title: Double-strand break-induced transcriptional silencing is associated with loss of tri-methylation at H3K4.
abstract: Epigenetic alterations induced by ionizing radiation may contribute to radiation carcinogenesis. To detect relative accumulations or losses of constitutive post-translational histone modifications in chromatin regions surrounding DNA double-strand breaks (DSB), we developed a method based on ion microirradiation and correlation of the signal intensities after immunofluorescence detection of the histone modification in question and the DSB marker γ-H2AX. We observed after ionizing irradiation markers for transcriptional silencing, such as accumulation of H3K27me3 and loss of active RNA polymerase II, at chromatin regions labeled by γ-H2AX. Confocal microscopy of whole nuclei and of ultrathin nuclear sections revealed that the histone modification H3K4me3, which labels transcriptionally active regions, is underrepresented in γ-H2AX foci. While some exclusion of H3K4me3 is already evident at the earliest time amenable to this kind of analysis, the anti-correlation apparently increases with time after irradiation, suggesting an active removal process. Focal accumulation of the H3K4me3 demethylase, JARID1A, was observed at damaged regions inflicted by laser irradiation, suggesting involvement of this enzyme in the DNA damage response. Since no accumulation of the repressive mark H3K9me2 was found at damaged sites, we suggest that DSB-induced transcriptional silencing resembles polycomb-mediated silencing rather than heterochromatic silencing.
authors: Department of Radiation Oncology, University Hospital of Munich, Schillerstr. 42, 80336, Munich, Germany.
authors: Doris M
authors: DM
authors: Seiler
authors: Jacques
authors: J
authors: Rouquette
authors: Volker J
authors: VJ
authors: Schmid
authors: Hilmar
authors: H
authors: Strickfaden
authors: Christian
authors: C
authors: Ottmann
authors: Guido A
authors: GA
authors: Drexler
authors: Belinda
authors: B
authors: Mazurek
authors: Christoph
authors: C
authors: Greubel
authors: Volker
authors: V
authors: Hable
authors: Günther
authors: G
authors: Dollinger
authors: Thomas
authors: T
authors: Cremer
authors: Anna A
authors: AA
authors: Friedl
mesh_terms: D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D002843:Chromatin / Q000737:chemistry / Q000235:genetics; D002875:Chromosomes / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects*; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004249:DNA Damage / Q000528:radiation effects*; D005260:Female; D005455:Fluorescent Antibody Technique; D005720:Gamma Rays / Q000009:adverse effects; D020868:Gene Silencing / Q000528:radiation effects*; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008745:Methylation / Q000528:radiation effects; D018613:Microscopy, Confocal; D012516:Osteosarcoma / Q000235:genetics / Q000473:pathology; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000528:radiation effects*; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D056485:Retinoblastoma-Binding Protein 2 / Q000235:genetics / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000473:pathology
chemical_list: D002843:Chromatin; D006657:Histones; C070050:KDM5A protein, human; D056485:Retinoblastoma-Binding Protein 2; D012319:RNA Polymerase II