Division of labor in insect societies : genetic components and physiological regulation

The shift from solitary to social organisms constitutes one of the major transitions in evolution. The highest level of sociality is found in social insects (ants, termites and some species of bees and wasps). Division of labor is central to the organization of insect societies and is thought to be at the root of their ecological success. There are two main levels of division of labor in social insect colonies. The first relates to reproduction and involves the coexistence of queen and worker castes: while reproduction is usually monopolized by one or several queens, functionally sterile workers perform all the tasks to maintain the colony, such as nest building, foraging or brood care. The second level of division of labor, relating to such non-reproductive duties, is characterized by the performance of different tasks or roles by different groups of workers. This PhD aims to better understand the mechanisms underlying division of labor in insect societies, by investigating how genes and physiology influence caste determination and worker behavior in ants. In the first axis of this PhD, we studied the nature of genetic effects on division of labor. We used the Argentine ant Linepithema humile to conduct controlled crosses in the laboratory, which revealed the existence of non-additive genetic effects, such as parent-of-origin and genetic compatibility effects, on caste determination and worker behavior. In the second axis, we focused on the physiological regulation of division of labor. Using Pogonomyrmex seed- harvester ants, we performed experimental manipulation of hibernation, hormonal treatments, gene expression analyses and protein quantification to identify the physiological pathways regulating maternal effects on caste determination. Finally, comparing gene expression between nurses and foragers allowed us to reveal the association between vitellogenin and worker behavior in Pogonomyrmex ants. This PhD provides important insights into the role of genes and physiology in the regulation of division of labor in social insect colonies, helping to better understand the organization, evolution and ecological success of insect societies. - L'une des principales transitions évolutives est le passage de la vie solitaire à la vie sociale. La socialité atteint son paroxysme chez les insectes sociaux que sont les fourmis, les termites et certaines espèces d'abeilles et de guêpes. La division du travail est la clé de voûte de l'organisation de ces sociétés d'insectes et la raison principale de leur succès écologique. La division du travail s'effectue à deux niveaux dans les colonies d'insectes sociaux. Le premier niveau concerne la reproduction et implique la coexistence de deux castes : les reines et les ouvrières. Tandis que la reproduction est le plus souvent monopolisée par une ou plusieurs reines, les ouvrières stériles effectuent les tâches nécessaires au bon fonctionnement de la colonie, telles que la construction du nid, la recherche de nourriture ou le soin au couvain. Le second niveau de division du travail, qui concerne les tâches autres que la reproduction, implique la réalisation de différents travaux par différents groupes d'ouvrières. Le but de ce doctorat est de mieux comprendre les mécanismes sous-jacents de la division du travail dans les sociétés d'insectes en étudiant comment les gènes et la physiologie influencent la détermination de la caste et le comportement des ouvrières chez les fourmis. Dans le premier axe de ce doctorat, nous avons étudié la nature des influences génétiques sur la division du travail. Nous avons utilisé la fourmi d'Argentine, Linepithema humile, pour effectuer des croisements contrôlés en laboratoire. Cette méthode nous a permis de révéler l'existence d'influences génétiques non additives, telles que des influences dépendantes de l'origine parentale ou des effets de compatibilité génétique, sur la détermination de la caste et le comportement des ouvrières. Dans le second axe, nous nous sommes intéressés à la régulation physiologique de la division du travail. Nous avons utilisé des fourmis moissonneuses du genre Pogonomyrmex pour effectuer des hibernations artificieHes, des traitements hormonaux, des analyses d'expression de gènes et des mesures de vitellogénine, ce qui nous a permis d'identifier les mécanismes physiologiques régulant les effets maternels sur la détermination de la caste. Enfin, la comparaison d'expression de gènes entre nourrices et fourrageuses suggère un rôle de la vitellogénine dans la régulation du comportement des ouvrières chez les fourmis moissonneuses. En détaillant les influences des gènes et de la physiologie dans la régulation de la division du travail dans les colonies d'insectes sociaux, ce doctorat fournit d'importantes informations permettant de mieux comprendre l'organisation, l'évolution et le succès écologique des sociétés d'insectes.

Maf1, a new player in the regulation of human RNA polymerase III transcription.

BACKGROUND: Human RNA polymerase III (pol III) transcription is regulated by several factors, including the tumor suppressors P53 and Rb, and the proto-oncogene c-Myc. In yeast, which lacks these proteins, a central regulator of pol III transcription, called Maf1, has been described. Maf1 is required for repression of pol III transcription in response to several signal transduction pathways and is broadly conserved in eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We show that human endogenous Maf1 can be co-immunoprecipitated with pol III and associates in vitro with two pol III subunits, the largest subunit RPC1 and the alpha-like subunit RPAC2. Maf1 represses pol III transcription in vitro and in vivo and is required for maximal pol III repression after exposure to MMS or rapamycin, treatments that both lead to Maf1 dephosphorylation. CONCLUSIONS/SIGNIFICANCE: These data suggest that Maf1 is a major regulator of pol III transcription in human cells.

Neuroligin-1 links neuronal activity to sleep-wake regulation.

Maintaining wakefulness is associated with a progressive increase in the need for sleep. This phenomenon has been linked to changes in synaptic function. The synaptic adhesion molecule Neuroligin-1 (NLG1) controls the activity and synaptic localization of N-methyl-d-aspartate receptors, which activity is impaired by prolonged wakefulness. We here highlight that this pathway may underlie both the adverse effects of sleep loss on cognition and the subsequent changes in cortical synchrony. We found that the expression of specific Nlg1 transcript variants is changed by sleep deprivation in three mouse strains. These observations were associated with strain-specific changes in synaptic NLG1 protein content. Importantly, we showed that Nlg1 knockout mice are not able to sustain wakefulness and spend more time in nonrapid eye movement sleep than wild-type mice. These changes occurred with modifications in waking quality as exemplified by low theta/alpha activity during wakefulness and poor preference for social novelty, as well as altered delta synchrony during sleep. Finally, we identified a transcriptional pathway that could underlie the sleep/wake-dependent changes in Nlg1 expression and that involves clock transcription factors. We thus suggest that NLG1 is an element that contributes to the coupling of neuronal activity to sleep/wake regulation.

Implication of DNA methylation, CTCF and BORIS factors in the transcriptional regulation of the human telomerase catalytic subunit hTERT

RESUME La télomérase confère une durée de vie illimitée et est réactivée dans la plupart des cellules tumorales. Sa sous-unité catalytique hTERT est définie comme le facteur limitant pour son activation. De l'identification de facteurs liant la région régulatrice d'hTERT, au rôle de la méthylation de l'ADN et de la modification des histones, de nombreux modèles de régulation ont été suggérés. Cependant, aucun de ces modèles n'a pu expliquer l'inactivation de la télomérase dans la plupart des cellules somatiques et sa réactivation dans la majorité des cellules tumorales. De plus, les observations contradictoires entre le faible niveau d'expression d'ARN messager d'hTERT dans les cellules télomérase-positives et la très forte activité transcriptionnelle du promoteur d'hTERT en transfection restent incomprises. Dans cette étude, nous avons montré que la région proximale du gène hTERT (exon 1 et 2) était impliquée dans la répression de l'activité de son promoteur. Nous avons identifié le facteur CTCF comme étant un inhibiteur du promoteur d'hTERT, en se liant au niveau de son premier exon. La méthylation de l'exon 1 du gène hTERT, couramment observée dans les tumeurs mais pas dans les cellules normales, empêcherait la liaison de CTCF. L'étude du profil de méthylation du promoteur d'hTERT indique qu'une partie du promoteur reste déméthylée et qu'elle semble suffisante pour permettre une faible activité transcriptionnelle du gène hTERT. Ainsi, la méthylation particulière des régions régulatrices d'hTERT inhibe la liaison de CTCF tout en permettant une faible transcription du gène. Cependant, dans certaines cellules tumorales, le promoteur et la région proximale du gène hTERT ne sont pas méthylés. Dans les lignées cellulaires tumorales de tesitcules et d'ovaires, l'inhibition de CTCF est contrée par son paralogue BORIS, qui se lie aussi au niveau de l'exon 1 d'hTERT, mais permet ainsi l'activation du promoteur. L'étude de l'expression du gène BORIS montre qu'il est exclusivement exprimé dans les tissus normaux de testicules et d'ovaires jeunes, ainsi qu'à différents niveaux dans la plupart des tumeurs. Sa transcription est sous le contrôle de deux promoteurs. Le promoteur proximal est régulé par méthylation et un transcrit alternatif majoritaire, délété de l'exon 6, est trouvé lorsque ce promoteur est actif. Tous ces résultats conduisent à un modèle de régulation du gène hTERT qui tient compte du profil épigénétique du gène et qui permet d'expliquer le faible taux de transcription observé in vivo. De plus, l'expression de BORIS dans les cancers et son implication dans l'activation du gène hTERT pourrait permettre de comprendre les phénomènes de dérégulation épigénétique et d'immortalisation qui ont lieu durant la tumorigenèse. SUMMARY Telomerase confers an unlimited lifespan, and is reactivated in most tumor cells. The catalytic subunit of telomerase, hTERT, is defined as the limiting factor for telomerase activity. Between activators and repressors that bind to the hTERT 5' regulatory region, and the role of CpG methylation and histone acetylation, an abundance of regulatory models have been suggested. None of these models can explain the silence of telomerase in most somatic cells and its reactivation in tumor cells. Moreover, the contradictory observations of the low level of hTERT mRNA in telomerase-positive cells and the high transcriptional activity of the hTERT promoter in transfection experiments remain unresolved. In this study, we demonstrated that the proximal exonic region of the hTERT gene (exon 1 and 2) is involved in the inhibition of its promoter. We identified the protein CTCF as the inhibitor of the hTERT promoter, through its binding to the first exon. The methylation of the first exon region, which is often observed in cancer cells but not in noimal cells, represses CTCF binding. Study of hTERT promoter methylation shows a partial demethylation sufficient to activate the transcription of the hTERT gene. Therefore, we demonstrated that the particular methylation profile of the hTERT regulatory sequences inhibits the binding of CTCF, while it allows a low transcription of the gene. Nevertheless, in some tumor cells, the promoter and the proximal exonic region of hTERT are unmethylated. In testicular and ovarian cancer cell lines, CTCF inhibition is counteracted by its BORIS paralogue that also binds the hTERT first exon but allows the promoter activation. The study of BORIS gene regulation showed that this factor is exclusively expressed in normal tissue of testis and ovary of young woman, as well as in almost all tumors with different levels. Two promoters were found to induce its transcription. The proximal promoter was regulated by methylation. Moreover, a major alternative transcript, deleted of the exon 6, is detected when this promoter is active. All these results lead to a model for hTERT regulation that takes into account the epigenetic profile of the gene and provides an explanation for the low transcriptional level observed in vivo. BORIS expression in cancers and its implication in hTERT activation might also permit the understanding of epigenetic deregulation and immortalization phenomena that occur during tumorigenesis.

Regulation of the expression of Nav1.5, the cardiac voltage-gated sodium channel

Abstract The cardiac sodium channel Nav1.5 plays a key role in cardiac excitability and conduction. Its importance for normal cardiac function has been highlighted by descriptions of numerous mutations of SCN5A (the gene encoding Nav1.5), causing cardiac arrhythmias which can lead to sudden cardiac death. The general aim of my PhD research project has been to investigate the regulation of Nav1.5 along two main axes: (1) We obtained experimental evidence revealing an interaction between Nav1.5 and a multiprotein complex comprising dystrophin. The first part of this study reports the characterization of this interaction. (2) The second part of the study is dedicated to the regulation of the cardiac sodium channel by the mineralocorticoid hormone named aldosterone. (1) Early in this study, we showed that Nav1.5 C-terminus was associated with dystrophin and that this interaction was mediated by syntrophin proteins. We used dystrophin-deficient mdx5cv mice to study the role of this interaction. We reported that dystrophin deficiency led to a reduction of both Nav1.5 protein level and the sodium current (INa). We also found that mdx5cv mice displayed atrial and ventricular conduction defects. Our results also indicated that proteasome inhibitor MG132 treatment of mdx5cv mice rescued Nav1.5 protein level and INa in cardiac tissue. (2) We showed that aldosterone treatment of mice cardiomyocytes led to an increase of the sodium current with no modification of Nav1.5 transcript and protein level. Altogether, these results suggest that the sodium current can be increased by distribution of intracellular pools of protein to the plasma membrane (e.g. upon aldosterone stimulation) and that interaction with dystrophin multiprotein complex is required for the stabilization of the channel at the plasma membrane. Finally, we obtained preliminary results suggesting that the proteasome could regulate Nav1.5 in mdx5cv mice. This study defines regulatory mechanisms of Nav1.5 which could play an important role in cardiac arrhythmia and bring new insight in cardiac conduction alterations observed in patients with dystrophinopathies. Moreover, this work suggests that Brugada syndrome, and some of the cardiac alterations seen in Duchenne patients may be caused by overlapping molecular mechanisms leading to a reduction of the cardiac sodium current.

Hormonal regulation of (Na+,K+)-ATPase biosynthesis in the toad bladder. Effect of aldosterone and 3,5,3'-triiodo-L-thyronine.

Aldosterone stimulates transepithelial Na+ transport in the toad bladder, and thyroid hormone antagonizes this mineralocorticoid action. In the present study, we assessed the influence of these two hormones on the biosynthesis of (Na+,K+)ATPase, the major driving force of Na+ transport. Rates of enzyme synthesis were estimated by immunoprecipitation with monospecific alpha (96,000 daltons) and beta (60,000 daltons) subunit antibodies. After a 30-min pulse of intact tissue with [35S]methionine, the anti-alpha-serum recognized the 96,000-dalton alpha subunit and the anti-beta-serum, a 42,000-dalton protein, in total cell extracts. The biosynthesis rates of both these proteins were increased 2.8- and 2.4-fold respectively, over controls by 80 nM aldosterone after 18 h of hormone treatment. The hormonal effect was not apparent up to 3 h of incubation and was dose dependent between 0.2 and 20 nM aldosterone. The hormonal induction was antagonized by spironolactone (500-fold excess) but not by amiloride. The action of aldosterone thus seems to be a receptor-mediated process and a primary event independent of the Na+ permeability of the apical membrane. Thyroid hormone, on the other hand, had no effect on either basal or aldosterone-stimulated synthesis rates of both enzyme proteins. The results demonstrate a direct effect of aldosterone on gene expression of the (Na+,K+)-ATPase. Ultimately, this phenomenon could be linked to the late mineralocorticoid action of this hormone. On the other hand, thyroid hormone, in contrast to the situation in mammals, does not stimulate de novo enzyme synthesis in amphibia. Neither can the antimineralocorticoid action of thyroid hormone in the toad bladder be explained by an inhibition of the (Na+,K+)-ATPase synthesis.

Cell cycle regulation of mitochondrial function.

Specific cellular functions, such as proliferation, survival, growth, or senescence, require a particular adaptive metabolic response, which is fine tuned by members of the cell cycle regulators families. Currently, proteins such as cyclins, CDKs, or E2Fs are being studied in the context of cell proliferation and survival, cell signaling, cell cycle regulation, and cancer. We show in this review that cellular, animal and molecular studies provided enough evidence to prove that these factors play, in addition, crucial roles in the control of mitochondrial function; finally resulting in a dual proliferative and metabolic response.

The monocarboxylate transporter MCT4 : mechanism of regulation in astrocytes and its putative role in cerebral ischemia

Despite its small fraction of the total body weight (2%), the brain contributes for 20% and 25% respectively of the total oxygen and glucose consumption of the whole body. Indeed, glucose has been considered the energy substrate par excellence for the brain. However, evidence accumulated over the last half century revealed an important role for the monocarboxylate lactate in fulfilling the energy needs of neurons. This is particularly true during physiological neuronal activation and in pathological conditions. Lactate transport into and out of the cell is mediated by a family of proton-linked transporters called monocarboxylate transporters (MCTs). In the central nervous system, only three of them have been well characterized: MCT2 is the predominant neuronal isoform, while the other non¬neuronal cell types of the brain express the ubiquitous isoform MCT1. Quite recently, the MCT4 isoform has been described in astrocytes. Due to its high transport capacity compared to the other two isoforms, MCT4 is particularly adapted for glycolytic cells. Because of its recent discovery in the brain, nothing was known about its regulation in the central nervous system. Here we show that MCT4 is regulated by oxygen levels in primary cultures of astrocytes in a time- and concentration-dependent manner via the hypoxia inducible factor-la (HIF-la). Moreover, we showed that MCT4 expression is essential for astrocyte survival under low oxygen conditions. In parallel, we investigated the possible implication of the pyruvate kinase isoform Pkm2, a strong enhancer of glycolysis, in its regulation. Then we showed that MCT4 expression, as well as the expression of the other two MCT isoforms, is altered in a murine model of stroke. Surprisingly, neurons started to express MCT4, as well as MCT1, under such conditions. Altogether, these data suggest that MCT4, due to its high transport capacity for lactate, may be the isoform that enables cells to operate a major metabolic adaptation in response to pathological situations that alter metabolic homeostasis of the brain. -- Le cerveau représente 2% du poids corporel total, mais il contribue pour 20% de la consommation totale d'oxygène et 25% de celle de glucose au repos. Le glucose est considéré comme le substrat énergétique par excellence pour le cerveau. Néanmoins, depuis un demi- siècle maintenant, de plus en plus de travaux ont démontré que le lactate joue un rôle majeur dans le métabolisme cérébral et est capable du subvenir aux besoins énergétiques des neurones. Le lactate est tout particulièrement nécessaire pendant l'activation neuronale ainsi qu'en situation pathologique. Le transport du lactate à travers la barrière hématoencéphalique ainsi qu'à travers les membranes cellulaires est assuré par la famille des transporteurs aux monocarboxylates (MCTs). Dans le système nerveux central, uniquement trois d'entre eux ont été décrits: MCT2 est considéré comme le transporteur neuronal, alors que les autres types cellulaires qui constituent le cerveau expriment l'isoforme ubiquitaire MCT1. Récemment, l'isoforme MCT4 a été rapportée sur les astrocytes. Dû à sa grande capacité de transport pour le lactate, MCT4 est tout particulièrement adapté pour soutenir le métabolisme des cellules hautement glycolytiques, comme les astrocytes. En raison de sa toute récente découverte, les aspects comprenant sa régulation et son rôle dans le cerveau sont pour l'instant méconnus. Les résultats exposés dans ce travail démontrent dans un premier temps que l'expression de MCT4 est régulée par les niveaux d'oxygène dans les cultures d'astrocytes corticaux par le biais du facteur de transcription HIF-la. De plus, nous avons démontré que l'expression de MCT4 est essentielle à la survie des astrocytes quand le niveau d'oxygénation baisse. En parallèle, des résultats préliminaires suggèrent que l'isoforme 2 de la pyruvate kinase, un puissant régulateur de la glycolyse, pourrait jouer un rôle dans la régulation de MCT4. Dans la deuxième partie du travail nous avons démontré que l'expression de MCT4, ainsi que celle de MCT1 et MCT2, est altérée dans un modèle murin d'ischémie cérébrale. De façon surprenante, les neurones expriment MCT4 dans cette condition, alors que ce n'est pas le cas en condition physiologique. En tenant compte de ces résultats, nous suggérons que MCT4, dû à sa particulièrement grande capacité de transport pour le lactate, représente le MCT qui permet aux cellules du système nerveux central, notamment les astrocytes et les neurones, de s'adapter à de très fortes perturbations de l'homéostasie métabolique du cerveau qui surviennent en condition pathologique.

Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability.

Secondary structure-forming DNA sequences such as CAG repeats interfere with replication and repair, provoking fork stalling, chromosome fragility, and recombination. In budding yeast, we found that expanded CAG repeats are more likely than unexpanded repeats to localize to the nuclear periphery. This positioning is transient, occurs in late S phase, requires replication, and is associated with decreased subnuclear mobility of the locus. In contrast to persistent double-stranded breaks, expanded CAG repeats at the nuclear envelope associate with pores but not with the inner nuclear membrane protein Mps3. Relocation requires Nup84 and the Slx5/8 SUMO-dependent ubiquitin ligase but not Rad51, Mec1, or Tel1. Importantly, the presence of the Nup84 pore subcomplex and Slx5/8 suppresses CAG repeat fragility and instability. Repeat instability in nup84, slx5, or slx8 mutant cells arises through aberrant homologous recombination and is distinct from instability arising from the loss of ligase 4-dependent end-joining. Genetic and physical analysis of Rad52 sumoylation and binding at the CAG tract suggests that Slx5/8 targets sumoylated Rad52 for degradation at the pore to facilitate recovery from acute replication stress by promoting replication fork restart. We thereby confirmed that the relocation of damage to nuclear pores plays an important role in a naturally occurring repair process.

Catechol-O-methyltransferase, dopamine, and sleep-wake regulation.

NlmCategory="UNASSIGNED">Sleep and sleep disorders are complex and highly variable phenotypes regulated by many genes and environment. The catechol-O-methyltransferase (COMT) gene is an interesting candidate, being one of the major mammalian enzymes involved in the catabolism of catecholamines. The activity of COMT enzyme is genetically polymorphic due to a guanine-to-adenine transition at codon 158, resulting in a valine (Val) to methionine (Met) substitution. Individuals homozygous for the Val allele show higher COMT activity, and lower dopaminergic signaling in prefrontal cortex (PFC) than subjects homozygous for the Met allele. Since COMT has a crucial role in metabolising dopamine, it was suggested that the common functional polymorphism in the COMT gene impacts on cognitive function related to PFC, sleep-wake regulation, and potentially on sleep pathologies. The COMT Val158Met polymorphism may predict inter-individual differences in brain electroencephalography (EEG) alpha oscillations and recovery processes resulting from partial sleep loss in healthy individuals. The Val158Met polymorphism also exerts a sexual dimorphism and has a strong effect on objective daytime sleepiness in patients with narcolepsy-cataplexy. Since the COMT enzyme inactivates catecholamines, it was hypothesized that the response to stimulant drugs differs between COMT genotypes. Modafinil maintained executive functioning performance and vigilant attention throughout sleep deprivation in subjects with Val/Val genotype, but less in those with Met/Met genotype. Also, homozygous Met/Met patients with narcolepsy responded to lower doses of modafinil compared to Val/Val carriers. We review here the critical role of the common functional COMT gene polymorphism, COMT enzyme activity, and the prefrontal dopamine levels in the regulation of sleep and wakefulness in normal subjects, in narcolepsy and other sleep-related disorders, and its impact on the response to psychostimulants.

Multilayered organization of jasmonate signalling in the regulation of root growth.

Physical damage can strongly affect plant growth, reducing the biomass of developing organs situated at a distance from wounds. These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling. Using a novel assay involving repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a function of JA in suppressing cell division and elongation in roots. Regulatory JA signalling components were then manipulated to delineate their relative impacts on root growth. The new transcription factor mutant myc2-322B was isolated. In vitro transcription assays and whole-plant approaches revealed that myc2-322B is a dosage-dependent gain-of-function mutant that can amplify JA growth responses. Moreover, myc2-322B displayed extreme hypersensitivity to JA that totally suppressed root elongation. The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation. Furthermore, in a JA-deficient mutant background, ninja1 myc2-322B still repressed root elongation, indicating that it is possible to generate JA-responses in the absence of JA. We show that NINJA forms a broadly expressed regulatory layer that is required to inhibit JA signalling in the apex of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4 displayed cell layer-specific localisations and MYC3 and MYC4 were expressed in mutually exclusive regions. In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth. Our data reveal how distinct negative regulatory layers, including both NINJA-dependent and -independent mechanisms, restrain JA responses to allow normal root growth. Mechanistic insights from this work underline the importance of mapping JA signalling components to specific cell types in order to understand and potentially engineer the growth reduction that follows physical damage.