TY - GEN A1 - Lee, Jin-Ho A1 - Bobkova, Elizaveta A1 - Gier, Theresa A1 - Gote, Martin A1 - Schmidt-kaler, Fanz A1 - Brieger, Emily A1 - Maus, Emanuel A1 - Krufczik, Matthias A1 - Chojowski, Robert A1 - Korn, Friederike A1 - Sarah, Schumann A1 - Scherthan, Harry A1 - Falkova, Iva A1 - Falk, Martin A1 - Hausmann, Michael A1 - Hildenbrand, Georg T1 - Mechanisms and Challenges for Understanding Radiation Induced Changes in Chromatin Nanoarchitecture T2 - DRO 2018 N2 - The three-dimensional architecture of genomes acts as an additional level of mode for fundamental biological processes such as DNA damage response. In this context, nanoprobing and super-resolution microscopy are powerful methods for structural analyses of genomic targets in native chromatin of single cells at resolutions of single antibodies, proteins, histones, short DNA stretches, etc. We used multi-color chromatin nanoprobing and single molecule localization microscopy of established DNA damage and chromatin markers in 3D-conserved nuclei of different cell types exposed to various types and doses of ionizing radiation. Similarly, effect of gold nanoparticles on extent and direction of cellular radiation response was assessed. Our studies revealed the nanoarchitecture of damage foci with respect to γH2AX, Mre11 or 53BP1 and their molecular rearrangements during repair processes. Nanoscopy of genomic Alu resulted in linear-quadratic dose-effects for low to higher dose ranges and in changes of H3K9me3 distribution around Alu clusters upon radiation exposure. Preliminary results show post-irradiation time dependent changes in Alu chromatin. Moreover, gold nanoparticles incorporated into cells seem to act by an interplay of radiation enhancement and chromatin remodeling leading to altered radiosensitivity. Our results contribute to the understanding of cellular radiation responses, thereby laying the basis for improved biological dosimetry and radiotherapies in future. N2 - Poster KW - Genom Y1 - 2019 U6 - https://doi.org/https://doi.org/10.13140/RG.2.2.31391.20647 ER - TY - JOUR A1 - Krufczik, Matthias A1 - Sievers, Aaron A1 - Hausmann, Annkathrin A1 - Lee, Jin-Ho A1 - Hildenbrand, Georg A1 - Schaufler, Wladimir A1 - Hausmann, Michael T1 - Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure JF - International Journal of Molecular Sciences N2 - Immunostaining and fluorescence in situ hybridization (FISH) are well established methods for specific labelling of chromatin in the cell nucleus. COMBO-FISH (combinatorial oligonucleotide fluorescence in situ hybridization) is a FISH method using computer designed oligonucleotide probes specifically co-localizing at given target sites. In combination with super resolution microscopy which achieves spatial resolution far beyond the Abbe Limit, it allows new insights into the nano-scaled structure and organization of the chromatin of the nucleus. To avoid nano-structural changes of the chromatin, the COMBO-FISH labelling protocol was optimized omitting heat treatment for denaturation of the target. As an example, this protocol was applied to ALU elements—dispersed short stretches of DNA which appear in different kinds in large numbers in primate genomes. These ALU elements seem to be involved in gene regulation, genomic diversity, disease induction, DNA repair, etc. By computer search, we developed a unique COMBO-FISH probe which specifically binds to ALU consensus elements and combined this DNA–DNA labelling procedure with heterochromatin immunostainings in formaldehyde-fixed cell specimens. By localization microscopy, the chromatin network-like arrangements of ALU oligonucleotide repeats and heterochromatin antibody labelling sites were simultaneously visualized and quantified. This novel approach which simultaneously combines COMBO-FISH and immunostaining was applied to chromatin analysis on the nanoscale after low-linear-energy-transfer (LET) radiation exposure at different doses. Dose-correlated curves were obtained from the amount of ALU representing signals, and the chromatin re-arrangements during DNA repair after irradiation were quantitatively studied on the nano-scale. Beyond applications in radiation research, the labelling strategy of immunostaining and COMBO-FISH with localization microscopy will also offer new potentials for analyses of subcellular elements in combination with other specific chromatin targets. KW - Genom Y1 - 2017 U6 - https://doi.org/https://doi.org/10.3390/ijms18051005 SN - 1422-0067 VL - 18 IS - 5 PB - MDPI AG ER - TY - CHAP A1 - Hausmann, Michael A1 - Lee, Jin-Ho A1 - Sievers, Aaron A1 - Krufczik, Matthias A1 - Hildenbrand, Georg T1 - COMBinatorial Oligonucleotide FISH (COMBO-FISH) with Uniquely Binding Repetitive DNA Probes T2 - The Nucleus KW - Genom Y1 - 2020 SN - 9781071607626 U6 - https://doi.org/https://doi.org/10.1007/978-1-0716-0763-3_6 SN - 1064-3745 SP - 65 EP - 77 PB - Springer US CY - New York, NY ER -