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

The contribution of the host stress response to the pathogenesis of Pseudomonas entomophila in the Drosophila intestine

Sveta Chakrabarti

Recently, several studies done in Drosophila have revealed that efficient and rapid recovery from bacterial infection in the gut is possible only when bacterial clearance by the immune system is coordinated with repair through renewal of the epithelium damaged by infection. In this thesis, I have analyzed how the entomopathogenic bacterium Pseudomonas entomophila affects the immune response and epithelium renewal. A microarray analysis first showed that P. entomophila is recognized by the Drosophila immune system since ingestion of this bacterium stimulated the expression of antimicrobial peptide genes by the Imd pathway. In addition, stress and damage related pathways were strongly induced by P. entomophila, which correlate with its capacity to inflict severe damage. While antibacterial genes were induced in the gut following infection with P. entomophila, the immune response was not productive due to a general inhibition of translation that affected all the newly synthesized transcripts. Furthermore, the blockage of translation also inhibited the repair program by blocking the production of JAK-STAT ligands (upd3, upd2) and growth factors, which stimulate the intestinal stem cells proliferation and differentiation. I next analyzed the pathways that link cellular damage to reduction of translation. Using a genetic approach, I showed that inhibition of translation was induced by two signaling pathways: i) the phosphorylation of elongation initiation factor 2α (eIF2α) by the stress kinase GCN2 and ii) the inhibition of the TOR pathway by the AMPK kinase. Both kinases sense metabolic deprivation, suggesting that cellular damages induced by P. entomophila induce a state of “starvation”. Inhibition of translation is usually an adaptive cellular response to adjust the metabolism to the energy status of the cells. The observation that GCN2-deficient flies survived better than wild-type flies to P. entomophila indicates that pathogenesis is linked to an over-activation of stress pathways that usually help to endure the consequence of an infection. In this in vivo model of infection, I also showed that the reduction of translation was a consequence of cellular damage to the intestine caused by host-derived reactive oxygen species (through the activity of Duox) and by the direct action of a pore-forming toxin produced by the pathogen. As a consequence of this translational arrest, flies succumbed P. entomophila infection because they were unable to repair gut damage. Finally, I showed that inhibition of translation also had a strong influence on innate immune responses observed upon P. entomophila infection. The specific activation of a systemic immune response (antimicrobial peptides produced by the fat body) observed upon P. entomophila infection could be recapitulated by feeding flies with a non-lethal pathogen and an inhibitor of translation. Hence, I showed translation inhibition could be an important feature that shapes the immune response. The p38 MAPK family is involved in stress and immunity in both mammals and Drosophila. In Drosophila, three p38-MAPK-encoding genes, p38a, p38b and p38c, have been identified but their function in the gut immune response is poorly characterized. In the second part of my thesis, I have analyzed the role of these three p38 MAPKs in Drosophila intestinal immunity, especially p38c, which is strongly enriched in the gut. I first confirmed that the three p38 are involved kinases are involved in the defense to oral infection with pathogenic (but non-lethal) bacteria such as Erwinia carotovora 15. Surprisingly, p38c mutant flies were more resistant to P. entomophila and did not show the reduction in translation observed in wild-type flies. Interestingly, the transcription of the ROS producing enzyme Duox was reduced in p38c raising the hypothesis that p38 contributes to P. entomophila pathogenicity by activating Duox. Furthermore, I have obtained preliminary data indicating that p38c and the transcription factor ATF-3 act in the same pathway contributing the host immune response and lipid metabolism. Together, my thesis reveals the complex cross-talks between stress and immune pathways during microbial infection. While, it shows that stress pathways usually contribute to endure the damage caused by infection, excessive activation of these pathways can contribute to pathogenesis.

EPFL2013

Hematopoietic Stem Cell Function and Survival Depend on c-Myc and N-Myc Activity

Armin Ehninger, Anne Wilson

Myc activity is emerging as a key element in acquisition and maintenance of stem cell properties. We have previously shown that c-Myc deficiency results in accumulation of defective hematopoietic stem cells (HSCs) due to niche-dependent differentiation defects. Here we report that immature HSCs coexpress c-myc and N-myc mRNA at similar levels. Although conditional deletion of N-myc in the bone marrow does not affect hematopoiesis, combined deficiency of c-Myc and N-Myc (dKO) results in pancytopenia and rapid lethality. Interestingly, proliferation of HSCs depends on both myc genes during homeostasis, but is c-Myc/N-Myc independent during bone marrow repair after injury. Strikingly, while most dKO hematopoietic cells undergo apoptosis, only self-renewing HSCs accumulate the cytotoxic molecule GranzymeB, normally employed by the innate immune system, thereby revealing an unexpected mechanism of stem cell apoptosis. Collectively, Myc activity (c-Myc and N-Myc) controls crucial aspects of HSC function including proliferation, differentiation, and survival.

2008

The role and transcriptional regulation of FAM46A in adipocyte differentiation

Ann-Kristin Hov

Obesity is currently a leading global health concern. Treatments include undergoing a weight-loss regimen. However, successful weight-loss is variable, where the heritability of obesity is considered part of the cause. This patient variability to weight-loss was addressed by Carayol et al. (2017), where protein plasma level changes was associated to genetic variability and changes in BMI under a low-calorie diet intervention. A highly significant association region was found to regulate downstream gene FAM46A; coding for a nucleotidyl-transferase protein. Loss of function mutations in FAM46A have previously been discovered in multiple human diseases, such as osteogenesis imperfecta. However, there is a lack of understanding of FAM46Aâs function, especially in the context of adipose tissue and metabolism. This thesis aimed to follow up on the Carayol et al. study by investigating the transcriptional regulation of FAM46A and how the associated region is influencing this regulation, as well as how FAM46A is involved in adipose tissue function. When mapping the physical DNA interaction network around the associated region using chromosome conformation capture (3C) in human subcutaneous adipocytes (SGBS cells), no interactions between the FAM46A promoter and selected loci of the associated region were found. Interestingly, significant interaction peaks downstream of the promoter overlapped with regions of open chromatin and were enriched for transcription factor (TF) motifs that are involved in stem cell differentiation and TGFÎ² signalling. SMAD4, part of the TGFÎ² signalling pathway, binds to this region, indicating a potential regulator of FAM46A. To address FAM46Aâs potential role in adipocyte function, FAM46A expression was disrupted in vitro in differentiating adipocytes. Reduction of FAM46A expression in SGBS cells resulted in decreased expression of PPARG and CEBPA, the master regulators of adipogenesis, complemented by a reduction in lipid accumulation. Follow up studies in murine mesenchymal stem cells where Fam46a was over-expressed were controversial as this led to a decrease in adipogenic markers (Adipoq and PPARðŸ). Overall, disruption of FAM46A expression led to opposing changes in adipogenic potential, suggesting the requirement for a more stable cell system to study FAM46A in vitro. To further characterise the role of Fam46a, metabolic phenotyping of a Fam46a knockout (KO) mouse model under hyper-caloric stress was performed in collaboration with the IMG in Prague. This model revealed a significantly smaller mouse, and increased plasma alkaline phosphatase, as previously observed. When fed a high-fat diet, KO mice had reduced overall fat mass, improved glucose clearance and reduced adipocyte size in both subcutaneous and visceral white adipose tissue (subWAT/visWAT) depots. Transcriptomic analysis of these two depots revealed an up-regulation of genes expressed in ribosome related pathways in the subWAT, whereas the visWAT showed the up-regulation of fatty acid metabolism pathways, suggesting that the KO adipose tissue is more metabolically active. Other down-regulated pathways involved stem cell differentiation and BMP/TGFÎ²-signalling. In conclusion, this study found the possible regulation of the FAM46A promoter by TFs involved in adipogenesis regulation, such as SMAD proteins, as well as strengthening its possible functional involvement in TGFÎ² signalling by enrichment of this pathway in KO studies.