Caractérisation de l’effet de mutations MODY sur la fonction de bookmarking de HNF1beta

HNF1beta is a POU transcription factor that is frequently mutated in patients that suffer from diabetes and renal cystic dysplasia. This protein has the peculiar ability to bind mitotic chromosomes and behave as a gene bookmarking. Here we show that the capacity of HNF1beta to bind to DNA plays an essential role for mitotic binding. A close homologue, HNF1alpha, shares the ability of HNF1beta to bind to mitotic chromosomes, and several MODY mutations (e.g P256S, V265L and C273Y) affect the ability of the protein to localize to mitotic chromatin. Interestingly, the phenotype induced by these mutations is very rapidly rescued by sudden temperature shifts. Temperature-sensitivity is probably linked to a conformational change that prevents DNA binding ability of P256S and V265L mutants at 37°C. Interestingly, the mitotic relocalization of these mutants induced by temperature shift was sensitive to energy depletion and importazole, suggesting an active mechanism involving the importin-beta system. Interestingly, C273Y mutant exhibited a significantly mitotic dispersion that is not correlated with any DNA or interphase chromatin binding defect, indicating that DNA binding function is necessary but not sufficient to accomplish bookmarking.HNF1beta est un facteur de transcription homeobox, dont les mutations sont fréquemment rencontrées chez des patients atteints d’anomalies congénitales du rein et du tractus urinaire (Congenital Abnomalities of the Kidney and the Urogenital Tract, CAKUT). HNF1beta est également impliqué dans le diabète de type Maturity Onset Diabetes of the Young 5 (MODY5). Le laboratoire d’accueil a démontré que HNF1beta était impliqué dans un mécanisme épigénétique, le Bookmarking, nécessaire à la réexpression post-mitotique de ses gènes cibles. En particulier, des expériences de vidéo-microscopie ont montré que la partie N-terminale de HNF1beta, contenant le domaine de liaison à l’ADN, en fusion avec la GFP (HNF1beta -GFP) est liée à la chromatine pendant la mitose. L’objectif de ma thèse était de caractériser les modalités biochimiques d’interaction de HNF1beta avec la chromatine mitotique. Nous avons mis en évidence le fait que la capacité de liaison à l’ADN est indispensable à la localisation mitotique de HNF1beta. En effet, la délétion de la troisième hélice alpha de l’homéo-domaine, responsable de l’interaction avec le grand sillon de l’ADN, entraîne la dissociation de la chromatine de HNF1beta pendant la mitose. Nous avons ensuite étudié l’effet de plusieurs mutations identifiées chez des patients MODY sur la localisation mitotique de HNF1beta. Nos résultats ont montré que certaines mutations faux-sens sont capables d’empêcher la fixation de la chromatine mitotique. Parmi ces mutations, certaines manifestent un phénotype dépendant de la température. Par exemple, à une température permissive, inférieure à 30°C, les mutations P256S et C273Y présentent une localisation mitotique normale. En revanche, à 37°C pour P256S et à 39°C pour le mutant C273Y, les protéines sont complètement dissociées, alors que dans toutes ces conditions de température, l’association de la protéine sauvage avec la chromatine mitotique n’est pas affectée. A température permissive (4°C), nous avons montré par retard sur gel (Electophoresis Mobility Shift Assay EMSA) que les mutants lient l’ADN avec un Kd apparent similaire à celui de la protéine sauvage. Par contre, à température restrictive, les mutants présentent des comportements différents. En effet, P256S perd sa capacité de liaison à l’ADN (de façon réversible), tandis que C273Y continue à lier l’ADN avec une affinité similaire à celui de la protéine sauvage. Le caractère thermosensible des mutants de HNF1beta nous a permis d’étudier les modalités de son recrutement sur la chromatine mitotique. Nos résultats ont montré que l’association des protéines à la chromatine mitotique présente une nature très dynamique. En effet, nous avons observé qu’une diminution rapide de température détermine la relocalisation mitotique réversible de la protéine, dans un délai de quelques secondes. Nous avons pu montrer que la relocalisation mitotique de HNF1beta induit par la température était affectée par une déplétion d’énergie, ainsi que par l’action d’un inhibiteur spécifique de l’importine-β (importazole). Nous avons enfin mis en évidence par immuno-précipitation de chromatine (ChIP) que la liaison de HNF1beta à la chromatine mitotique est séquence-spécifique. Nos résultats suggèrent que le recrutement de HNF1beta à la chromatine mitotique est énergie-dépendante, et nécessite le bon fonctionnement du système de transport lié à l’importine-beta. Mes résultats suggèrent que des mutations trouvées chez des patients MODY3 et MODY5 inactivent ou affaiblissent la capacité de HNF1beta de remplir son activité de Bookmarking

A biome-dependent distribution gradient of tree species range edges is strongly dictated by climate spatial heterogeneity

Altres ajuts: M.F.M. was supported by a postdoctoral fellowship from 'la Caixa' Foundation (ID 100010434), code: LCF/BQ/PI21/11830010.Understanding the causes of the arrest of species distributions has been a fundamental question in ecology and evolution. These questions are of particular interest for trees owing to their long lifespan and sessile nature. A surge in data availability evokes a macro-ecological analysis to determine the underlying forces limiting distributions. Here we analyse the spatial distribution of >3,600 major tree species to determine geographical areas of range-edge hotspots and find drivers for their arrest. We confirmed biome edges to be strong delineators of distributions. Importantly, we identified a stronger contribution of temperate than tropical biomes to range edges, adding strength to the notion that tropical areas are centres of radiation. We subsequently identified a strong association of range-edge hotspots with steep spatial climatic gradients. We linked spatial and temporal homogeneity and high potential evapotranspiration in the tropics as the strongest predictors of this phenomenon. We propose that the poleward migration of species in light of climate change might be hindered because of steep climatic gradients

High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly- and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used o increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications

Carm1-arginine methylation of the transcription factor C/EBPα regulates transdifferentiation velocity

Developmental biology; Gene regulation; Transcription factorBiologia del desenvolupament; Regulació gènica; Factor de transcripcióBiología del desarrollo; Regulación génica; Factor de transcripciónHere, we describe how the speed of C/EBPα-induced B cell to macrophage transdifferentiation (BMT) can be regulated, using both mouse and human models. The identification of a mutant of C/EBPα (C/EBPαR35A) that greatly accelerates BMT helped to illuminate the mechanism. Thus, incoming C/EBPα binds to PU.1, an obligate partner expressed in B cells, leading to the release of PU.1 from B cell enhancers, chromatin closing and silencing of the B cell program. Released PU.1 redistributes to macrophage enhancers newly occupied by C/EBPα, causing chromatin opening and activation of macrophage genes. All these steps are accelerated by C/EBPαR35A, initiated by its increased affinity for PU.1. Wild-type C/EBPα is methylated by Carm1 at arginine 35 and the enzyme’s perturbations modulate BMT velocity as predicted from the observations with the mutant. Increasing the proportion of unmethylated C/EBPα in granulocyte/macrophage progenitors by inhibiting Carm1 biases the cell’s differentiation toward macrophages, suggesting that cell fate decision velocity and lineage directionality are closely linked processes.TG was supported by the Center for Genomic Regulation, Barcelona, the Spanish Ministry of Economy, Industry and Competitiveness, (Plan Estatal PID2019-109354GB-100), AGAUR (SGR 006713) and the 4D-Genome European Research Council Synergy grant. KSZ was supported by the NIH grant R01GM36477. We have used ChatGPT to improve parts of the text

Doing Well by Doing Good? Community Development Venture Capital

This paper examines the investments and performance of community development venture capital (CDVC). We find substantial differences between CDVC and traditional venture capital (VC) investments: CDVC investments are far more likely to be in nonmetropolitan regions and in regions with little prior venture capital activity. Moreover, CDVC is likely to be in earlier-stage investments and in industries outside the venture capital mainstream that have lower probabilities of successful exit. Even after we control for this unattractive transaction mix, the probability of a CDVC investment being successfully exited is lower. One benefit of CDVCs may be their effect in bringing traditional VC investment to underserved regions: When we control for the presence of traditional VC investments, each additional CDVC investment results in an additional 0.06 new traditional VC fi rm in a region

Perceiving Others’ Feelings: The Importance of Personality and Social Structure

Recent research has explored the relationship between social hierarchy and empathic accuracy— the ability to accurately infer others’ mental states. In the current research, we tested the hypothesis that, regardless of one’s personal level of status and power, simply believing that social inequality is natural and morally acceptable (e.g., endorsement of social dominance orientation, or SDO), would be negatively associated with empathic accuracy. In a sample of managers, a group for whom empathic accuracy is a valuable skill, empathic accuracy was lower for those who possessed structural power and also for those who endorsed social dominance, regardless of their structural power. Moreover, men were less empathically accurate than women, a relationship statistically mediated by men’s higher SDO and greater structural power. These findings suggest that for empathic abilities, it matters just as much what you think about social hierarchies as it does where you stand within them

The Interaction of Testosterone and Cortisol Is Associated With Attained Status in Male Executives.

Are hormone levels associated with the attainment of social status? Although endogenous testosterone predicts status-seeking social behaviors, research suggests that the stress hormone cortisol may inhibit testosterone’s effects. Thus, individuals with both high testosterone and low cortisol may be especially likely to occupy high-status positions in social hierarchies while individuals with high testosterone and high cortisol may not. We tested this hypothesis by recruiting a sample of real executives and examining testosterone, cortisol, and a concrete indicator of attained status: the number of subordinates over which the executive has authority. Despite the myriad nonhormonal factors that determine organizational promotion, the executives’ endogenous testosterone and cortisol interacted to significantly predict hierarchical position: Testosterone positively predicted executives’ number of subordinates, but only among low-cortisol executives. The results imply that reducing cortisol levels via stress reduction may be a critical goal not only because doing so will improve health but also because doing so may enhance leadership potential

High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly- and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used o increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications

Leptin Stimulates Cellular Glycolysis Through a STAT3 Dependent Mechanism in Tilapia

We assessed if leptin, a cytokine hormone known to enhance energy expenditure by promoting lipid and carbohydrate catabolism in response to physiologic stress, might directly regulate cellular glycolysis. A transcriptomic analysis of prolactin cells in the tilapia (Oreochromis mossambicus) pituitary rostral pars distalis (RPD) revealed that recombinant leptin (rtLep) differentially regulates 1,995 genes, in vitro. Machine learning algorithms and clustering analyses show leptin influences numerous cellular gene networks including metabolism; protein processing, transport, and metabolism; cell cycle and the hypoxia response. Leptin stimulates transcript abundance of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (gapdh) in a covariate manner to the hypoxic stress gene network. Orthogonal tests confirm that rtLepA dose-dependently increases gapdh gene expression in the RPD along with transcript abundance of 6-phosphofructo-1-kinase (pfk1), the rate limiting glycolytic enzyme. Functional testing demonstrated that leptin stimulates PFK activity and glycolytic output, while Stattic (a STAT3 blocker) was sufficient to suppress these responses, indicating leptin stimulates glycolysis through a STAT3-dependent mechanism. Leptin also stimulated pfk1 gene expression and lactate production in primary hepatocyte incubations in a similar manner to those shown for the pituitary RPD. This work characterizes a critical metabolic action of leptin to directly stimulate glycolysis across tissue types in a teleost model system, and suggest that leptin may promote energy expenditure, in part, by stimulating glycolysis. These data in a teleost fish, suggest that one of leptin's ancient, highly-conserved functions among vertebrates may be stimulation of glycolysis to facilitate the energetic needs associated with various stressors