title: Epigenetic control of EMT/MET dynamics: HNF4α impacts DNMT3s through miRs-29.
abstract: OBJECTIVE
  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.
authors.affiliation: 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.
authors.forename: Carla
authors.initials: C
authors.lastname: Cicchini
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Hematology, Section of Molecular Genetics, Sapienza University of Rome, Rome, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: de Nonno
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Hematology, Section of Molecular Genetics, Sapienza University of Rome, Rome, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Battistelli
authors.affiliation: National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy.
authors.forename: Angela Maria
authors.initials: AM
authors.lastname: Cozzolino
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Hematology, Section of Molecular Genetics, Sapienza University of Rome, Rome, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: De Santis Puzzonia
authors.affiliation: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Silvia Anna
authors.initials: SA
authors.lastname: Ciafrè
authors.affiliation: Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Chad
authors.initials: C
authors.lastname: Brocker
authors.affiliation: Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Gonzalez
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Hematology, Section of Molecular Genetics, Sapienza University of Rome, Rome, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Amicone
authors.affiliation: 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.
authors.forename: Marco
authors.initials: M
authors.lastname: Tripodi
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D000090205:DNA Methyltransferase 3A; D044127:Epigenesis, Genetic / Q000502:physiology*; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D051557:Hepatocyte Nuclear Factor 4 / Q000502:physiology*; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D035683:MicroRNAs / Q000502:physiology*
chemical_list: C000715535:Dnmt3a protein, mouse; D051557:Hepatocyte Nuclear Factor 4; C496593:Hnf4a protein, mouse; C547014:MIRN29 microRNA, mouse; D035683:MicroRNAs; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A
keywords: De novo DNA methylation; Epithelial–mesenchymal transition; Hepatocyte; TGFβ; miR-29