Investigation of transcriptional partners and genes downstream of Foxn1

The thymus is the second fundamental organ of the immune system after the bone marrow; it is the essential for T cell maturation and repertoire selection. The function of the thymus critically depends on the thymic epithelium, which is structured in two distinct regions: The cortex and the medulla. The knowledge about molecular mechanisms involved in thymus development is limited. However, some evidences suggest that the Hox, Pax, Six, Eya, Gcm2, FGFs and Foxn1 are key players in thymus organogenesis. Foxn1 is essential for thymic epithelial cell development but its exact role is still unknown. The null mutation in the transcription factor Foxn1 generates the nude phenotype of athymia and hairlessness. The aim of our laboratory is to better understand thymus development and specially focuses on the Foxn1 function. The aim of this project is then to investigate target genes and transcription partners of Foxn1 as a transcriptional factor. As a transcription factor, Foxn1 protein should be able to bind with DNA but should also interact with other proteins such as cofactors in order to activate its target genes. At present, the transcriptional partners of Foxn1 are completely unknown in the literature and the proteinprotein interaction databases. The experimental approach proposed here is the standard method for identification of unknown transcriptional partners: Immunoprecipitation (IP) followed by a mass spectrometry analysis. The list of protein candidates can then be validated by Western blotting. We identified the right antibodies to use for this procedure and optimised protocols for the IP. We also performed a mass spectrometry analysis that was not conclusive, however, the platform is now established to complete this analysis. Only little is known about genes up- or down-stream Foxn1. Some genes such as FgfR2IIIb, DLL4 and CCL25 are believed to be up-regulated by Foxn1. Our laboratory has generated a revertible hypomorphic allele of Foxn1 and have used this to obtain a series of six allelic combinations at Foxn1. These mice, expressing different levels of Foxn1, are a very useful tool in order to investigate target genes of Foxn1. In particular, we wish to investigate whether different Foxn1 targets are activated at different Foxn1 levels. 6 To reach this objective, the proposed approach is the comparison of gene expression profiles of thymic epithelial and mesenchymal cells of the allelic series at two different time points E13.5 and E15.5 using a Solexa tool called SAGE (Serial Analysis of Gene Expression). As preliminary analysis before the global gene expression analysis, we have determined expression profiles of several genes across the allelic series by quantitative PCR. Our findings suggest that DLL4, CCL25, FgfR2IIIb, FGF10 and possible Pax1 are direct or indirect targets of Foxn1 activated at different levels of Foxn1. DLL4 and CCL25 seem to be involved in lymphocyte attraction and maturation. FgfR2IIIb appears to be important for thymic epithelium differentiation and development by increasing the sensitivity of epithelial cells to FGF10 signalling. Finally, Pax1 is possibly required downstream of Foxn1 for terminal differentiation of cortical thymic epithelial cells