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Regulation et fonction des clusters Hox pendant l'extension de l'axe corporel in vivo et in vitro

English title Gene regulation and function during axial extension in vivo and in vitro
Applicant Duboule Denis
Number 196868
Funding scheme Project funding (Div. I-III)
Research institution Département de Génétique et Evolution Faculté des Sciences Université de Genève
Institution of higher education EPF Lausanne - EPFL
Main discipline Genetics
Start/End 01.03.2021 - 28.02.2025
Approved amount 1'208'000.00
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All Disciplines (2)

Discipline
Genetics
Embryology, Developmental Biology

Keywords (7)

Transcription; enhancers; Gastruloids; Gene regulation; CRISPR; ES cells; 3R

Lay Summary (French)

Lead
Le développement des axes corporels principaux est un phénomène essentiel dans la construction des animaux. Comment les gènes qui contrôlent ces processus sont-ils activés au bon moment et dans les bonnes cellules est une question fondamentale de la biologie qui reste à résoudre.
Lay summary

Contenu et objectifs du travail de recherche

Les génomes des mammifères contiennent 39 gènes Hox dont les protéines identifient les différentes parties des axes du corps par des combinatoires différentes. Des erreurs dans la distribution de ces protéines conduisent à des syndromes génétiques congénitaux et il est donc essentiel de comprendre les mécanismes qui contrôlent l’activation de ces gènes. C’est le but de ce projet qui utilisera les développements technologiques récents dans le domaine de la post-génomique ainsi que les outils CRISPR-cas9 permettant des modifications génétiques rapides et précises soit d’animaux, soit de cellules souches en culture. Ce projet prévoit également l’étude de pseudo-embryons produits in vitro et récapitulant plusieurs aspects du développement embryonnaire normal.

Nous souhaitons modifier dans le génome de la souris certains composants de ces mécanismes afin de déterminer leur rôle dans l’activation des gènes Hox. Une attention particulière est portée sur les relations entre ces mécanismes de contrôle génétique et la structure de la chromatine, à savoir comment un génome empaqueté dans les noyaux cellulaires peut se rendre accessible de façon sélective pendant le développement. Les résultats et conclusions obtenus devraient s’appliquer à l’ensemble des phénomènes de régulation génétiques à l’œuvre chez l’embryon.

 

Contexte scientifique et social du projet de recherche

Ce projet relève de la recherche fondamentale et les connaissances acquises contribuent à la compréhension de la question essentielle de la mise en action de notre génome. Il est patent que les erreurs de régulations conduisent souvent à des maladies génétiques sévères et comprendre ces mécanismes est donc une priorité de la recherche actuelle en sciences de la vie.

Direct link to Lay Summary Last update: 29.03.2021

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
176395 Collinear regulation of HoxD genes during development and evolution 01.11.2017 Project funding (Div. I-III)

Abstract

Background and rationale: The Hox genes family encode series of transcription factors with a key function in organizing various aspects of the developing bodies of all animals displaying a bilateral symmetry, including vertebrates and hence mammals. Ever since their discovery in the mid 1980’s, both their function and their transcriptional regulation have been heavily studied in many different systems, and critical concepts have derived from these studies, which has made this gene family a spectacular epistemic system. However, due to their intrinsic properties, two of the most essential questions have remained unanswered. The first one has to do with the mechanism underlying the time sequence of Hox genes transcriptional activation following their physical order along the different clusters. Thus far, the understanding of this fundamental mechanism has resisted many years of experimental approaches. The second question is that of the basic function of this gene family when taken as a whole rather than as a collection of separate elements. Indeed, while we know quite well the function of each Hox gene individually, we do not know what would be the effect on embryo development of removing either large parts of- or all of this gene family. The reasons for this failure are numerous but mostly based on the experimental difficulties to study a complex mechanism acting right at the worst development stage to work with the mouse embryo. The accessibility of the material, the small number of cells concerned, the heterogeneity in the ages of littermates as well as all kind of experimental hurdles have thus hampered the solutions to these questions. Recently, various systems were developed which allow for pseudo-embryos to be produce in vitro starting exclusively from ES cells, which can thus be easily modifiable through CRISPR-Cas9 approaches. We thus believe that these two major questions can now be answered by using this alternative system, within the 48 months covered by this grant application.Overall objectives: The two overarching aims of this application are 1) to decipher the mechanism implementing the time-sequenced transcriptional activation of Hox genes along their respective clusters (the ‘Hox clock’) and 2) to reveal the deep functional importance of HOX proteins during the development of vertebrates. We believe these are the two main fundamental questions left to be answered concerning the Hox gene family and we trust that a drastic switch in our experimental model and tools may help us to eventually achieve this task.Specific aims: For overarching aim 1, we shall first calibrate gastruloids (pseudo-embryos extending a ‘body axis’, see below) to precisely describe the Hox clock in these biological objects (a clock, which we know is perfectly implemented). Once this will be done, we will start a series of mutations in ES cells, either affecting the genomic structure of Hox clusters or modifying (adding, deleting) CTCF binding sites and produce mutant gastruloids, which will be analyzed using a pipeline of readouts (RNA-seq, ATAC-seq and ChIP-seq or Cut&Run of PolII, Rad21, CTCF, Nipbl as well as a variety of histone H3 tail modifications). In this experimental configuration, even minor changes in timing will be scored. For overarching aim 2, we shall produce series of ES cell lines carrying massive variations in their number of Hox gene and clusters. Two specific aims will be targeted, the first being the functional removal of entire paralogy groups (i.e. all Hox gene derived from ancestral duplication events), in particular all Hox1 and all Hox13 genes. The second will aim at removing the entire Hox gene family (i.e. 4 ca. 100Kb large deletions on different chromosomes) to assess whether Hox function is only required to identify body parts (as in invertebrates) or if is it also involved in the production thereof, as is the case during vertebrate limb development. The experimental readout will consist of single cells RNA-seq, which should give us a molecular readout of the perturbations, as gastruloids do not develop full embryonic structures.Expected results: From the first set of experiments, we expect to see whether or not the various mutations impact upon the timing of transcriptional activation in gastruloids. If yes, we will refine experiments through an iterative prediction-validation process and eventually build a mechanistic model for temporal collinearity. From the second set of experiments, we expect to learn whether the deep function of Hox genes in vertebrates is similar to that of invertebrates or alternatively, whether vertebrates have evolved additional functional features related to this gene familyImpact for the field: The impact of this research will be mostly visible in fundamental research, at the boundary between developmental genetics and the study of global gene regulation in the context of 3D chromatin structure. This is an emerging interface where studies on the Hox gene family can bring important information and concepts, as in the recent past.
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