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Transcription In The Presomitic Mesoderm Of The Mouse Embryo: In Vitro And In Vivo Approaches

Reference : Thesis Subject

Publication de l'offre : 7 avril 2014

PROJECT : Downstream of the cascades that control gene expression in cells, RNA pol II transcription requires the formation of a multi protein complex called the pre-initiation complex (PIC) at the level of the promoters. One of the subunits ; TFIID, is composed of TBP (TATA binding protein) and 13 TAF (TBP associated factors). TFIID composition is not fixed and some of the TAFs are actually tissue-specific, suggesting that the basal transcriptional machinery can be regulated. In particular, Taf10 is necessary for transcription in the embryo but not in the adult. During development, the embryo elongates via the repeated addition of bilateral structures ; the somites, from the presomitic mesoderm (PSM). These structures will contribute later to the trunk and limb muscles and axial skeleton. Somite formation (65 pairs in mouse) is under the control of the clock and wave front. The determination front is localized at the intersection of a FGF/Wnt gradient that maintains the progenitor state of the cells and a counter-gradient of retinoic acid that promotes differentiation. The clock, characterized by the cyclic expression of genes, determines the ability of a group of cells that have reached the determination front to respond to signals of differentiation. The PSM is an excellent paradigm to study transcription in vivo. Indeed, not only these cells are the place of transcription downstream of signaling pathways, but also, in the posterior PSM, these cells undergo cyclic waves of transcription that are absolutely required for the implementation of the developmental program of the somites. Surprisingly, in the absence of TAF10 protein in the PSM, transcription is not immediateley blocked, the expressio of some genes being even up-regulated (Vincent SD, et al., in preparation). The goal of the PhD proposal is to couple developmental biology and biochemistry to study transcription during embryogenesis. The candidate will use models that have already been established in the lab, in collaboration with the team of Laszlo Tora (IGBMC) that has expertise in the analysis of the transcriptional complexes under the co-supervision of S. Vincent.

-characterization of the embryonic transcriptional machinery, in normal conditions and in the absence of Taf10 : conditional animal models are already available in the lab (Taf10 deletion in the PSM : TCre or in the whole embryo : RosaCreERT2). In order to simplify the biochemical approach, the candidate will use a ES cell differentiation protocol in PSM-like cells (Chal J. et al., submitted). RosaCreERT2/+ ;Taf10flox/flox ES cell lines have already been established.TFIID complex will be immunopurified from WT embryos or differentiated ES cells in order to compare its composition with the published data from cell lines. Then, in order to understand how transcription is maintained in the absence of TAF10, TFIID will be immunopurified from mutant embryos and differentiated ES cells.

-Characterization of the transcriptional landscape during somitogenesis : if the the signaling pathways have been well characterized, the transcription process is not well documented during somitogenesis. The RNA pol II occupancy and the chromatin state profiles will be established from differentiated ES cells and later on, from microdissected PSM, focusing in particular on the cyclic genes.

- WISHED SKILLS : - knowledges in molecular biology and biochemsitry

- knowledges in developmental biology

- cell culture skills

- critical thinking, ability to synthesize

- curiosity

an experience using ChIP would be a plus.


-ES cell culture (derivation, maintenance and differentiation)

-embryo microdissection

-MudPit proteomic analysis

-genomic analysis


-conduction of a research project


- oral and written (publications redaction) presentations

Votre candidature

Imprimer Envoyer

Université de Strasbourg

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