RESEARCH ARTICLE Radiation Induced Chromatin Conformation Changes Analysed by Fluorescent Localization Microscopy, Statistical Physics, and Graph Theory

It has been well established that the architecture of chromatin in cell nuclei is not random but functionally correlated. Chromatin damage caused by ionizing radiation raises complex repair machineries. This is accompanied by local chromatin rearrangements and structural changes which may for instance improve the accessibility of damaged sites for repair protein com-plexes. Using stably transfected HeLa cells expressing either green fluorescent protein (GFP) labelled histone H2B or yellow fluorescent protein (YFP) labelled histone H2A, we in-vestigated the positioning of individual histone proteins in cell nuclei by means of high resolu-tion localization microscopy (Spectral Position Determination Microscopy = SPDM). The cells were exposed to ionizing radiation of different doses and aliquots were fixed after different repair times for SPDM imaging. In addition to the repair dependent histone protein pattern, the positioning of antibodies specific for heterochromatin and euchromatin was separately re-corded by SPDM. The present paper aims to provide a quantitative description of structural changes of chromatin after irradiation and during repair. It introduces a novel approach to an-alyse SPDM images by means of statistical physics and graph theory. The method is base