|Director : Moshe YANIV (firstname.lastname@example.org)|
Our research unit studies the interplay between transcription factors and chromatin remodeling in activating and repressing genes in mammalian cells. Our research examines the importance of several families of transcription factors in controling cell differentiation and organogenesis, cell growth or apoptosis. We use complete or conditional gene inactivation in mice , micro-array analysis and bioinformatics. Our work has implications for understanding viral and non-viral malignant cell transformation and for deciphering the mechanisms underlying diseases such as Type II diabetes, kidney or liver disorders.
Proto-oncogenes Jun and Fos : a family of transcription factors
Head of the group : Fatima Mechta-Grigoriou and Jonathan Weitzman. Others members of the group : Maya Ameyar-Zazoua, Damien Gérald, Stéphane Girardin, Chaouki Miled, Marta Wisniewska.
The AP1 transcription factor is central to the cell's ability to integrate multiple extracellular signals and initiate the appropriate genetic response. AP1 is composed of dimers of Jun (c-Jun, JunB or JunD) and Fos (c-Fos, FosB, Fra-1 and Fra-2) proto-oncoproteins. Our laboratory focuses on unravelling the functions of the different Jun proteins using genetic and biochemical approaches. We have defined roles for the cytoskeleton and for an intracellular receptor for LPS in the initiation of a kinase signalling pathway that leads to Jun activation following cellular stress or bacterial infection. We have shown that the balance between different AP1 dimers controls progression through the cell cycle. The different Jun proteins also determine cellular transformation by the RAS oncogene. We have demonstrated that JunD can protect cells from p53-dependent senescence and apoptosis induced by inflammatory cytokines or genotoxic stress. These observations place AP1 at a key position linking the Ras/pRb and p53 regulatory pathways. By crossing mice with mutations in the c-jun and junD genes we have found evidence for genetic redundancy and cooperation between these proto-oncogenes. Mice lacking both jun genes display cardiovascular and angiogenic defects during embryonic development. We are currently investigating the gene programs that underlie these cellular and developmental phenotypes.
HNF1alpha and HNF1beta : development and diseases
Head of the group : Marco Pontoglio .Others members of the group : Claire Chéret, Antonia Doyen, Lionel Gresh, Andreas Reimann
Hepatocyte Nuclear Factors 1alpha and beta (HNF1alpha and HNF1beta) are two homologous atypical dimeric homeoproteins that appeared during evolution with the first vertebrates. They are expressed in polarized epithelia of different organs including liver, kidney, pancreas and intestine, where they control the expression of numerous tissue-specific genes. Mice lacking HNF1beta die in utero at embryonic day 7.5 because of a defect in visceral endoderm differentiation. In contrast, the conditional inactivation of HNF1beta in the liver leads to defective intrahepatic bile duct differentiation and cholestasis. Inactivation of HNF1alpha leads to postnatal dysfunctions of liver, kidney and pancreas. These mice suffer from hypercholesterolemia, hyperphenylalaninemia, renal Fanconi syndrome and a drastic defect in insulin secretion. Mutations in both, HNF1alpha and beta are associated with familial type II diabetes in humans (MODY3 and MODY5). We have also shown that the hepatic expression of several complement components is severely affected (C5 and C8 alpha). This leads to a defective membrane attack complex assembly which is normally induced by complement activation. Whereas the inactivation of HNF1alpha per se does not involve any developmental defect, mice that are homozygous mutant for HNF1alpha and heterozygous for HNF1beta die in utero. The mechanismes for this embryonic lethality are currently being investigated. Our aim is to try to understand the mechanisms by which HNF1alpha and HNF1beta control cellular differentiation and organogenesis. To reach this goal, we are using microarray and in silico approaches to caraterize the HNF1 transcriptome.
Chromatin remodelling and cell growth control
Head of the group : Christian Muchardt. Other members of the group: Brigitte Bourachot, Marie Guillemé, Peggy Rematier
In eukaryotes, the genomic DNA associates with histones to form chromatin. This environment renders the DNA partially inaccessible to other proteins. To overcome the chromatin barrier, the cell contains large multi-subunit machines that use the energy of ATP hydrolysis to locally modify the histone-DNA interactions. One of these machineries is known as the SWI/SNF complex. This complex is specifically involved in transcription and facilitates the recruitment of transcriptional regulators to a limited number of promoters. We have shown that this complex is essential for early development of the mouse. In addition, several lines of evidence suggest that the SWI/SNF complex is generally associated with the control of cell growth. We have shown that overexpression of Brm, the catalytic subunit of the complex, slows down the growth of cancer cells. Conversely, inactivation of the Brm gene in mice causes increased cell proliferation. Another subunit of the complex, known as SNF5/INI1, is encoded by a "tumor suppressor" gene inactivated in rhabdoid tumors, a very aggressive form of cancer of young children. We have shown that inactivation of one copy of this gene in mice leads to the formation of tumors presenting many similarities with the human rhabdoid tumors. These mice will facilitate the study of this form of cancer. Currently, we are characterizing the role of the SWI/SNF complex in cells in quiescence (G0) and during re-entry into the cell cycle (G0/G1 transition).
Control of papillomavirus HPV18 transcription and replication : role of carcinogenesis
Head of the group : Françoise Thierry. Other members of the group : Sophie Bellanger, Stéphanie Blachon, Caroline Demeret, Sébastien Teissier.
Our research focuses on the mechanisms of malignant conversion of cervical cells infected with the Human Papillomavirus type 18 (HPV18). Cervical cancer is the second cause of mortality in women from cancer, after breast cancer. The HPV virus infects specifically the genital tract where it replicates in the upper layers of the epidermis. The virus encodes two oncogenic proteins E6 and E7, which alter the normal proliferation control of the cells by interfering with the two regulatory proteins of the cell cycle, p53 and pRB. During carcinogenic progression, the viral genome integrates into the cellular genome. Transcription of the two viral oncogenes, E6 and E7, is strongly activated by a complex transcriptional control element called an "enhanceosome". This enhanceosome is specific for cervical carcinoma cells and is composed exclusively of cellular proteins. We are currently searching for the proteins involved in its activity and for the basis of its cell specificity by chromatin immunoprecipitation. In contrast, transcription of the viral oncogenes is repressed by the viral protein E2, whose coding sequences are inactivated by integration of the viral DNA into the cellular genome in cervical carcinomas. Reintroduction of the viral E2 protein into these cells induces a strong anti-proliferative effect due to both cell cycle arrest and cell death by apoptosis. We are currently studying the mechanism by which E2 induces apoptosis and its interactions with cellular components.
Photo 1: Expression of c-jun during early mouse embryonic development.
c-jun expression profile was analysed using whole-mount in situ hybridisation. Dorsal (A-C) or lateral (D) views are presented to show c-jun labelling. c-jun expression is first detected in the posterior hindbrain (A, B), but expands rapidly to other sites including the cephalic mesoderm (B), the dorsal part of the neural tube (C), the heart and the foregut (D). c-jun expression is also detected in the intersomitic blood vessels and the developing somites, the strongest expression being in the most recently formed somites (D). To define precisely this expression profile, transverse sections along the antero-posterior (AP) axis were performed on a 12 somite stage embryo. The AP axis level of the sections is indicated by double arrows and the corresponding letters in D. c-jun expression is restricted to the medial part of the neural tube in the hindbrain (a). c-jun is also specifically expressed in the endothelial cells of the heart (b). The staining lining the posterior half of the foregut, immediately behind the heart, corresponds to the septum transversum (c). c-jun is also expressed in the dorsal part of the spinal cord, in the region of neural tube closure (nt) (d). Finally, c-jun is expressed in the hindgut and in the presumptive sclerotomal derivative of the somitic mesoderm (e).
hb, hindbrain; cm, cephalic mesoderm; h, heart; f, foregut; isv, intersomitic vessels; hb, hindbrain; ec, endothelial cells; st, septum transversum; nt, neural tube; s, somite; scl, schlerotome; hg, hindgut. Scale bars: 60 mm (A-D), 100mm (a-e).
Photo 2: Complement synthesis defects in HNF1-deficient mice
Northern blot analysis of total hepatic RNA from wild-type, heterozygous and mutant homozygous animals for a null-mutation in HNF1alpha. Four animals for each genotype were analyzed. Parallel filters were hybridized with probes specific for the different cDNAs as indicated to the left. C6 mRNA expression along with an internal control for beta-actin were monitored with RT-PCR with 12 cycles and subsequently revealed by Southern blot analysis. Histograms to the right indicate the average expression levels for each genotype expressed in arbitrary units and normalized on the expression of the beta actin. The value of 100 was attributed to the expression level in the wild-type animals). Error bars represent Standard Error of the Mean and the significance of the differences between wild-type and heterozygotes vs. homozygotes is indicated with (*) for p<0.05 and (**) for p<0.01 in a t Student test (-/- vs. control animals) (Student t test).
A)Real-time microscopy of HeLa cells transfected with the HPV18 E2 protein fused to the GFP protein. Pictures were taken at the times after transfection indicated at the top of the figure. Appearance of fluorescence occurred in two sister cells after mitosis and accumulated until 23 hr post transfection, then disappeared, indicating instability of the protein. E2 induces apoptosis in a fraction of the transfected HeLa cells, indicated by white arrows.
B) Reappearance of the fluorescence in GFP-E2 transfected HeLa cells, upon treatment with inhibitors of the proteasome, Lactacystine and MG132, 40 hr post transfection, indicating that E2 is degraded by the ubiquitin-proteasome pathway (Bellanger et al. 2001).
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
OLLIVIER Edith, Institut Pasteur
DEMERET Caroline, Institut Pasteur, Assistant de Recherche (email@example.com)
LAVIGNE MARC, Institut Pasteur, Chargé de Recherche (firstname.lastname@example.org)
MECHTA-GRIGORIOU Fatima, Institut Pasteur, Chargé de Recherche (email@example.com)
THIERRY Françoise, Institut pasteur, Chef de laboratoire (firstname.lastname@example.org)
WEITZMAN Jonathan, Institut Pasteur, Chargé de Recherche (email@example.com)
AMEYAR-ZAZOUA Maya, Postdoc
BELLANGER Sophie, PhD student
BLACHON Stéphanie, PhD student
CHERET Claire, PhD student
GERALD Damien, PhD student
GIRARDIN Stéphane, PhD student
GRESH Lionel, PhD student
GUILLEME Marie, DEA
MILED Chaouki, Postdoc
REIMANN Andreas, PhD student
TEISSIER Sébastien, DEA
WISNIEWSKA Marta, Postdoc
BOURACHOT Brigitte, CNRS, Engineer (firstname.lastname@example.org)
DOYEN Antonia, Institut Pasteur, Technician
GARBAY Serge, CNRS, Engineer (email@example.com)