title: Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene.
abstract: We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15-20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression.
authors.affiliation: Dipartimento di Medicina Molecolare e Biotecnologie mediche, Istituto di Endocrinologia ed Oncologia Sperimentale del C.N.R., Università Federico II, 80131 Napoli, Italy, IRCCS CROB, Dipartimento di Oncologia Sperimentale, via Padre Pio, 1 85028 Rionero in Vulture, Italy, Dipartimento di Medicina e di Scienze della Salute, Università del Molise, 86100 Campobasso, Itay, Dipartimento di Biologia, Università Federico II, 80126 Napoli, Italy, Dipartimento di Biologia Strutturale e Funzionale, Università dell'Insubria, Varese 21100, Italy, Department of Molecular Biology and Microbiology and Biomolecular Science Center, University of Central Florida, 12722 Research Parkway, Orlando, FL 32826, USA and Institute of Cancer Research, Departments of Microbiology and Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, NY 10032, USA.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Morano
authors.forename: Tiziana
authors.initials: T
authors.lastname: Angrisano
authors.forename: Giusi
authors.initials: G
authors.lastname: Russo
authors.forename: Rosaria
authors.initials: R
authors.lastname: Landi
authors.forename: Antonio
authors.initials: A
authors.lastname: Pezone
authors.forename: Silvia
authors.initials: S
authors.lastname: Bartollino
authors.forename: Candida
authors.initials: C
authors.lastname: Zuchegna
authors.forename: Federica
authors.initials: F
authors.lastname: Babbio
authors.forename: Ian Marc
authors.initials: IM
authors.lastname: Bonapace
authors.forename: Brittany
authors.initials: B
authors.lastname: Allen
authors.forename: Mark T
authors.initials: MT
authors.lastname: Muller
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Chiariotti
authors.forename: Max E
authors.initials: ME
authors.lastname: Gottesman
authors.forename: Antonio
authors.initials: A
authors.lastname: Porcellini
authors.forename: Enrico V
authors.initials: EV
authors.lastname: Avvedimento
mesh_terms: D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D059767:Recombinational DNA Repair; D014158:Transcription, Genetic; D044767:Ubiquitin-Protein Ligases; D000097642:GADD45 Proteins
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D018797:Cell Cycle Proteins; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D049452:Green Fluorescent Proteins; D009687:Nuclear Proteins; D044767:Ubiquitin-Protein Ligases; C000717967:DNMT3A protein, human; C439070:GADD45A protein, human; D000097642:GADD45 Proteins; C406364:UHRF1 protein, human