeishIF4E1 and LeishIF4E2, do not bind any identified LeishIF4Gs, thus their roles are intriguing. Here, we combine structural prediction, proteomic analysis, and interaction assays to shed light on LeishIF4E2 function. A nonconserved C-terminal extension was identified through structure prediction and sequence alignment. m7 GTP-binding assays involving both recombinant and transgenic LeishIF4E2 with and without the C-terminal extension revealed that this extension functions as a regulatory gate, modulating the cap-binding activity of LeishIF4E2. The interactomes of the two LeishIF4E2 versions were investigated, highlighting the role of the C-terminal extension in binding to SLBP2. SLBP2 is known to interact with a stem-loop structure in the 3' UTRs of histone mRNAs. Consistent with the predicted inhibitory effect of SLBP2 on histone expression in Xenopus laevis, a hemizygous deletion mutant of LeishIF4E2, exhibited an upregulation of several histones. We therefore propose that LeishIF4E2 is involved in histone expression, possibly through its interaction between SLBP2 and LeishIF4E2, thus affecting cell cycle progression. In addition, cell synchronization showed that LeishIF4E2 expression decreased during the S-phase, when histones are known to be synthesized. Previous studies in T. brucei also highlighted an association between TbEIF4E2 and SLBP2, and further reported on an interaction between TbIF4E2 and S-phase-abundant mRNAs. Our results show that overexpression of LeishIF4E2 correlates with upregulation of cell cycle and chromosome maintenance proteins. Along with its effect on histone expression, we propose that LeishIF4E2 is involved in cell cycle progression.

Choroid plexus volume is increased in mood disorders and associates with circulating inflammatory cytokines

Depressed patients exhibit altered levels of immune-inflammatory markers both in the peripheral blood and in the cerebrospinal fluid (CSF) and inflammatory processes have been widely implicated in the pathophysiology of mood disorders. The Choroid Plexus (ChP), located at the base of each of the four brain ventricles, regulates the exchange of substances between the blood and CSF and several evidence supported a key role for ChP as a neuro-immunological interface between the brain and circulating immune cells. Given the role of ChP as a regulatory gate between periphery, CSF spaces and the brain, we compared ChP volumes in patients with bipolar disorder (BP) or major depressive disorder (MDD) and healthy controls, exploring their association with history of illness and levels of circulating cytokines. Plasma levels of inflammatory markers and MRI scans were acquired for 73 MDD, 79 BD and 72 age- and sex-matched healthy controls (HC). Patients with either BD or MDD had higher ChP volumes than HC. With increasing age, the bilateral ChP volume was larger in patients, an effect driven by the duration of illness; while only minor effects were observed in HC. Right ChP volumes were proportional to higher levels of circulating cytokines in the clinical groups, including IFN-γ, IL-13 and IL-17. Specific effects in the two diagnostic groups were observed when considering the left ChP, with positive association with IL-1ra, IL-13, IL-17, and CCL3 in BD, and negative associations with IL-2, IL-4, IL-1ra, and IFN-γ in MDD. These results suggest that ChP could represent a reliable and easy-to-assess biomarker to evaluate the brain effects of inflammatory status in mood disorders, contributing to personalized diagnosis and tailored treatment strategies.

Genome-wide characterization of gap junction (connexins and pannexins) genes in turbot (Scophthalmus maximus L.): evolution and immune response following Vibrio anguillarum infection.

Gap junction (GJ), a special intercellular junction between different cell types, directly connects the cytoplasm of adjacent cells, allows various molecules, ions and electrical impulses to pass through the intercellular regulatory gate, and plays vital roles in response to bacterial infection. Up to date, the information about the GJ in turbot (Scophthalmus maximus L.) is still limited. In current study, 43 gap junction genes were identified in turbot, phylogeny analysis suggested that gap junctions from turbot and other species were clustered into six groups, GJA, GJB, GJC, GJD, GJE and PANX, and turbot GJs together with respective GJs from Japanese flounder, half-smooth tongue sole and large yellow croaker, sharing same ancestors. In addition, these 43 GJ genes distributed in different chromosomes unevenly. According to gene structure and domain analysis, these genes (in GJA-GJE group) were highly conserved in that most of them contain the transmembrane area, connexin domain (CNX) and cysteine-rich domain (connexin CCC), while PANXs contain Pfam Innexin. Although only one tandem duplication was identified in turbot gap junction gene, 235 pairs of segmental duplications were identified in the turbot genome. To further investigate their evolutionary relationships, Ka/Ks was calculated, and results showed that most ratios were lower than 1, indicating they had undergone negative selection. Finally, expression analysis showed that gap junction genes were widely distributed in turbot tissues and significantly regulated after Vibrio anguillarum infection. Taken together, our research could provide valuable information for further exploration of the function of gap junction genes in teleost.

Abiotic processes dominate soil organic matter mineralization: Investigating the regulatory gate hypothesis by inoculating a previously fumigated soil with increasing fresh soil inocula

The Regulatory Gate Hypothesis proposes that the rate of soil organic C mineralization is largely controlled by abiotic processes rather than biological ones. To further test this hypothesis, a repeatedly fumigated soil was inoculated with increasing weights of the initial fresh soil (0, 2, 10 and 25%) and then incubated for 30 days. The CO2-C evolved, soil microbial biomass C and adenosine 5′-triphosphate were measured at days 1, 5, and 30. The fresh soil was incubated as a control. Our aim was to further test the Regulatory Gate hypothesis that the rate of mineralization of stabilized soil organic matter is not regulated by the size, activity or community structure of the microbial biomass.Cumulative CO2 evolved changed little, irrespective of weight of inoculum, in the inoculated soils during the incubation. Soil respiration rates of inoculated soils (21 µg CO2-C g−1 soil) were significantly higher than those of non-inoculated-fumigated soil (18 µg CO2-C g−1 soil) and fresh soil (18.5 µg CO2-C g−1 soil) at the beginning of the incubation (day 0–1). However, they were not significantly different (6.5 µg CO2-C g−1 soil in inoculated soils) from those in non-inoculated-fumigated and fresh soil (5.5 and 7.1 µg CO2-C g−1 soil respectively) at the end of the incubation. Soil adenosine 5′-triphosphate (ATP) proportionally increased in the inoculated soils, but remained much lower (0.49–0.78 nmol g−1 soil) than that in fresh soil (2.28 nmol g−1 soil) until the end of the incubation. The recoveries of the microbial communities and diversity in the inoculated soils were proportional to the inoculation rates. Proteobacteria, mainly Gammaproteobacteria, had the highest recovery in inoculated soils after incubation, and Nitrospira the lowest. We conclude that soil organic matter mineralization is not regulated by the size or composition of the soil microbial population, but mainly depends on the conversion of non-bioavailable SOC to bioavailable substrate C. The precise processes involved now require further research to improve our understanding of the mechanisms driving the mineralization of soil organic matter.

Soil biochemical properties and bacteria community in a repeatedly fumigated-incubated soil

The regulatory gate hypothesis considers that soil organic C mineralization is a two-step process, where stable C is firstly transformed abiotically and is only then a microbially available substrate. The mechanisms involved in the abiotic conversion of non-microbially available to available soil organic C remain largely unknown. We conducted a perfusion experiment using a repeated fumigated-incubated soil and the corresponding fresh soil. We found that repeated fumigation-incubation significantly decreased soil microbial ATP to 0.22 nmol g−1 soil, 10% of that in the fresh soil, and significantly destroyed microbial composition and diversity. However, it had little influence on the soil CO2-C evolution rate after the flush of fumigant-killed dead biomass (8 μg CO2-C g−1 soil day−1) or dissolved organic C (DOC) concentration (about 7 μg C g−1 soil) and composition during long-term perfusion. We conclude that soil CO2 evolution rate and DOC generation were not regulated by the size or composition of the soil microbial communities. This is in support of the regulatory gate hypothesis. We suggest that abiotic processes in soil organic C mineralization need to be considered more and studied further.

Is the rate of mineralization of soil organic carbon under microbiological control?

A theory called the Regulatory Gate Hypothesis was previously proposed to considers that the rate limiting step in soil organic carbon (SOC) mineralization is independent of the size, community stricture or specific activity (mg CO2-C evolved g−1 biomass C) of the soil microbial biomass. Here we report new experiments to test this hypothesis. In the first experiment, six different soils were perfused with CHCl3-saturated water to model SOC release and to stop microbial activity. Apart from one highly organic soil, they all released SOC at low and roughly constant rates, over sixty three days. In the second experiment, when the freeze-dried perfusates were returned to the parent soils, their % mineralization ranged from 17 to 35% over ten days, in contrast to bulk SOC (range 0.46–0.77%). In the third experiment, two soils were given three consecutive fumigations, each followed by 10 days aerobic incubation. The microbial biomass was decreased by > 90%, yet SOC mineralization proceeded at the same rate as in nonfumigated soil. In the fourth experiment, the six soils were subjected to various perturbations, including non-perturbed controls, fumigation-incubation, air-drying rewetting, freeze-thaw (−20 °C and −80 °C) and sieving < 0.3 mm. After an initial flush due to the perturbations, the rates of mineralization became roughly equal in nearly all soil treatments and comparable to the control, despite significant declines in biomass. This shows that basal respiration was little affected by the perturbations. In Experiment five the effects of the perturbations on the microbial communities in the different soils and perturbations were determined. The bacterial community was significantly modified by both fumigation and air drying-rewetting, due mainly to increased Firmiculites and decreased Proteobacteria populations. Our findings suggest that mineralization of SOC is a two-stage process: firstly, non-bioavailable forms are converted abiologically to bioavailable forms (termed the Regulatory Gate), which, only then, undergo second process, biological mineralization. This finding has serious implications for theories of e.g. SOC dynamics, effects of global warming and soil nutrient cycling.

Precise cis-regulatory control of spatial and temporal expression of the alx-1 gene in the skeletogenic lineage of s. purpuratus

Deployment of the gene-regulatory network (GRN) responsible for skeletogenesis in the embryo of the sea urchin Strongylocentrotus purpuratus is restricted to the large micromere lineage by a double negative regulatory gate. The gate consists of a GRN subcircuit composed of the pmar1 and hesC genes, which encode repressors and are wired in tandem, plus a set of target regulatory genes under hesC control. The skeletogenic cell state is specified initially by micromere-specific expression of these regulatory genes, viz. alx1, ets1, tbrain and tel, plus the gene encoding the Notch ligand Delta. Here we use a recently developed high throughput methodology for experimental cis-regulatory analysis to elucidate the genomic regulatory system controlling alx1 expression in time and embryonic space. The results entirely confirm the double negative gate control system at the cis-regulatory level, including definition of the functional HesC target sites, and add the crucial new information that the drivers of alx1 expression are initially Ets1, and then Alx1 itself plus Ets1. Cis-regulatory analysis demonstrates that these inputs quantitatively account for the magnitude of alx1 expression. Furthermore, the Alx1 gene product not only performs an auto-regulatory role, promoting a fast rise in alx1 expression, but also, when at high levels, it behaves as an auto-repressor. A synthetic experiment indicates that this behavior is probably due to dimerization. In summary, the results we report provide the sequence level basis for control of alx1 spatial expression by the double negative gate GRN architecture, and explain the rising, then falling temporal expression profile of the alx1 gene in terms of its auto-regulatory genetic wiring.

Tyrosine Latching of a Regulatory Gate Affords Allosteric Control of Aromatic Amino Acid Biosynthesis

The first step of the shikimate pathway for aromatic amino acid biosynthesis is catalyzed by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Thermotoga maritima DAH7PS (TmaDAH7PS) is tetrameric, with monomer units comprised of a core catalytic (β/α)(8) barrel and an N-terminal domain. This enzyme is inhibited strongly by tyrosine and to a lesser extent by the presence of phenylalanine. A truncated mutant of TmaDAH7PS lacking the N-terminal domain was catalytically more active and completely insensitive to tyrosine and phenylalanine, consistent with a role for this domain in allosteric inhibition. The structure of this protein was determined to 2.0 Å. In contrast to the wild-type enzyme, this enzyme is dimeric. Wild-type TmaDAH7PS was co-crystallized with tyrosine, and the structure of this complex was determined to a resolution of 2.35 Å. Tyrosine was found to bind at the interface between two regulatory N-terminal domains, formed from diagonally located monomers of the tetramer, revealing a major reorganization of the regulatory domain with respect to the barrel relative to unliganded enzyme. This significant conformational rearrangement observed in the crystal structures was also clearly evident from small angle X-ray scattering measurements recorded in the presence and absence of tyrosine. The closed conformation adopted by the protein on tyrosine binding impedes substrate entry into the neighboring barrel, revealing an unusual tyrosine-controlled gating mechanism for allosteric control of this enzyme.

Brain Traffic

Brain Traffic

ISG15 modification of filamin B negatively regulates the type I interferon‐induced JNK signalling pathway

Interferon (IFN)-induced signalling pathways have essential functions in innate immune responses. In response to type I IFNs, filamin B tethers RAC1 and a Jun N-terminal kinase (JNK)-specific mitogen-activated protein kinase (MAPK) module--MEKK1, MKK4 and JNK--and thereby promotes the activation of JNK and JNK-mediated apoptosis. Here, we show that type I IFNs induce the conjugation of filamin B by interferon-stimulated gene 15 (ISG15). ISGylation of filamin B led to the release of RAC1, MEKK1 and MKK4 from the scaffold protein and thus to the prevention of sequential activation of the JNK cascade. By contrast, blockade of filamin B ISGylation by substitution of Lys 2467 with arginine or by knockdown of ubiquitin-activating enzyme E1-like (UBEL1) prevented the release of the signalling molecules from filamin B, resulting in persistent promotion of JNK activation and JNK-mediated apoptosis. These results indicate that filamin B ISGylation acts as a negative feedback regulatory gate for the desensitization of type I IFN-induced JNK signalling.

A perturbation model of the gene regulatory network for oral and aboral ectoderm specification in the sea urchin embryo

The current gene regulatory network (GRN) for the sea urchin embryo pertains to pregastrular specification functions in the endomesodermal territories. Here we extend gene regulatory network analysis to the adjacent oral and aboral ectoderm territories over the same period. A large fraction of the regulatory genes predicted by the sea urchin genome project and shown in ancillary studies to be expressed in either oral or aboral ectoderm by 24 h are included, though universally expressed and pan-ectodermal regulatory genes are in general not. The loci of expression of these genes have been determined by whole mount in situ hybridization. We have carried out a global perturbation analysis in which expression of each gene was interrupted by introduction of morpholino antisense oligonucleotide, and the effects on all other genes were measured quantitatively, both by QPCR and by a new instrumental technology (NanoString Technologies nCounter Analysis System). At its current stage the network model, built in BioTapestry, includes 22 genes encoding transcription factors, 4 genes encoding known signaling ligands, and 3 genes that are yet unknown but are predicted to perform specific roles. Evidence emerged from the analysis pointing to distinctive subcircuit features observed earlier in other parts of the GRN, including a double negative transcriptional regulatory gate, and dynamic state lockdowns by feedback interactions. While much of the regulatory apparatus is downstream of Nodal signaling, as expected from previous observations, there are also cohorts of independently activated oral and aboral ectoderm regulatory genes, and we predict yet unidentified signaling interactions between oral and aboral territories.

Analysis of Npl4 deletion mutants in mammalian cells unravels new Ufd1-interacting motifs and suggests a regulatory role of Npl4 in ERAD

Npl4 is a 67 kDa protein forming a stable heterodimer with Ufd1, which in turn binds the ubiquitous p97/VCP ATPase. According to a widely accepted model, VCP Ufd1–Npl4 promotes the retrotranslocation of emerging ER proteins, their ubiquitination by associated ligases, and handling to the 26S proteasome for degradation in a process known as ERAD (ER-associated degradation). Using a series of Npl4 deletion mutants we have revealed that the binding of Ufd1 to Npl4 is mediated by two regions: a conserved stretch of amino acids from 113 to 255 within the zf-Npl4 domain and by the Npl4 homology domain between amino acids 263 and 344. Within the first region, we have identified two discrete subdomains: one involved in Ufd1 binding and one regulating VCP binding. Expression of any one of the mutants failed to induce any changes in the morphology of the ER or Golgi compartments. Moreover, we have observed that overexpression of all the analyzed mutants induced mild ER stress, as evidenced by increased Grp74/BiP expression without associated XBP1 splicing or induction of apoptosis. Surprisingly, we have not observed any accumulation of the typical ERAD substrate αTCR. This favors the model where the Ufd1–Npl4 dimer forms a regulatory gate at the exit from the retrotranslocone, rather than actively promoting retrotranslocation like the p97VCP ATPase.

Alzheimer's disease: advances in trafficking

Alzheimer's disease: advances in trafficking

The mineralisation of fresh and humified soil organic matter by the soil microbial biomass

Soil organic matter comprises all dead plant and animal residues, from the most recent inputs to the most intensively humified. We have found that traces of fresh substrates at microg g(-1) soil concentrations (termed 'trigger molecules') activate the biomass to expend more energy than is contained in the original 'trigger molecules'. In contrast, we suggest that the rate limiting step in soil organic matter mineralisation is independent of microbial activity, but is governed by abiological processes (which we term the Regulatory Gate theory). These two findings have important implications for our understanding of carbon mineralisation in soil, a fundamental process in the sequestration of soil organic matter.

Mineralization of native soil organic matter is not regulated by the size, activity or composition of the soil microbial biomass—a new perspective

Soil organic matter is extensively humified; some fractions existing for more than 1000 years. The soil microbial biomass is surrounded by about 50 times its mass of soil organic matter, but can only metabolize it very slowly. Paradoxically, even if more than 90% of the soil microbial biomass is killed, the mineralization of soil organic matter proceeds at the same rate as in an unperturbed soil. Here we show that soil organic matter mineralization is independent of microbial biomass size, community structure or specific activity. We suggest that the rate limiting step is governed by abiological processes (which we term the Regulatory Gate hypothesis), which convert non-bioavailable soil organic matter into bioavailable soil organic matter, and cannot be affected by the microbial population. This work challenges one of the long held theories in soil microbiology proposed by Winogradsky, of the existence of autochthonous and zymogenous microbial populations. This has significant implications for our understanding of carbon mineralization in soils and the role of soil micro-organisms in the global carbon cycle. Here we describe experiments designed to determine if the Regulatory Gate operates. We conclude that there is sufficient experimental evidence for it to be offered as a working hypothesis.

CaMKII Structure—An Elegant Design

Crystal structures of protein kinases continue to reveal new mechanisms for the regulation of catalytic activity of these enzymes. In this issue of Cell, Rosenberg et al. (2005) report the structure of the catalytic and regulatory domains of CaMKII, a protein kinase important in the cellular response to changes in intracellular calcium ion concentration. This study provides new mechanistic insights into the workings of this finely tuned molecular machine.

Involvement of oxidative stress in the rapid Akt1 regulating a JNK scaffold during ischemia in rat hippocampus

It has been well documented that the activation of Akt1 and JNK pathways are involved in the neuronal cell death in cerebral ischemia. In this study, we describe a novel interaction between Akt1 and JNK interacting protein 1 (JIP-1). We first detected the interaction of Akt1 and JIP-1 in hippocampus at various time points of ischemia. In the basal state, JIP-1 bind to Akt1, MLK3 at maximum while JIP-1 binds to JNK3 at minimum. Ischemia stimulus decreased the Akt1–JIP-1 interaction and concomitantly increased association between JIP-1 and JNK3. While MLK3 binding to JIP-1 decreased, similar to Akt1–JIP-1 interaction during ischemia. These results indicated that Akt1 interaction with JIP-1 inhibited JIP-1-mediated potentiation of JNK activity by decreasing JIP-1 binding to specific JNK pathway kinases. Akt1 binding to JIP-1 acts as a regulatory gate preventing JNK activation, which is opened under conditions ischemia injury. Administration of antioxidant N-acetylcysteine (NAC) can obviously affected the level of MLK3, JNK3 and Akt1 binding to JIP-1 and JNK3 activation in the hippocampus at 15 min ischemia. The findings suggest that Akt1 regulating JNK scaffold and then regulating JNK activation were closely associated with reactive oxygen species (ROS) during cerebral ischemia.

Akt1 Regulates a JNK Scaffold during Excitotoxic Apoptosis

Cell survival is determined by a balance among signaling cascades, including those that recruit the Akt and JNK pathways. Here we describe a novel interaction between Akt1 and JNK interacting protein 1 (JIP1), a JNK pathway scaffold. Direct association between Akt1 and JIP1 was observed in primary neurons. Neuronal exposure to an excitotoxic stimulus decreased the Akt1-JIP1 interaction and concomitantly increased association between JIP1 and JNK. Akt1 interaction with JIP1 inhibited JIP1-mediated potentiation of JNK activity by decreasing JIP1 binding to specific JNK pathway kinases. Consistent with this view, neurons from Akt1-deficient mice exhibited higher susceptibility to kainate than wild-type littermates. Overexpression of Akt1 mutants that bind JIP1 reduced excitotoxic apoptosis. These results suggest that Akt1 binding to JIP1 acts as a regulatory gate preventing JNK activation, which is released under conditions of excitotoxic injury.