An ultraconserved Hox–Pbx responsive element resides in the coding sequence of Hoxa2 and is active in rhombomere 4

The Hoxa2 gene has a fundamental role in vertebrate craniofacial and hindbrain patterning. Segmental control of Hoxa2 expression is crucial to its function and several studies have highlighted transcriptional regulatory elements governing its activity in distinct rhombomeres. Here, we identify a putative Hox–Pbx responsive cis-regulatory sequence, which resides in the coding sequence of Hoxa2 and is an important component of Hoxa2 regulation in rhombomere (r) 4. By using cell transfection and chromatin immunoprecipitation (ChIP) assays, we show that this regulatory sequence is responsive to paralogue group 1 and 2 Hox proteins and to their Pbx co-factors. Importantly, we also show that the Hox–Pbx element cooperates with a previously reported Hoxa2 r4 intronic enhancer and that its integrity is required to drive specific reporter gene expression in r4 upon electroporation in the chick embryo hindbrain. Thus, both intronic as well as exonic regulatory sequences are involved in Hoxa2 segmental regulation in the developing r4. Finally, we found that the Hox–Pbx exonic element is embedded in a larger 205-bp long ultraconserved genomic element (UCE) shared by all vertebrate genomes. In this respect, our data further support the idea that extreme conservation of UCE sequences may be the result of multiple superposed functional and evolutionary constraints

Etude de gènes cibles pour l'homéoprotéine Hoxa2 de la souris

The Hox homeoproteins are transcription factors involved in important numerous biological processes during embryonic development. Although the functions fulfilled by these proteins have been well documented by knock-out or missexpression analysis, the target genes regulated by these transcription factors remain largely unknown. Our aims consisted in searching and characterizing target genes of the Hoxa2 protein. We have designed an experimental approach based on transfection assays. This approach involved the transfection of expression vectors for Hoxa2 or inactive Hoxa2 variant, and expression vectors for its Pbx1a and Prep1 cofactors. The differential analysis of mRNA from both cellular populations has allowed the identification of 60 candidate target genes. Among these, eight have been selected on the basis of available functional data that may contribute to the understanding of the role of Hoxa2 during embryonic development. We have shown that these eight genes were specifically modulated by Hoxa2 and that this modulation was enhanced by Pbx1a and Prep1 cofactors. Two genes, Lmo1 and Pepp2, have been retained to characterize their expression pattern during mouse embryonic development between E8,5 and E10,5 by whole-mount in situ hybridization.Les homéoprotéines Hox sont des facteurs de transcription impliqués dans de nombreux processus biologiques importants durant le développement embryonnaire. Bien que les fonctions assurées par ces protéines soient bien documentées par les analyses de perte de fonction ou de surexpression, les données relatives aux gènes cibles régulés par ces facteurs de transcription restent très fragmentaires. Nous avons donc pris pour objectifs de rechercher et de caractériser des gènes cibles pour la protéine Hoxa2. Pour ce faire, nous avons défini une approche expérimentale basée sur des transfections cellulaires. Cette approche implique la transfection de vecteurs d'expression pour Hoxa2 ou un variant inactif de celle-ci, ainsi que des vecteurs d'expression pour ses cofacteurs Pbx1a et Prep1. L'analyse différentielle du contenu en ARNm de telles populations a permis d'identifier 60 gènes cibles candidats. Parmi ceux-ci, nous en avons sélectionné 8 sur base des données fonctionnelles partielles disponibles pouvant contribuer à expliquer le rôle de la protéine Hoxa2 au cours du développement embryonnaire. Pour ces huit gènes, nous avons établi qu'ils répondaient de manière spécifique à Hoxa2, et que cette réponse était amplifiée par les cofacteurs Pbx1a et Prep. Deux gènes, Lmo1 et Pepp2 ont été retenus afin de caractériser leurs profils d'expression au cours du développement embryonnaire murin entre les jours 8,5 et 10,5jpc, par hybridation in situ en « whole-mount ».Doctorat en sciences (sciences biologiques) (BIOL 3)--UCL, 200

Decision-Making in Rats is Sensitive to Rare and Extreme Events: the Black Swan Avoidance

In Brief Most studies assessing decision-making under uncertainty use events with probabilities that are above 10-20 %. Here, to study decision-making in radical uncertainty conditions, Degoulet, Willem, Baunez, Luchini and Pintus provide a novel experimental design that aims at measuring the extent to which rats are sensitive - and how they respond - to extremely rare (below 1% of probability) but extreme events in a four-armed bandit task. Gains (sugar pellets) and losses (time-out punishments) are such that large - but rare - values materialize or not depending on the option chosen. The results show that all rats diversify their choices across options. However, most rats exhibit sensitivity to rare and extreme events despite their sparse occurrence, by combining more often options with extreme gains (Jackpots) and/or avoidance of extreme losses (Black Swans). In general, most rats’ choices feature one-sided sensitivity in favor of trying more often to avoid extreme losses than to seek extreme gains - that is, they feature Black Swan Avoidance. Highlights - A novel experiment is proposed to measure whether rats are sensitive - and how they respond - to radical uncertainty materialized by extremely rare and extreme events in a four-armed bandit task; - Most rats exhibit sensitivity to rare and extreme events; - Most rats opt more often to avoid rare and extreme losses than to seek rare and extreme gains: they feature Black Swan avoidance

Sensibilité spécifique aux évènements rares et extrêmes: Evitement quasi-complet des Black Swans et recherche partielle des Jackpots dans la prise de décision chez le rat

International audienceMost studies assessing animal decision-making under risk rely on probabilities that are typically larger than 10%. To study Decision-Making in uncertain conditions, we explore a novel experimental and modelling approach that aims at measuring the extent to which rats are sensitive - and how they respond - to outcomes that are both rare (probabilities smaller than 1%) and extreme in their consequences (deviations larger than 10 times the standard error). In a four-armed bandit task, stochastic gains (sugar pellets) and losses (time-out punishments) are such that extremely large - but rare - outcomes materialize or not depending on the chosen options. All rats feature both limited diversification, mixing two options out of four, and sensitivity to rare and extreme outcomes despite their infrequent occurrence, by combining options with avoidance of extreme losses (Black Swans) and exposure to extreme gains (Jackpots). Notably, this sensitivity turns out to be one-sided for the main phenotype in our sample: it features a quasi-complete avoidance of Black Swans, so as to escape extreme losses almost completely, which contrasts with an exposure to Jackpots that is partial only. The flip side of observed choices is that they entail smaller gains and larger losses in the frequent domain compared to alternatives. We have introduced sensitivity to Black Swans and Jackpots in a new class of augmented Reinforcement Learning models and we have estimated their parameters using observed choices and outcomes for each rat. Adding such specific sensitivity results in a good fit of the selected model - and simulated behaviors that are close - to behavioral observations, whereas a standard Q-Learning model without sensitivity is rejected for almost all rats. This model reproducing the main phenotype suggests that frequent outcomes are treated separately from rare and extreme ones through different weights in Decision-Making

Decision-Making in Rats is Sensitive to Rare and Extreme Events: the Black Swan Avoidance

In BriefMost studies assessing decision-making under uncertainty exploit events with probabilities that are typically above 10%. Here, to study decision-making in radical uncertainty conditions, Degoulet, Willem, Baunez, Luchini and Pintus provide a novel modelling and experimental approach that aims at measuring the extent to which rats are sensitive - and how they respond - to events that are both very rare (probability below 1%) and very extreme in their consequences, in a novel four-armed bandit task. Gains (sugar pellets) and losses (time-out punishments) are such that large - but rare - outcomes materialize or not depending on the option chosen. The results show that all rats diversify their choices mainly across two options, out of four, and that most rats feature sensitivity to rare and extreme events despite their infrequent occurrence. They do so by combining more often options with extreme gains (Jackpots) and/or avoidance of extreme losses (Black Swans). Notably, most rats’ choices feature one-sided sensitivity in favor of trying more often to avoid extreme losses than to seek extreme gains - that is, they feature the Black Swan Avoidance.Highlights-Novel modelling and experimental design are proposed to measure the extent to which whether rats are sensitive - and how they respond - to radical uncertainty materialized by events that are both very rare and extreme in their consequences, in a novel four-armed bandit task;-Most rats exhibit moderate to high sensitivity to rare and extreme events;-Most rats opt more often to avoid rare and extreme losses than to seek rare and extreme gains: they feature the Black Swan avoidance.</jats:sec

Changing homeodomain residues 2 and 3 of Hoxa1 alters its activity in a cell-type and enhancer dependent manner

The second and third amino acid residues of the N-terminal arm of most Hox protein homeodomains are basic (lysine or arginine), whereas they are asparagine and alanine, respectively, in the Hoxa1 homeodomain. Previous reports pinpointed these residues as specificity determinants in the function of Hoxa1 when it is acting as a monomer. However, in vitro data supported that these residues do not influence the target specificity of Hoxa1 in Pbx1a–Hoxa1 heterodimers. Here, we have analysed the transcriptional activity of a Hoxa1(NA-KR) mutant for which the asparagine and alanine residues of the homeodomain have been replaced by lysine and arginine, respectively. Comparison between the wild-type and mutant Hoxa1 reveals that they show distinct activity on the TSEII enhancer of the somatostatin gene, but that they are equally active in the presence of Pbx and Prep cofactors. This therefore corroborates the biochemical evidence having shown that the second and third residues of the homeodomain do not contribute to the DNA binding of Hoxa1–Pbx dimers. However, on the hoxb1 autoregulatory enhancer, Hoxa1 and Hoxa1(NA-KR) may display distinct activity despite the presence of Pbx, in a cell-type dependent manner. Therefore, our data suggest that, depending on the enhancer, these residues may contribute to the functional specificity of Hoxa1 and that this contribution may not be abrogated by the interaction with Pbx

Identification of Lmo1 as part of a Hox-dependent regulatory network for hindbrain patterning.

The embryonic functions of Hox proteins have been extensively investigated in several animal phyla. These transcription factors act as selectors of developmental programmes, to govern the morphogenesis of multiple structures and organs. However, despite the variety of morphogenetic processes Hox proteins are involved in, only a limited set of their target genes has been identified so far. To find additional targets, we used a strategy based upon the simultaneous overexpression of Hoxa2 and its cofactors Pbx1 and Prep in a cellular model. Among genes whose expression was upregulated, we identified LMO1, which codes for an intertwining LIM-only factor involved in protein-DNA oligomeric complexes. By analysing its expression in Hox knockout mice, we show that Lmo1 is differentially regulated by Hoxa2 and Hoxb2, in specific columns of hindbrain neuronal progenitors. These results suggest that Lmo1 takes part in a Hox paralogue 2-dependent network regulating anteroposterior and dorsoventral hindbrain patterning

Cellular uptake of Antennapedia Penetratin peptides is a two-step process in which phase transfer precedes a tryptophan-dependent translocation

Several homeodomains and homeodomain-containing proteins enter live cells through a receptor- and energy-independent mechanism. Translocation through biological membranes is conferred by the third α-helix of the homeodomain, also known as Penetratin. Biophysical studies demonstrate that entry of Penetratin into cells requires its binding to surface lipids but that binding and translocation are differentially affected by modifications of some physico-chemical properties of the peptide, like helical amphipathicity or net charge. This suggests that the plasma membrane lipid composition affects the internalization of Penetratin and that internalization requires both lipid binding and other specific properties. Using a phase transfer assay, it is shown that negatively charged lipids promote the transfer of Penetratin from a hydrophilic into a hydrophobic environment, probably through charge neutralization. Accordingly, transfer into a hydrophobic milieu can also be obtained in the absence of negatively charged lipids, by the addition of DNA oligonucleotides. Strikingly, phase transfer by charge neutralization was also observed with a variant peptide of same charge and hydrophobicity in which the tryptophan at position 6 was replaced by a phenylalanine. However, Penetratin, but not its mutant version, is internalized by live cells. This underscores that charge neutralization and phase transfer represent only a first step in the internalization process and that further crossing of a biological membrane necessitates the critical tryptophan residue at position 6

Differential and opposed transcriptional effects of protein fusions containing the VP16 activation domain

AbstractOverexpression of strong transcriptional activators like herpes simplex virion protein 16 (VP16) may lead to non-specific inhibition of gene expression as a result of the titration of transcription factors. Here we report that a fusion between the homeoprotein Hoxa2 and the VP16 activation domain inhibits transcription from the strong promoter/enhancers of cytomegalovirus (CMV) and Rous sarcoma virus (RSV). A similar fusion involving a Hoxa2 mutant protein that is defective in DNA binding has no effect on the CMV promoter but increases, rather than inhibits, the RSV promoter activity. This suggests that depending on its ability to bind DNA, the VP16 activator can interact with different sets of cofactors, giving rise to distinct transcriptional effects