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, 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 the extracellular environment 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.
HNF1 alpha and HNF1 beta : development and diseases. Head of the group : Marco Pontoglio. Others members of the group : Claire Chéret, Antonia Doyen, Lionel Gresh
Hepatocyte Nuclear Factors 1 a and b (HNF1a and HNF1b) 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 HNF1b die in utero at embryonic day 7.5 because of a defect in visceral endoderm differentiation. In contrast, the conditional inactivation of HNF1b in the liver leads to defective intrahepatic bile duct differentiation and cholestasis. Inactivation of HNF1a 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. We have shown that reactivation of the phenylalanine hydroxylage gene, that is silenced in HNF1a-/- mice, by viral mediated transfer of HNF1a can be achieved within a defined time window during development. Autosomal dominant mutations in the genes encoding HNF1a and HNF1b have been shown to give rise to a particular form of diabetes called Maturity Onset Diabetes of the Young (MODY3 and MODY5, respectively). Some MODY3 missense mutations in the transactivation domain of HNF1a generate a dominant negative effector. This is mediated by an aberrant interaction of the mutant protein with CBP whose acetyltransferase activity becomes severely compromised. Our aim is to understand the way HNF1a and HNF1b control the expression of their target genes and how they fashion the correct cellular differentiation (organogenesis) program.
Chromatin remodelling and cell growth control. Head of the group : Christian Muchardt. Other members of the group: Brigitte Bourachot, 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 machineries 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. At the present time, only few of these promoters have been identified. However, 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 identifying new SWI/SNF target genes and molecular partners to achieve better understanding of the role of the complex in the control of cell growth.
Control of papillomavirus HPV18 transcription and replication : role of carcinogenesis. Head of the group : Françoise Thierry. Other members of the group : Sophie Bellanger, Isabelle Bouallaga, Caroline Demeret
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 structural element called an "enhanceosome". This enhanceosome is specific for cervical carcinoma cells and interacts exclusively with cellular proteins. We are currently searching for the proteins involved in its activity within the cellular genome 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 antiproliferative effect due to both cell cycle arrest and cell death by apoptosis. We are currently trying to discover the interactions of E2 with cellular components and the mechanism by which it induces apoptosis.
Assymetric cell division. Head of the group : Benoît Arcangioli. Other members of the group : Atanas Kaykov, Raynald de Lahondès (this group became Unité Postulante in 2001 and joined the Department of Molecular Biology)
We are investigating the chromosomal imprinting mechanism responsible for the asymmetric division in the yeast Schizosaccharomyces pombe. The efficiency of this process leads to a clonal haploid cell population containing the same proportion of both mating-types (P/M). Our previous studies has shown that this process is initiated by a single-strand DNA lesion at the mating-type locus, and involves DNA replication. The lesion is introduced during lagging strand synthesis, on only one of the two sister chromatids, is maintained during the entire length of the cell cycle and triggers mating-type switching during the following S-phase. The mechanism responsible for the formation and maintenance of this single-strand DNA lesion is unknown. Recently, we have shown, by density labeling, that the mating-type locus is not replicated by the semi-conservative mode, but instead both DNA strands are synthesized de novo. This finding establishes a link between the phenotype and genotype in the process of asymmetric mating-type switching. We are also studying the function of the sap1 gene, initially implicated in mating-type switching. Recently, we have shown that this gene plays an important role in the chromosomal cohesion/condensation processes which is essential for post-replicative recombination and segregation of the genetic material.
Figure 1 : HNF1beta mutation affects intrahepatic bile duct development. Immunostaining for cytokeratin (CK) on 10 days postnatal liver sections. (A) Differentiated bile ducts are seen on control mice. (B-D) Mutants show no or very few CK-positive cells (B), or ductal plate remnants and abnormal CK-positive structures within the portal mesenchyme (C), or abnormal bile ducts (D). hp: hepatic parenchyma; pv: portal vein; pm: portal mesenchyme; dp: ductal plate; bd: bile ducts. Scale bars: 100 mm.
Figure 2 : Blastocyst outgrowth and apoptosis studies. (A) Day 3.5 p.c. embryos were isolated from SNF5+/lacZ intercrosses and cultured in 96-well plates for 4 days. The phase contrast vew shows a growing inner cell mass (ICM) node and a single layer of trophoblastic giant cells (TGC) in wild-type and heterozygous blastocysts after 1,2 and 4 days in cluture, while homozygous mutants are impaired in both trophectoderm and ICM outgrowth. After 4 days of culture, TUNEL assay was performed. Fluorescein (TUNEL) and 4'-6-diamidino-2-phenylindole (DAPI) fluorescent images are shown.
Figure 3 :Real-time microscopy of HeLa cells transfected with a GFP-E2 fusion protein. Times after transfection are writed at the top and arrows indicate apoptotic cells.