TIF1gamma, nouveau régulateur négatif de la voie de signalisation du TGFbeta

The cytokine TGFbeta regulates several cellular processes such as proliferation, differentiation, migration and apoptosis, from embryonic development to adulthood. TGFbeta is well described for its tumor suppressor role through antiproliferative and proapoptotic activities, in particular in epithelial cells. During tumor progression however, TGFbeta becomes a tumor promotor, favoring angiogenesis, immune suppression and inducing the epitheliomesenchymal transition. Binding of TGFbeta ligand to its receptors activate cytoplasmic messenger Smad2 and Smad3 to complex with Smad4 and shuttle into the nucleus to regulate TGFbetatarget genes expression. Recently, TIF1gamma has been described as a new negative regulator of TGFbeta signaling, through monoubiquitination of Smad4 or direct competition with Smad4 to bind activated Smad2/3. This signaling pathway has to be finely tuned to target an action dependent on a cellular context, which is why we analyze here the regulation of functional interactions between the TGFbeta canonical signaling and TIF1gamma. In this study, we show that TIF1gamma acts as a negative regulator of Smad4 functions in TGFbetasignaling during the epithelio-mesenchymal transition and during terminal differentiation of mammary epithelial cells and lactation. We are also interested in studying TIF1gamma SUMOylation as additional level of regulation of cell response to TGFbeta. Thus we characterized four functional SUMOylation sites in TIF1gamma and we found that this post-translational modification inhibits the formation of Smads transcriptional complex and is needed to temporally restrict Smad4 residence on the promoter of TGFbetatarget genes. Our results show the critical role of TIF1gamma SUMOylation in the regulation of TGFbeta- induced epithelio-mesenchymal transition. As a conclusion, our study unveils the major role of TIF1gamma in the regulation of TGFbeta transcriptional responses. Moreover, we show that TIF1gamma requires SUMOylation to exert its repressive activity on TGFbetasignalingLe TGFbeta intervient dans la régulation de nombreux processus cellulaires comme la prolifération, la différenciation, la migration, l'apoptose, du développement embryonnaire jusqu'à la vie adulte. Le TGFbeta est aujourd'hui bien décrit pour son rôle de suppresseur de tumeur de par ses activités anti-prolifératives et pro-apoptotiques, en particulier sur les cellules épithéliales. Cependant, au cours de la progression tumorale, le TGFbeta devient un promoteur de tumeur en favorisant l'angiogenèse, l'échappement de la tumeur vis-à-vis du système immunitaire et en induisant la transition épithélio-mésenchymateuse. Après fixation du ligand TGFbeta , le complexe de récepteurs active les protéines cytoplasmiques Smad2 et Smad3 qui s'associent à Smad4 pour former le complexe transcriptionnel qui se transloque alors dans le noyau pour réguler la transcription de nombreux gènes cibles. Récemment, la protéine TIF1gamma a été décrite pour intervenir dans la régulation négative de la voie du TGFbeta , en monoubiquitinant Smad4 ou en interagissant avec Smad2/3 en compétition avec Smad4. Cette voie de signalisation devant être finement contrôlée pour cibler son action en fonction du contexte cellulaire, nous analysons ici la régulation des interactions fonctionnelles entre la voie canonique du TGFbeta et la protéine TIF1gamma. Dans cette étude, nous montrons que TIF1gamma agit comme un régulateur négatif des fonctions de Smad4 dans la voie de signalisation du TGFbeta au cours du processus de transition épithélio-mésenchymateuse et au cours de la différenciation terminale des cellules épithéliales mammaires et de la lactation. Nous étudions également la SUMOylation de TIF1gamma comme nouveau niveau de régulation de la réponse cellulaire au TGFbeta . Nous avons ainsi caractérisé les sites fonctionnels de SUMOylation de TIF1gamma et montré que cette modification post-traductionnelle inhibe la formation du complexe transcriptionnel Smad et est nécessaire pour réguler temporellement la résidence de Smad4 au niveau du promoteur de gènes cibles du TGFbeta . Nos résultats montrent le rôle important de la SUMOylation de TIF1gamma dans la régulation de la transition épithélio-mésenchymateuse induite par le TGFbeta . En conclusion, notre travail met en avant le rôle majeur de TIF1gamma dans la régulation de la réponse transcriptionnelle au TGFbeta . De plus, nous montrons que la SUMOylation de TIF1gamma est nécessaire à son activité répressive sur Smad

Molecular and functional analysis of the gene snoo in Drosophila melanogaster

The gene snoo forms part of the Ski/Sno protein family, which is characterised by a conserved SAND domain and a Dachshund homology domain. Snoo interacts with the Dpp/Activin sig-nalling pathways. However, there are serious doubts on some of the published experimental evidence of snoo mutants. By combining the CRISPR/Cas9 system with homology directed repair two mutant lines with precisely defined deletions in the gene snoo and two Gal4 driver lines for this gene were gen-erated. Using these driver lines, it could be revealed that snoo has a relatively broad expression pattern. During almost all stages of development, snoo is expressed in many different tissues and organs, including the central nervous system. Furthermore, it was revealed that a knockout of snoo reduces lifespan and negative geotaxis and that the deletion impairs locomotion in general. By specifically inducing a CRISPR/Cas9 dependent conditional knockout in different cell types using the Gal4/UAS system, it could be shown that these phenotypes are most likely caused by the lack of Snoo in neurons. Finally, the transcriptomic analysis in the brains of the generated deletion lines by RNAseq and qPCR led to the identification of a set of possible target genes or interaction partners of snoo. These include genes that are involved in metabolic homeostasis, circadian rhythm or the resistance to xenobiotics

Regulation and function of AGR2 and p53 pathways

Inactivation of p53 by mutation occurs in half of human tumours. The majority of these mutations affect the DNA-binding core domain and hence impair DNA binding and hinder transcription of p53 target genes. A wealth of data indicates that even cancers carrying wild type p53 protein, evolve mechanisms to render the p53 pathway inactive. Thus, inactivation of the p53 response, either by mutation or the alternative mechanisms, allows unpurturbed tumour growth. Recent work identified Anterior Gradient-2 (AGR2) as a protein overexpressed in wild type p53 expressing tumours and it was subsequently shown that AGR2 inhibits p53 pathway. In this study I confirmed that AGR2 protein inhibits p53 and AGR2 depletion potentiates p53-dependent DNA damage response. As there were no physiological signals known that regulate the AGR2-p53 axis, here I set out to identify pathways that activate or inhibit AGR2. I found that transforming growth factor β(TGF- β) triggers AGR2 protein reduction and this is concomitant with the stabilisation and increased activity of p53 protein. TGF-β halts AGR2 transcription in a SMAD4- dependent manner and triggers AGR2 protein degradation involving an ATM kinase. I found that SMAD nuclear interacting protein (SNIP1) mediated the ATMdependent AGR2 degradation. Interestingly, SNIP1 overexpression by itself promoted AGR2 protein degradation. I found that AGR2 protein degradation was proteasome independent and involed autophagy-lysosomal degradation pathway. As the mechanism of p53 inhibition by AGR2 is not known, I reasoned that identifying interactors of AGR2 may potentially further our understanding of the mechanism accounting for AGR2-mediated p53 inhibtion. I isolated the ATP binding protein Reptin in the yeast two-hybrid system and subsequently validated it as an AGR2 binding partner. Mutations of the two ATP binding motifs in Reptin resulted in altered oligomerization and thermostability of Reptin and affected the AGR2-Reptin complex stability. I also identified the Reptin docking site and it was mapped to a divergent octapeptide loop. I found that AGR2-Reptin complex coimmunoprecipitated with the p53 protein. Subsequently, I showed that Reptin protein can influence p53 activity, and depending on local concentration, either inhibit the transcription of p53-genes or chaperone its DNA binding activity. Interestingly, I found that Reptin formed a stable complex, independent of AGR2, with p53 R175H, p53 F270A, p53 S269D and p53 S269A, which has implication for the Reptin function in cancers bearing mutant form of p53 protein

Function of the Myc-binding protein Miz1 in the mouse mammary gland

The study of the expression and function of proteins important for human health in normal development provides valuable information for the design of therapeutical opportunities in the context of disease. Myc is one of the current most promising targets for a number of cancer types including triple-negative breast cancer and Miz1 has been shown to play an important role in Myc-mediated tumorigenesis. In the present work, the function of the Myc-binding protein Miz1 in the mammary gland is investigated for the first time using two different lines of transgenic mice expressing Cre-recombinase to conditionally knockout the POZ domain of Miz1 in the murine mammary gland. Deletion of this evolutionary-conserved region impedes multimerization and stable association of Miz1 with chromatin. MMTV-Cre mediated deletion was used to investigate Miz1 function in the virgin gland, considering branching morphogenesis and mammary stem/progenitor biology. Ablation under the Wap-Cre promoter provided information about alveologenesis and mammary differentiation. The mammary gland is a very suitable organ for stem cell and developmental studies as rounds of proliferation, differentiation and apoptosis occur after each pregnancy. POZ domain deletion using MMTV-Cre (Line A), already active in the embryo, led to a delayed ductal tree formation, less cellularity in knockout ducts and a Myc-independent accumulation of stem/progenitor cells in virgin mammary glands of Miz1DPOZ animals. No differences in the expression of luminal and myoepithelial markers were observed between control and Miz1DPOZ virgin mice. In addition, the delay in the development of the mammary ductal tree in knockout mice is rescued at around two months of age. Endogenous Miz1 expression in the mammary gland of control animals was found to be highly boosted during lactation by immunohistochemistry and Western blotting. Very low Miz1 levels were detected at the end of pregnancy, which increased after parturition and diminished upon cessation of pup suckling at around 48 hours of forced involution. Miz1 POZ domain ablation in luminal alveolar mammary cells during pregnancy using the WAP-Cre transgenic line resulted in a lactation defect in mutant dams during the first two pregnancies analysed. Mutant lactating glands display a reduced alveologenesis as a result of a diminished mammary cell proliferation and differentiation. These data were also confirmed in vitro using the HC11 murine mammary cell line after retroviral infection for stable knockdown of Miz1. HC11 cells with low levels of Miz1 show a reduced proliferation and a decreased expression of ß-casein after inducing differentiation by addition of a lactogenic hormone cocktail containing prolactin. Apoptosis is unaffected after either Miz1 POZ domain ablation in vivo or stable knockdown of Miz1 in vitro. Mutant glands display lower levels of activated Stat5 which lead to a reduced expression of its transcriptional targets, mainly genes which code for milk proteins like a-casein, b-casein or whey acidic protein (WAP). Gene expression of negative regulators of the Jak2/Stat5 pathway like Socs (Socs1, Socs2 and Socs3) or Caveolin-1 (Cav1) is not upregulated in Miz1DPOZ lactating glands. In contrast, the expression of receptors important for a proper phosphorylation of Stat5, like the prolactin receptor or ErbB4, is decreased in lactating mutant glands. ChIP-Seq experiments revealed that genes encoding the prolactin receptor and ErbB4 are not direct targets of Miz1. Rather, Miz1 binds to genes which regulate vesicular transport and thus alters processes like endocytosis and autophagy in mammary gland cells. A model in which the vesicular transport of these receptors in mutant glands could be disrupted is proposed. In conclusion, this work shows for the first time that Miz1 is important for mammary stem/progenitor cell regulation in the virgin gland and for a proper proliferation and differentiation in the lactating mammary gland

Examination of the expression of genes and proteins controlling M. longissimus thoracis et lumborum growth in steers

The first study conducted investigated the effect of sire breed and genetic merit for growth potential of the transcriptional regulation of the somatotropic axis followed by a proteomic approach to assess differentially abundant proteins. Following this, a second study was set up to examine the compensatory growth phenomena in cattle which aimed to investigate the effect of feed restriction and feed realimentation on animal production and physiological variables and the residual effects on meat quality attributes. The final chapter in this thesis focused on the transcriptional regulation of compensatory growth in M. longissimus thoracis et lumborum in crossbred Aberdeen Angus steers. It is evident from this thesis that genetic merit for growth potential in cattle is under molecular control and chapters 3 and 4 offer revealing insight into the somatotropic axis and glucose metabolism. RNAseq, a highly sensitive approach to transcriptome sequencing, was used to conduct the transcriptional sequencing analysis in chapter 7. During the differential feeding period, gene pathways relating to lipid metabolism were significantly different between the two treatments and consistent with plasma leptin concentrations and ultrasonically scanned fat depth data (chapter 5). During the realimentation period, when previously restricted steers were experiencing compensatory growth, the TGF-βR1 gene involved in the TGF-β signalling pathway, a negative regulator of growth, was down-regulated in expression. The results obtained from this study offer a novel insight into key regulatory genes and pathways controlling compensatory growth in skeletal muscle of cattle which following appropriate validation may be incorporated into genomically assisted selection strategies for beef cattle. Overall, this thesis has offered significant insight into key pathways regulating growth in cattle such as the somatotropic, glycolytic and TGF-β signalling pathways

Examination of the expression of genes and proteins controlling M. longissimus thoracis et lumborum growth in steers

The first study conducted investigated the effect of sire breed and genetic merit for growth potential of the transcriptional regulation of the somatotropic axis followed by a proteomic approach to assess differentially abundant proteins. Following this, a second study was set up to examine the compensatory growth phenomena in cattle which aimed to investigate the effect of feed restriction and feed realimentation on animal production and physiological variables and the residual effects on meat quality attributes. The final chapter in this thesis focused on the transcriptional regulation of compensatory growth in M. longissimus thoracis et lumborum in crossbred Aberdeen Angus steers. It is evident from this thesis that genetic merit for growth potential in cattle is under molecular control and chapters 3 and 4 offer revealing insight into the somatotropic axis and glucose metabolism. RNAseq, a highly sensitive approach to transcriptome sequencing, was used to conduct the transcriptional sequencing analysis in chapter 7. During the differential feeding period, gene pathways relating to lipid metabolism were significantly different between the two treatments and consistent with plasma leptin concentrations and ultrasonically scanned fat depth data (chapter 5). During the realimentation period, when previously restricted steers were experiencing compensatory growth, the TGF-βR1 gene involved in the TGF-β signalling pathway, a negative regulator of growth, was down-regulated in expression. The results obtained from this study offer a novel insight into key regulatory genes and pathways controlling compensatory growth in skeletal muscle of cattle which following appropriate validation may be incorporated into genomically assisted selection strategies for beef cattle. Overall, this thesis has offered significant insight into key pathways regulating growth in cattle such as the somatotropic, glycolytic and TGF-β signalling pathways