Project description:C/EBPβ plays a major role in numerous biological processes but unfortunately its precise role is not still clear and conflicting studies showed that this transcription factor could have contradictory functions. These latter arise from the complexity of mechanisms regulating C/EBPβ activity. Indeed, C/EBPβ encodes an intronless gene that generates a single mRNA that is alternatively translated into two major isoforms of 35kDa (liver-enriched activator protein: LAP) and 20kDa (liver-enriched inhibitory protein: LIP). LAP is the active isoform of this transcription factor whereas LIP, a truncated isoform negatively regulate C/EBPβ-LAP-mediated gene expression. The main goal of our research was to understand how LAP and LIP isoforms governe C/EBPβ cellular functions with the identification of new genes and pathways regulated by these isoforms in the human Hep3B hepatoma cell line. For this purpose an original in vitro system characterized by a target genes induction with the activatory C/EBPß isoform LAP and a target genes repression with the inhibitory C/EBPß isoform (LIP) was used to identify the genuine C/EBPβ molecular signature. Using a cDNA microarray which provides a complete coverage of the liver transcriptome, we identified 676 genes inversely regulated by LAP and LIP. These selected genes are involved in many biological processes as the hepatic metabolism (cholesterol), detoxification, induction of apoptosis and negative regulation of the cell proliferation. According to the involvement of C/EBPβ in hepatic carcinogenesis we focused on cell cycle regulation and apoptosis. Through functional studies, we proved for the first time that LIP plays in favor of the survival of the Hep3B cells whereas LAP makes the cells more sensitive to staurosporine-induced cell death. Moreover, a lot of studies demonstrated that the anti-proliferative action of C/EBPβ mainly depends on RB protein. By studying the rate of Hep3B cells proliferation which overexpress LAP, we brought to the fore that this isoform would be able to induce a repression of the Hep3B cells line proliferation - a RB- and p53- negative cell line. Thus, LAP seems able to induce the repression of proliferation by a different metabolic way from the RB one. Keywords: comparison of cells expressing LAP or LIP RNA were extracted from 5 Hep3BLAPexpressing LAP, 5 Hep3BLAP control without expression of LAP, 5 Hep3BLIP expressing LIP and 5 Hep3BLIP control without epression of LIP. Each sample was hybridized once in 5 different nylon membranes.

Project description:BACKGROUND & AIMS: C/EBPbeta is involved in numerous process as carcinogenesis but its role is still not clear due to the existence of an active form (LAP) and an inhibitory form (LIP) of this transcription factor. The main goals of the present research were (i) the identification of genes inversely regulated by LAP and LIP i-e the genuine C/EBPbeta molecular signature in the Hep3B human hepatoma cell line (ii) a better understanding of LAP and LIP respective role in hepatic cells survival and proliferation (iii) the search of the C/EBPbeta signature among hepatocellular carcinomas. METHODS: Using Tet-off expression system we engineered Hep3BLAP and Hep3BLIP cells, in which LAP and LIP were over-expressed respectively. Then, using both expression profiling (DNA arrays) and ChIP-on-chip analysis, we identified genes inversely and/or directly regulated by each of the C/EBPbeta isoforms. The expression levels of these genes regulated by LAP/LIP were compared in controls and HCCs patients. RESULTS: We identified 676 genes inversely regulated by LAP and LIP and among these, 45 are direct targets. Using functional studies, we displayed the opposite role of LAP and LIP in staurosporine-induced cell death and the implication of LAP in the repression of Hep3B cells proliferation. Finally we identified a subgroup of HCCs with a deregulation of 165 genes belonging to C/EBPbeta signature and coding for proteins involved in chemoresistance and metastasis formation. CONCLUSIONS: Our study increases knowledge on LAP and LIP functions and provides first evidence that their molecular signature in the HCCs could predict tumor evolution. Total genomic DNA were extracted from 3 Hep3BLAP expressing LAP and were labelled Cy3 fluorochrome. Genomic DNA were extracted from 3 Hep3BLAP expressing LAP, were immunoprecipited with anti-CEBPbeta antibody and were labelled with Cy5 fluorochrome. Each sample was hybridized on an Agilent two-color microarray G4489A (Human Promoter ChIP-on-Chip Set 244K).

Project description:C/EBPβ plays a major role in numerous biological processes but unfortunately its precise role is not still clear and conflicting studies showed that this transcription factor could have contradictory functions. These latter arise from the complexity of mechanisms regulating C/EBPβ activity. Indeed, C/EBPβ encodes an intronless gene that generates a single mRNA that is alternatively translated into two major isoforms of 35kDa (liver-enriched activator protein: LAP) and 20kDa (liver-enriched inhibitory protein: LIP). LAP is the active isoform of this transcription factor whereas LIP, a truncated isoform negatively regulate C/EBPβ-LAP-mediated gene expression. The main goal of our research was to understand how LAP and LIP isoforms governe C/EBPβ cellular functions with the identification of new genes and pathways regulated by these isoforms in the human Hep3B hepatoma cell line. For this purpose an original in vitro system characterized by a target genes induction with the activatory C/EBPß isoform LAP and a target genes repression with the inhibitory C/EBPß isoform (LIP) was used to identify the genuine C/EBPβ molecular signature. Using a cDNA microarray which provides a complete coverage of the liver transcriptome, we identified 676 genes inversely regulated by LAP and LIP. These selected genes are involved in many biological processes as the hepatic metabolism (cholesterol), detoxification, induction of apoptosis and negative regulation of the cell proliferation. According to the involvement of C/EBPβ in hepatic carcinogenesis we focused on cell cycle regulation and apoptosis. Through functional studies, we proved for the first time that LIP plays in favor of the survival of the Hep3B cells whereas LAP makes the cells more sensitive to staurosporine-induced cell death. Moreover, a lot of studies demonstrated that the anti-proliferative action of C/EBPβ mainly depends on RB protein. By studying the rate of Hep3B cells proliferation which overexpress LAP, we brought to the fore that this isoform would be able to induce a repression of the Hep3B cells line proliferation - a RB- and p53- negative cell line. Thus, LAP seems able to induce the repression of proliferation by a different metabolic way from the RB one. Keywords: comparison of cells expressing LAP or LIP

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Transcriptomes of Effector Memory T cells undergoing lymphopenia induced proliferation

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