title: DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation.
abstract: Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.
authors.affiliation: Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Lafaye
authors.affiliation: Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054, France.
authors.forename: Ewa
authors.initials: E
authors.lastname: Barbier
authors.affiliation: Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Miscioscia
authors.affiliation: Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Saint-Pierre
authors.affiliation: Etude de la Dynamique des Protéomes, Biologie à Grande Echelle, UMR S_1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054, France.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Kraut
authors.affiliation: Etude de la Dynamique des Protéomes, Biologie à Grande Echelle, UMR S_1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054, France.
authors.forename: Yohann
authors.initials: Y
authors.lastname: Couté
authors.affiliation: INSERM, U1009, Institut Gustave Roussy, Université Paris 11, 114 rue Edouard Vaillant, Villejuif F-94805, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Plo
authors.affiliation: Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Gasparutto
authors.affiliation: Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054, France.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Ravanat
authors.affiliation: Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054, France. Electronic address: jean.breton@cea.fr.
authors.forename: Jean
authors.initials: J
authors.lastname: Breton
mesh_terms: D044503:5-Methylcytosine / Q000378:metabolism / Q000031:analogs & derivatives; D045744:Cell Line, Tumor; D018899:CpG Islands; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D044127:Epigenesis, Genetic; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*
chemical_list: C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004273:DNA, Neoplasm; D012097:Repressor Proteins; C544396:ZBTB2 protein, human; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine
keywords: 5-hydroxymethylcytosine; Protein–DNA interactions; ZBTB2