Myc et compétitions intercellulaires chez la drosophile

La différenciation cellulaire repose en partie sur des mécanismes de sélection, notamment par compétition vis-à-vis de facteurs extrinsèques vitaux présents en quantité limitante ; mais quels sont les déterminants internes gouvernant l’aptitude des cellules dans ce type de compétition ? De récents travaux menés sur le disque imaginal de l’aile de drosophile en identifie un, la protéine d-Myc. En effet, le niveau relatif d’expression de d-Myc par les cellules de cet épithélium détermine l’issue de leur compétition, les cellules les mieux pourvues tendant à surcroître, au détriment de leur voisines qui disparaissent par apoptose. Un niveau relativement élevé de d-Myc confère donc un avantage compétitif décisif, probablement en stimulant la biogenèse des ribosomes, ce qui augmenterait la capacité des cellules à recevoir, voire à accaparer, des facteurs extrinsèques vitaux, en particulier Dpp (decapentaplegic). Ces résultats pourraient expliquer comment une surexpression des protéines Myc de vertébrés contribue à la tumorigenèse et, par conséquent, éclairer les liens unissant métabolisme et cancer.Cell differentiation and organ shaping proceed not only upon instructive but also upon competitive cell-cell interactions. In the proliferating epithelium forming the larval Drosophila wing disc, cell competition contributes to the fidelity of the organogenesis. Several recent studies show how d-myc, encoding a bHLH/LZ transcription factor homologous to vertebrate Myc proteins, controls cell competition during wing development. In this model, any experiment leading to the confrontation of two populations differing by their levels of d-Myc expression, even in a two-fold ratio, gives rise to a competition characterized both by an overgrowth of the population having the highest level and an apoptotic elimination of the neighbouring « weakly » expressing cells. As a consequence of the mutually compensating nature of these two processes, the final size of the wing remains unchanged. Importantly, lowering or elevating d-Myc expression to the same extent in all cells of the disc does not trigger competition. This indicates that competition is linked to a spatial heterogeneity in, and not to the absolute level of, d-Myc expression. Both vertebrate and Drosophila Myc proteins stimulate ribosome biogenesis, and genetic evidence in Drosophila suggests that this property underlies the strong competitive advantage imparted by its relatively high expression. Accordingly, it is proposed, although not proved, that the more the wing cells express d-Myc and amplify their protein synthesis apparatus, the more they bind, internalize, and transduce the vital and limiting growth factor Dpp, which in turn is presumed to increase d-Myc protein level. These findings suggest that wing organogenesis is a self-corrected process whereby d-Myc induction in overgrowing cells ensures the compensatory elimination of their neighbours. Moreover, they have important implications for the oncogenic role of vertebrate Myc proteins and possibly of related transcription factors

A new generation of pPRIG-based retroviral vectors

<p>Abstract</p> <p>Background</p> <p>Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c).</p> <p>Results</p> <p>The convenience of pPRIG makes it a good basic vector to generate additional derivatives for an extended range of use. Here we present several novel pPRIG-based vectors (collectively referred to as PRIGs) in which : i) the HA tag sequence was inserted in the three reading frames 3' of the MCS (3'HA PRIGs); ii) a functional domain (ER, VP16 or KRAB) was inserted either 5' or 3' of the MCS (« modular » PRIGs); iii) eGFP was replaced by either eCFP, eYFP, mCherry or puro-R (« single color/resistance » PRIGs); and iv) mCherry, eYFP or eGFP was inserted 5' of the MCS of the IRES-eGFP, IRES-eCFP or IRES-Puro-R containing PRIGs, respectively (« dual color/selection » PRIGs). Additionally, some of these PRIGs were also constructed in a pMigR MSCV background which has been widely used in pluripotent cells.</p> <p>Conclusion</p> <p>These novel vectors allow for straightforward detection of any expressed protein (3'HA PRIGs), for functional studies of chimeric proteins (« modular » PRIGs), for multiple transductions and fluorescence analyses of transduced cells (« single color/resistance » PRIGs), or for quantitative detection of studied proteins in independently identified/selected transduced cells (« dual color/selection » PRIGs). They maintain the original advantages of pPRIG and provide suitable tools for either transient or stable expression and functional studies in a large range of experimental settings.</p

Development of a new bicistronic retroviral vector with strong IRES activity

BACKGROUND: Internal Ribosome Entry Site (IRES)-based bicistronic vectors are important tools in today's cell biology. Among applications, the expression of two proteins under the control of a unique promoter permits the monitoring of expression of a protein whose biological function is being investigated through the observation of an easily detectable tracer, such as Green Fluorescent Protein (GFP). However, analysis of published results making use of bicistronic vectors indicates that the efficiency of the IRES-controlled expression can vary widely from one vector to another, despite their apparent identical IRES sequences. We investigated the molecular basis for these discrepancies. RESULTS: We observed up to a 10 fold difference in IRES-controlled expression from distinct bicistronic expression vectors harboring the same apparent IRES sequences. We show that the insertion of a HindIII site, in place of the initiating AUG codon of the wild type EMCV IRES, is responsible for the dramatic loss of expression from the second cistron, whereas expression from the first cistron remains unaffected. Thus, while the replacement of the authentic viral initiating AUG by a HindIII site results in the theoretical usage of the initiation codon of the HindIII-subcloned cDNA, the subsequent drop of expression dramatically diminishes the interest of the bicistronic structure. Indeed, insertion of the HindIII site has such a negative effect on IRES function that detection of the IRES-controlled product can be difficult, and sometimes even below the levels of detection. It is striking to observe that this deleterious modification is widely found in available IRES-containing vectors, including commercial ones, despite early reports in the literature stating the importance of the integrity of the initiation codon for optimal IRES function. CONCLUSION: From these observations, we engineered a new vector family, pPRIG, which respects the EMCV IRES structure, and permits easy cloning, tagging, sequencing, and expression of any cDNA in the first cistron, while keeping a high level of expression from its IRES-dependent second cistron (here encoding eGFP)

Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse Yolk Sac: a comparative study between in situ electroporation and retroviral transduction

<p>Abstract</p> <p>Background</p> <p>Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.</p> <p>Results</p> <p>One transduction protocol involves transient modification of gene expression through <it>in situ </it>electroporation of the prospective blood islands, which allows the evolution of transfected mesodermal cells in their "normal" environment, upon organ culture. Following <it>in situ </it>electroporation of a GFP reporter construct into the YS cavity of embryos at post-streak (mesodermal/pre-hematopoietic precursors) or early somite (hematopoietic precursors) stages, high GFP expression levels as well as a good preservation of cell viability is observed in YS explants. Moreover, the erythro-myeloid progeny typical of the YS arises from GFP⁺mesodermal cells or hematopoietic precursors, even if the number of targeted precursors is low. The second approach, based on retroviral transduction allows a very efficient transduction of large precursor numbers, but may only be used to target 8 dpc YS hematopoietic precursors. Again, transduced cells generate a progeny quantitatively and qualitatively similar to that of control YS.</p> <p>Conclusion</p> <p>We thus provide two protocols whose combination may allow a thorough study of both early and late events of hematopoietic development in the murine YS. <it>In situ </it>electroporation constitutes the only possible gene transfer method to transduce mesodermal/pre-hematopoietic precursors and analyze the earliest steps of hematopoietic development. Both <it>in situ </it>electroporation and retroviral transduction may be used to target early hematopoietic precursors, but the latter appears more convenient if a large pool of stably transduced cells is required. We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.</p

Extracellular acidification stimulates GPR68 mediated IL-8 production in human pancreatic β cells.

International audienceAcute or chronic metabolic complications such as diabetic ketoacidosis are often associated with extracellular acidification and pancreatic β-cell dysfunction. However, the mechanisms by which human β-cells sense and respond to acidic pH remain elusive. In this study, using the recently developed human β-cell line EndoC-βH2, we demonstrate that β-cells respond to extracellular acidification through GPR68, which is the predominant proton sensing receptor of human β-cells. Using gain- and loss-of-function studies, we provide evidence that the β-cell enriched transcription factor RFX6 is a major regulator of GPR68. Further, we show that acidic pH stimulates the production and secretion of the chemokine IL-8 by β-cells through NF-кB activation. Blocking of GPR68 or NF-кB activity severely attenuated acidification induced IL-8 production. Thus, we provide mechanistic insights into GPR68 mediated β-cell response to acidic microenvironment, which could be a new target to protect β-cell against acidosis induced inflammation

Modeling human pancreatic beta cell dedifferentiation

Objective: Dedifferentiation could explain reduced functional pancreatic β-cell mass in type 2 diabetes (T2D). Methods: Here we model human β-cell dedifferentiation using growth factor stimulation in the human β-cell line, EndoC-βH1, and human pancreatic islets. Results: Fibroblast growth factor 2 (FGF2) treatment reduced expression of β-cell markers, (INS, MAFB, SLC2A2, SLC30A8, and GCK) and activated ectopic expression of MYC, HES1, SOX9, and NEUROG3. FGF2-induced dedifferentiation was time- and dose-dependent and reversible upon wash-out. Furthermore, FGF2 treatment induced expression of TNFRSF11B, a decoy receptor for RANKL and protected β-cells against RANKL signaling. Finally, analyses of transcriptomic data revealed increased FGF2 expression in ductal, endothelial, and stellate cells in pancreas from T2D patients, whereas FGFR1, SOX,9 and HES1 expression increased in islets from T2D patients. Conclusions: We thus developed an FGF2-induced model of human β-cell dedifferentiation, identified new markers of dedifferentiation, and found evidence for increased pancreatic FGF2, FGFR1, and β-cell dedifferentiation in T2D

Protéger et sévir : p53, métabolisme et suppression tumorale

Le gène p53 joue un rôle fondamental dans la suppression tumorale. Ce rôle est généralement expliqué par les trois réponses (le triumvirat)- arrêt transitoire du cycle cellulaire, sénescence ou apoptose - induites par p53 dans les cellules exposées à un stress génotoxique ou oncogénique. Cependant, des résultats récents indiquent que ce triumvirat n’est pas toujours nécessaire à la suppression tumorale. Ils suggèrent, en revanche, que la régulation du métabolisme énergétique par p53, plus précisément sa capacité à diminuer la glycolyse et à stimuler la phosphorylation oxydative, c’est-à-dire à inhiber l’effet Warburg, est un mécanisme important de la suppression tumorale. Une analyse de ces données suggère que les fonctions « conservatrices » de p53, en particulier l’inhibition de l’effet Warburg et l’activité anti-oxydante, mécanistiquement reliées et plutôt associées à une faible expression de p53, préviennent « quotidiennement » la transformation tumorale des cellules normales. Quand cette première barrière est franchie, les fonctions « destructrices » de p53, déclenchées par son expression plus élevée et conduisant à la sénescence ou à l’apoptose, permettent la suppression tumorale vis-à-vis des cellules partiellement ou complètement transformées

Ki67 : un surfactant des chromosomes mitotiques

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La protéine neuronale Arc : une capside de rétrotransposon recyclée pour des fonctions clés dans les synapses

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