scholarly journals 3 minutes to precisely measure morphogen concentration

2018 ◽  
Author(s):  
Tanguy Lucas ◽  
Huy Tran ◽  
Carmina Angelica Perez Romero ◽  
Aurélien Guillou ◽  
Cécile Fradin ◽  
...  
Keyword(s):  
Binding Sites ◽  
Transcriptional Response ◽  
Positional Information ◽  
Fruit Fly ◽  
Fluorescent Labeling ◽  
Hill Coefficient ◽  
Activation Process ◽  
Endogenous Expression ◽  
Time Period ◽  
Zygotic Transcription

AbstractMorphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the main Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Remarkably, despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to measure subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on cooperativity between the 6 known Bicoid binding sites in the hb promoter region and assuming rate limiting concentrations of the Bicoid transcription factor at the boundary is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that additional mechanisms are involved in the steepness of the response.

scholarly journals Synthetic deconstruction of hunchback regulation by Bicoid

2021 ◽  
Author(s):  
Gonçalo Fernandes ◽  
Huy Tran ◽  
Maxime Andrieu ◽  
Youssoupha Diaw ◽  
Carmina Perez Romero ◽  
...  
Keyword(s):  
Steady State ◽  
Binding Sites ◽  
Positional Information ◽  
Fruit Fly ◽  
Decay Length ◽  
Activation Process ◽  
Modeling Tools ◽  
Spatial Features ◽  
Correct Location ◽  
Almost All

During development, cell identity is established reproducibly among individuals through the expression of specific genes at the correct time and correct location in space. How genes extract and combine both positional and temporal information from different transcription factor (TF) profiles along polarity axes remain largely unexplored. Here, we showcase the classic hunchback gene in fruit fly embryos, with focus on 3 of its main TFs: Bicoid, Zelda and Hunchback proteins. We constructed a series of synthetic MS2 reporters, where the numbers and combination of binding sites for each TF are varied. Using live imaging of transcription dynamics by these synthetic reporters and modeling tools, we show that i) a Bicoid-only synthetic reporter needs 3 more Bicoid binding sites than found in the hunchback promoter to recapitulates almost all spatial features of early hb expression but takes more time to reach steady state; ii) Hunchback and Zelda binding sites combined with Bicoid sites both reduce the time to reach steady state and increase expression at a different step in the activation process: Zld sites lower the Bicoid threshold required for activation while Hb sites increase the polymerase firing rate and reduce bursting; iii) the shift of the Bicoid-only reporter induced by a reduction by half of Bicoid concentrations indicates that the decay length of the Bicoid activity gradient is lower than the decay length of the Bicoid protein gradient. Altogether, this work indicates that Bicoid is the main source of positional information for hunchback expression and places back the Bicoid system within the physical limits of an equilibrium model.

scholarly journals Allosteric effects of Mg2+ on the gating of Ca2+-activated K+ channels from mammalian skeletal muscle

1986 ◽  
Vol 124 (1) ◽  
pp. 5-13
Author(s):  
J. Golowasch ◽  
A. Kirkwood ◽  
C. Miller
Keyword(s):  
Binding Sites ◽  
Hill Coefficient ◽  
K Channel ◽  
Activation Process ◽  
Mammalian Skeletal Muscle ◽  
Activation Curve ◽  
K Channels ◽  
The Hill ◽  
Cytoplasmic Side ◽  
High Conductance

Ca2+-activated K+ channels from rat muscle transverse tubule membranes were inserted into planar phospholipid bilayers, and the activation of these channels by Ca2+ was studied. On the cytoplasmic side of the channel, calcium ions (in the range 10–100 mumol l-1) increase the opening probability of the channel in a graded way. This ‘activation curve’ is sigmoid, with an average Hill coefficient of about 2. Magnesium ions, in the range 1–10 mmol l-1, increase the apparent affinity of the channel for Ca2+ and greatly enhance the sigmoidicity of the Ca2+ activation curve. In the presence of 10 mmol l-1 Mg2+, the Hill coefficient for Ca2+ activation is about 4.5. This effect depends upon Mg2+ concentration but not upon applied voltage. Mg2+ is effective only when added to the cytoplasmic side of the channel. The results argue that this high-conductance, Ca2+-activated K+ channel contains at least six Ca2+-binding sites involved in the activation process.

scholarly journals Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites

2005 ◽  
Vol 288 (2) ◽  
pp. F327-F333 ◽  
Author(s):  
Rémon A. M. H. Van Aubel ◽  
Pascal H. E. Smeets ◽  
Jeroen J. M. W. van den Heuvel ◽  
Frans G. M. Russel
Keyword(s):  
Binding Sites ◽  
Efflux Pump ◽  
Substrate Binding ◽  
Organic Anion ◽  
Apical Cell ◽  
Cell Uptake ◽  
Hek293 Cells ◽  
Hill Coefficient ◽  
Anion Transporter ◽  
Substrate Binding Sites

The end product of human purine metabolism is urate, which is produced primarily in the liver and excreted by the kidney through a well-defined basolateral blood-to-cell uptake step. However, the apical cell-to-urine efflux mechanism is as yet unidentified. Here, we show that the renal apical organic anion efflux transporter human multidrug resistance protein 4 (MRP4), but not apical MRP2, mediates ATP-dependent urate transport via a positive cooperative mechanism ( Km of 1.5 ± 0.3 mM, Vmax of 47 ± 7 pmol·mg−1·min−1, and Hill coefficient of 1.7 ± 0.2). In HEK293 cells overexpressing MRP4, intracellular urate levels were lower than in control cells. Urate inhibited methotrexate transport (IC50 of 235 ± 8 μM) by MRP4, did not affect cAMP transport, whereas cGMP transport was stimulated. Urate shifted cGMP transport by MRP4 from positive cooperativity ( Km and Vmax value of 180 ± 20 μM and 58 ± 4 pmol·mg−1·min−1, respectively, Hill coefficient of 1.4 ± 0.1) to single binding site kinetics ( Km and Vmax value of 2.2 ± 0.9 mM and 280 ± 50 pmol·mg−1·min−1, respectively). Finally, MRP4 could transport urate simultaneously with cAMP or cGMP. We conclude that human MRP4 is a unidirectional efflux pump for urate with multiple allosteric substrate binding sites. We propose MRP4 as a candidate transporter for urinary urate excretion and suggest that MRP4 may also mediate hepatic export of urate into the circulation, because of its basolateral expression in the liver.

scholarly journals Low-affinity transcription factor binding sites shape morphogen responses and enhancer evolution

2013 ◽  
Vol 368 (1632) ◽  
pp. 20130018 ◽  
Author(s):  
Andrea I. Ramos ◽  
Scott Barolo
Keyword(s):  
Transcription Factor ◽  
Binding Affinity ◽  
Binding Sites ◽  
Target Genes ◽  
Gene Activation ◽  
Transcriptional Response ◽  
Morphogen Gradients ◽  
Weak Binding ◽  
Initial Hypothesis

In the era of functional genomics, the role of transcription factor (TF)–DNA binding affinity is of increasing interest: for example, it has recently been proposed that low-affinity genomic binding events, though frequent, are functionally irrelevant. Here, we investigate the role of binding site affinity in the transcriptional interpretation of Hedgehog (Hh) morphogen gradients . We noted that enhancers of several Hh-responsive Drosophila genes have low predicted affinity for Ci, the Gli family TF that transduces Hh signalling in the fly. Contrary to our initial hypothesis, improving the affinity of Ci/Gli sites in enhancers of dpp , wingless and stripe , by transplanting optimal sites from the patched gene, did not result in ectopic responses to Hh signalling. Instead, we found that these enhancers require low-affinity binding sites for normal activation in regions of relatively low signalling. When Ci/Gli sites in these enhancers were altered to improve their binding affinity, we observed patterning defects in the transcriptional response that are consistent with a switch from Ci-mediated activation to Ci-mediated repression. Synthetic transgenic reporters containing isolated Ci/Gli sites confirmed this finding in imaginal discs. We propose that the requirement for gene activation by Ci in the regions of low-to-moderate Hh signalling results in evolutionary pressure favouring weak binding sites in enhancers of certain Hh target genes.

scholarly journals Transcriptional response of Hoxb genes to retinoid signalling is regionally restricted along the neural tube rostrocaudal axis

2017 ◽  
Vol 4 (4) ◽  
pp. 160913 ◽  
Author(s):  
Nicoletta Carucci ◽  
Emanuele Cacci ◽  
Paola S. Nisi ◽  
Valerio Licursi ◽  
Yu-Lee Paul ◽  
...  
Keyword(s):  
Neural Tube ◽  
Neural Development ◽  
Transcriptional Response ◽  
Neural Tissue ◽  
Positional Information ◽  
Multiple Roles ◽  
Chromatin Activation ◽  
Neural Stem Progenitor Cells ◽  
Rostrocaudal Axis

During vertebrate neural development, positional information is largely specified by extracellular morphogens. Their distribution, however, is very dynamic due to the multiple roles played by the same signals in the developing and adult neural tissue. This suggests that neural progenitors are able to modify their competence to respond to morphogen signalling and autonomously maintain positional identities after their initial specification. In this work, we take advantage of in vitro culture systems of mouse neural stem/progenitor cells (NSPCs) to show that NSPCs isolated from rostral or caudal regions of the mouse neural tube are differentially responsive to retinoic acid (RA), a pivotal morphogen for the specification of posterior neural fates. Hoxb genes are among the best known RA direct targets in the neural tissue, yet we found that RA could promote their transcription only in caudal but not in rostral NSPCs. Correlating with these effects, key RA-responsive regulatory regions in the Hoxb cluster displayed opposite enrichment of activating or repressing histone marks in rostral and caudal NSPCs. Finally, RA was able to strengthen Hoxb chromatin activation in caudal NSPCs, but was ineffective on the repressed Hoxb chromatin of rostral NSPCs. These results suggest that the response of NSPCs to morphogen signalling across the rostrocaudal axis of the neural tube may be gated by the epigenetic configuration of target patterning genes, allowing long-term maintenance of intrinsic positional values in spite of continuously changing extrinsic signals.

Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements

Development ◽  
1998 ◽  
Vol 125 (22) ◽  
pp. 4349-4358 ◽  
Author(s):  
J. Charite ◽  
W. de Graaff ◽  
D. Consten ◽  
M.J. Reijnen ◽  
J. Korving ◽  
...  
Keyword(s):  
Binding Sites ◽  
Hox Genes ◽  
Regulatory Element ◽  
Ectopic Expression ◽  
Positional Information ◽  
Regulatory Elements ◽  
Gene Products ◽  
Anteroposterior Patterning

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

scholarly journals A study of novel lectins and their involvement in the activation of the prophenoloxidase system in Blaberus discoidalis

1995 ◽  
Vol 310 (1) ◽  
pp. 23-31 ◽  
Author(s):  
C Chen ◽  
H J Durrant ◽  
R P Newton ◽  
N A Ratcliffe
Keyword(s):  
Binding Sites ◽  
Serine Proteases ◽  
Time Course ◽  
Lectin Binding ◽  
Active Form ◽  
Reaction System ◽  
Activation Process ◽  
Blaberus Discoidalis ◽  
Sequence Of Events ◽  
Prophenoloxidase System

Endogenous and exogenous lectins have been found to activate the prophenoloxidase (proPO) system of the cockroach, Blaberus discoidalis, to the same extent as laminarin, a previously known microbial activator of proPO. The lectins can also further enhance this laminarin activation of the proPO system. Non-lectin proteins did not display any activation properties. The time course of proPO activation was studied after reconstitution of the reaction system using purified lectins, a trypsin-like enzyme, a trypsin inhibitor and partially purified lectin-binding proteins from the cockroach haemolymph. Lectin activation of the proPO system is probably not mediated by the lectin sugar-binding sites, as specific inhibitory sugars failed to abrogate the enhanced effect. The results suggest that alternative binding site(s) on the lectins may be involved in the proPO activation process. Evidence also suggests that several different lectins are involved in the regulation of the proPO system through separate receptors or binding molecules on the haemocytes, and that they exert their effects early in the sequence of events leading to conversion of proPO into its active form, possibly via regulation of serine proteases and protease inhibitors.

scholarly journals Genetic Composition of the Bacillus subtilis SOS System

2005 ◽  
Vol 187 (22) ◽  
pp. 7655-7666 ◽  
Author(s):  
Nora Au ◽  
Elke Kuester-Schoeck ◽  
Veena Mandava ◽  
Laura E. Bothwell ◽  
Susan P. Canny ◽  
...  
Keyword(s):  
Dna Damage ◽  
Bacillus Subtilis ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Transcriptional Response ◽  
Open Reading Frames ◽  
Mobility Shift ◽  
E Coli ◽  
Recombination Protein ◽  
Lexa Binding

ABSTRACT The SOS response in bacteria includes a global transcriptional response to DNA damage. DNA damage is sensed by the highly conserved recombination protein RecA, which facilitates inactivation of the transcriptional repressor LexA. Inactivation of LexA causes induction (derepression) of genes of the LexA regulon, many of which are involved in DNA repair and survival after DNA damage. To identify potential RecA-LexA-regulated genes in Bacillus subtilis, we searched the genome for putative LexA binding sites within 300 bp upstream of the start codons of all annotated open reading frames. We found 62 genes that could be regulated by putative LexA binding sites. Using mobility shift assays, we found that LexA binds specifically to DNA in the regulatory regions of 54 of these genes, which are organized in 34 putative operons. Using DNA microarray analyses, we found that 33 of the genes with LexA binding sites exhibit RecA-dependent induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of the distinct LexA binding sites reveals an expanded consensus sequence for the B. subtilis operator: 5′-CGAACATATGTTCG-3′. Although the number of genes controlled by RecA and LexA in B. subtilis is similar to that of Escherichia coli, only eight B. subtilis RecA-dependent SOS genes have homologous counterparts in E. coli.

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