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Identification Of The Mechanism Of Action Of Retinoic Acid In Germ Cell Differentiation

Reference : PhD Norbert GHYSELINCK

Offer publication : April 5, 2016

Retinoic acid (RA), the active metabolite of vitamin A, is indispensable for embryonic development (Mark et al. 2006), and after birth for maintenance of differentiation of various epithelia, including the seminiferous epithelium of the testis (Gely-Pernot et al. 2012). RA is used to treat cancers, notably because of its ability to inhibit tumor progression by triggering differentiation of stem cells carrying oncogenic mutations. Understanding the RA signaling pathways is therefore highly important. At the molecular level, RA acts through binding to nuclear receptors called
RAR, which function as ligand-dependent transcriptional factors and act, in general, heterodimerized with another nuclear receptor called RXR. However, we have demonstrated that, in the mouse, genetic ablation of all RXR in Sertoli cells (SC, the supporting cell of the seminiferous epithelium), does not reproduce the hallmarks of RAR ablation in SC, such as abnormal germ cell differentiation (Vernet et al. 2006). This finding demonstrates that RAR can act in vivo by non-canonical mechanisms, i.e., independently of RXR. Accordingly, the SC represents an excellent model to characterize
these new pathways through which RA control cell differentiation.

The thesis project is aimed at identifying, at the molecular level, the mechanisms that are operating in SC. The first aspect of the thesis work will be to test the possibility that RAR acts through repressing transcription because many genes appear
overexpressed in the testis of mutants bearing a RAR-null mutation in SC (unpublished data). Structural analyses of RAR/RXR complexes have highlighted the fact that replacing Isoleucine (I) located at position 396 in RAR by glutamic acid (E) abrogates, in vitro, the transcriptional repression activity of RAR (Le Maire et al. 2010). This I396E mutation has been introduced in the mouse and the line is already available in the laboratory.

The thesis work will consist in analyzing, notably by means of histology, immunohistochemistry, in-situ hybridization and candidate gene expression analyses, the phenotype induced by I396E mutation the mouse. The second aspect of the work will consist in identifying all the factors interacting with RAR by analyzing RAR-containing protein complexes immunoprecipitated from SC. This part of the project will first require the generation of a SC line expressing a tagged RAR, which will permit sequential immunoprecipitations to high purity using appropriate combinations of affinity resins. The factors interacting with RAR will subsequently be identified by mass spectrometry, and their functional relevance tested first in cultured SC and then in
vivo in the mouse. As cell differentiation in the seminiferous epithelium tightly depends on RAR in SC, the experimental model that we are exploring and the related thesis project will allow identifying novel molecular mechanisms governing, in a broad sense, the ability of RA to promote cell differentiation.

- EXPERTISES WHICH WILL BE ACQUIRED DURING THE TRAINING : Techniques in molecular and cellular biology, and analysis of gene expression (organ culture, cell culture and transfection, shRNA, purification of cells by FACS, histology, immunohistochemistry, in situ hybridization, DNA, RNA and protein extraction, quantitative real-time PCR, immunoprecipitation, mass spectrometry).

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Application Deadline : Dec. 31, 2016

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