Carla Cicchini1, Valeria de Nonno2, Cecilia Battistelli2, Angela Maria Cozzolino3, Marco De Santis Puzzonia2, Silvia Anna Ciafrè4, Chad Brocker5, Frank J Gonzalez5, Laura Amicone2, Marco Tripodi6. 1. Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Hematology, Section of Molecular Genetics, Sapienza University of Rome, Rome, Italy. Electronic address: cicchini@bce.uniroma1.it. 2. Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Hematology, Section of Molecular Genetics, Sapienza University of Rome, Rome, Italy. 3. National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy. 4. Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy. 5. Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. 6. Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Hematology, Section of Molecular Genetics, Sapienza University of Rome, Rome, Italy; National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy. Electronic address: tripodi@bce.uniroma1.it.
Abstract
BACKGROUND AND AIMS: Epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-to-epithelial transition (MET) are manifestations of cellular plasticity that imply a dynamic and profound gene expression reprogramming. While a major epigenetic code controlling the coordinated regulation of a whole transcriptional profile is guaranteed by DNA methylation, DNA methyltransferase (DNMT) activities in EMT/MET dynamics are still largely unexplored. Here, we investigated the molecular mechanisms directly linking HNF4α, the master effector of MET, to the regulation of both de novo of DNMT 3A and 3B. METHODS: Correlation among EMT/MET markers, microRNA29 and DNMT3s expression was evaluated by RT-qPCR, Western blotting and immunocytochemical analysis. Functional roles of microRNAs and DNMT3s were tested by anti-miRs, microRNA precursors and chemical inhibitors. ChIP was utilized for investigating HNF4α DNA binding activity. RESULTS: HNF4α silencing was sufficient to induce positive modulation of DNMT3B, in in vitro differentiated hepatocytes as well as in vivo hepatocyte-specific Hnf4α knockout mice, and DNMT3A, in vitro, but not DNMT1. In exploring the molecular mechanisms underlying these observations, evidence have been gathered for (i) the inverse correlation between DNMT3 levels and the expression of their regulators miR-29a and miR-29b and (ii) the role of HNF4α as a direct regulator of miR-29a-b transcription. Notably, during TGFβ-induced EMT, DNMT3s' pivotal function has been proved, thus suggesting the need for the repression of these DNMTs in the maintenance of a differentiated phenotype. CONCLUSIONS: HNF4α maintains hepatocyte identity by regulating miR-29a and -29b expression, which in turn control epigenetic modifications by limiting DNMT3A and DNMT3B levels.
BACKGROUND AND AIMS: Epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-to-epithelial transition (MET) are manifestations of cellular plasticity that imply a dynamic and profound gene expression reprogramming. While a major epigenetic code controlling the coordinated regulation of a whole transcriptional profile is guaranteed by DNA methylation, DNA methyltransferase (DNMT) activities in EMT/MET dynamics are still largely unexplored. Here, we investigated the molecular mechanisms directly linking HNF4α, the master effector of MET, to the regulation of both de novo of DNMT 3A and 3B. METHODS: Correlation among EMT/MET markers, microRNA29 and DNMT3s expression was evaluated by RT-qPCR, Western blotting and immunocytochemical analysis. Functional roles of microRNAs and DNMT3s were tested by anti-miRs, microRNA precursors and chemical inhibitors. ChIP was utilized for investigating HNF4α DNA binding activity. RESULTS:HNF4α silencing was sufficient to induce positive modulation of DNMT3B, in in vitro differentiated hepatocytes as well as in vivo hepatocyte-specific Hnf4α knockout mice, and DNMT3A, in vitro, but not DNMT1. In exploring the molecular mechanisms underlying these observations, evidence have been gathered for (i) the inverse correlation between DNMT3 levels and the expression of their regulators miR-29a and miR-29b and (ii) the role of HNF4α as a direct regulator of miR-29a-b transcription. Notably, during TGFβ-induced EMT, DNMT3s' pivotal function has been proved, thus suggesting the need for the repression of these DNMTs in the maintenance of a differentiated phenotype. CONCLUSIONS:HNF4α maintains hepatocyte identity by regulating miR-29a and -29b expression, which in turn control epigenetic modifications by limiting DNMT3A and DNMT3B levels.
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