Régulation de l'activité de Krox20 au cours de la mise en place du sytème nerveux central et périphérique

Le gène Krox20, codant un facteur de transcription, joue un rôle majeur dans le processus de segmentation du rhombencéphale. Il est nécessaire à la formation et au maintien des segments où il est exprimé, ainsi qu à l organisation correcte des nerfs crâniens. Une étude structure-fonction de Krox20 m a permis de montrer que son activité dans le rhombencéphale implique différents domaines de la protéine selon les cibles transcriptionnelles considérées et de mieux caractériser le rôle de ses co-facteurs Nab et Hcf-1. Krox20 intervient de plus dans la myélinisation du système nerveux périphérique et des mutations l affectant ont été caractérisées dans des cas de maladie de Charcot-Marie-Tooth. J ai généré une lignée de souris portant l une de ces mutations, abolissant l interaction entre Krox20 et les Nab. Les mutants présentent des défauts de locomotion et une altération complexe de la myélinisation, avec un maintien de la prolifération et une dérégulation génique des cellules de Schwann. Ces mutants présentent de plus une fusion de certains nerfs crâniens.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

A receptor protein tyrosine kinase implicated in the segmental patterning of the hindbrain and mesoderm

14 páginas, 9 figuras.Pattern formation in the hindbrain and paraxial mesoderm of vertebrates occurs by the formation of a series of repeated segments. These processes of segmentation appear different at the morphological level, since hindbrain segments, the rhombomeres, form by the subdivision of the neural epithelium into compartments, whereas the mesodermal somites form by the sequential aggregation of mesenchymal cells into epithelial balls. Previous studies have implicated genes encoding transcription factors in the development of hindbrain segments, but nothing is known of genes involved in the formation of somites. Cellular interactions and signal transduction must be an important aspect of hindbrain segmentation, so we have screened for tyrosine kinases expressed in rhombomere-restricted patterns in the developing mouse embryo. We have identified a receptor protein tyrosine kinase, Sek, that has high relative levels of expression in rhombomeres 3 and 5. This alternating pattern is established coincidentally, both spatially and temporally, with the expression of Krox-20, a zinc-finger gene expressed prior to the morphological formation of rhombomeres. In addition, Sek expression occurs in several other developing tissues, including a dynamic regulation in the developing forebrain, spinal cord, early mesoderm and anterior presomitic mesoderm (segmental plate). The latter expression occurs in two stripes that correlate with, and presage, the formation of somites. Sek expression initially occurs throughout the presumptive somite, then becomes restricted anteriorly, and finally is down-regulated as the definitive somite is formed. These data suggest that despite the morphological differences in the segmentation of the hindbrain and mesoderm, Sek is involved in the segmental patterning of both of these tissues.M. A. N. was supported by an EEC Science Program Postdoctoral Fellowship.Peer reviewe

Enhancer–gene maps in the human and zebrafish genomes using evolutionary linkage conservation

International audienceThe spatiotemporal expression of genes is controlled by enhancer sequences that bind transcription factors. Identifying the target genes of enhancers remains difficult because enhancers regulate gene expression over long genomic distances. To address this, we used an evolutionary approach to build two genome-wide maps of predicted enhancer-gene associations in the human and zebrafish genomes. Evolutionary conserved sequences were linked to their predicted target genes using PEGASUS, a bioinformatics method that relies on evolutionary conservation of synteny. The analysis of these maps revealed that the number of predicted enhancers linked to a gene correlate with its expression breadth. Comparison of both maps identified hundreds of putative vertebrate ancestral regulatory relationships from which we could determine that predicted enhancergene distances scale with genome size despite strong positional conservation. The two maps represent a resource for further studies, including the prioritization of sequence variants in whole genome sequence of patients affected by genetic diseases

Disruption of Krox20-Nab interaction in the mouse leads to peripheral neuropathy with biphasic evolution.

International audienc

Segmental expression of the EphA4 (Sek-1) receptor tyrosine kinase in the hindbrain is under direct transcriptional control of Krox-20.

International audienceSegmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres (r) with distinct identities. Recent studies have uncovered regulatory links between transcription factors governing this process, but little is known of how these relate to molecules mediating cell-cell signalling. The Eph receptor tyrosine kinase gene EphA4 (Sek-1) is expressed in r3 and r5, and function-blocking experiments suggest that it is involved in restricting intermingling of cells between odd- and even-numbered rhombomeres. We have analysed the cis-acting regulatory sequences of the EphA4 gene in transgenic mice and identified a 470 bp enhancer element that drives specific expression in r3 and r5. Within this element, we have identified eight binding sites for the Krox-20 transcription factor that is also expressed in r3 and r5. Mutation of these binding sites abolishes r3/r5 enhancer activity and ectopic expression of Krox-20 leads to ectopic activation of the enhancer. These data indicate that Krox-20 is a direct transcriptional activator of EphA4. Together with evidence that Krox-20 regulates Hox gene expression, our findings reveal a mechanism by which the identity and movement of cells are coupled such that sharply restricted segmental domains are generated

Neural crest patterning: autoregulatory and crest-specific elements co-operate for Krox20 transcriptional control

International audienceNeural crest patterning constitutes an important element in the control of the morphogenesis of craniofacial structures. Krox20, a transcription factor gene that plays a critical role in the development of the segmented hindbrain, is expressed in rhombomeres (r) 3 and 5 and in a stream of neural crest cells migrating from r5 toward the third branchial arch. We have investigated the basis of the specific neural crest expression of Krox20 and identified a cis-acting enhancer element (NCE) located 26 kb upstream of the gene that is conserved between mouse, man and chick and can recapitulate the Krox20 neural crest pattern in transgenic mice. Functional dissection of the enhancer revealed the presence of two conserved Krox20 binding sites mediating direct Krox20 autoregulation in the neural crest. In addition, the enhancer included another essential element containing conserved binding sites for high mobility group (HMG) box proteins and which responded to factors expressed throughout the neural crest. Consistent with this the NCE was strongly activated in vitro by Sox10, a crest-specific HMG box protein, in synergism with Krox20, and the inactivation of Sox10 prevented the maintenance of Krox20 expression in the migrating neural crest. These results suggest that the dependency of the enhancer on both crest- (Sox10) and r5- (Krox20) specific factors limits its activity to the r5-derived neural crest. This organisation also suggests a mechanism for the transfer and maintenance of rhombomere-specific gene expression from the hindbrain neuroepithelium to the emerging neural crest and may be of more general significance for neural crest patterning

Divergent transcriptional and transforming properties of PAX3-FOXO1 and PAX7-FOXO1 paralogs

Abstract The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Both PAX-FOXO1s result in related cell transformation in animal models, but both mutations are associated with distinct pathological manifestations in patients. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 causes stronger de novo transactivation of its bound regions than PAX3-FOXO1, resulting in greater transcriptomic dynamics involving genes regulating cell shape and cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into M phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into S phase, and causes more genomic instabilities. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the transcriptional activities of PAX3-FOXO1 and PAX7-FOXO1. Furthermore, the identified pronounced deleterious effects of PAX7-FOXO1 provide an explanation for the low frequency of the translocation generating this factor in patients with rhabdomyosarcoma

Segmental expression of the EphA4 (Sek-1) receptor tyrosine kinase in the hindbrain is under direct transcriptional control of Krox-20.

International audienceSegmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres (r) with distinct identities. Recent studies have uncovered regulatory links between transcription factors governing this process, but little is known of how these relate to molecules mediating cell-cell signalling. The Eph receptor tyrosine kinase gene EphA4 (Sek-1) is expressed in r3 and r5, and function-blocking experiments suggest that it is involved in restricting intermingling of cells between odd- and even-numbered rhombomeres. We have analysed the cis-acting regulatory sequences of the EphA4 gene in transgenic mice and identified a 470 bp enhancer element that drives specific expression in r3 and r5. Within this element, we have identified eight binding sites for the Krox-20 transcription factor that is also expressed in r3 and r5. Mutation of these binding sites abolishes r3/r5 enhancer activity and ectopic expression of Krox-20 leads to ectopic activation of the enhancer. These data indicate that Krox-20 is a direct transcriptional activator of EphA4. Together with evidence that Krox-20 regulates Hox gene expression, our findings reveal a mechanism by which the identity and movement of cells are coupled such that sharply restricted segmental domains are generated