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

Zhang, Yang; Máté, Gabriell; Müller, Patrick; Hillebrandt, Sabina; Krufczik, Matthias; Bach, Margund; Kaufmann, Rainer; Hausmann, Michael; Heermann, Dieter W.

doi:10.1371/journal.pone.0128555

Cited by 47 publications

(73 citation statements)

References 70 publications

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“…The pointillist localization image can be used for further analyses based on distance and frequency measurements (e.g., [45,46]). Usually, density images (see Figure 1 for the different possible methods to produce images from the localization data) are used.…”

Section: Introductionmentioning

confidence: 99%

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

Krufczik

Sievers

Hausmann

et al. 2017

IJMS

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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.

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Section: Introductionmentioning

confidence: 99%

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

Krufczik

Sievers

Hausmann

et al. 2017

IJMS

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“…We used combinatorial oligonucleotide fluorescence in situ hybridization (COMBO-FISH) 9 in combination with super-resolution single molecule localization microscopy (SPDM). 20 We extracted multiplets of 6 trinucleotide units [(CGG) 6 or (CCG) 6 probes] referred to as expansion-probes, showing the highest specificity to the (CGG)-repeat expansion region within the 5′ untranslated region of the FMR1 gene with a minimum of accessory binding sites within the whole genome. By data bank based analysis for accessory binding sites within the whole genome, the design of the probes is optimized so that the specificity of the used probe combination is increased by co-localization within the target region.…”

Section: Resultsmentioning

confidence: 99%

“…By simulating the binding of probes with a different number of (CGG)-repeats on targets of different lengths and a simultaneous search for accessory binding sites of these probes within the human genome we ascertained that the optimal length for the COMBO-FISH probes is (CGG) 6 . This requires an optimized probe design of multiplets of these triplets so that the specificity rises by co-localization within the target region.…”

Section: Probe Designmentioning

confidence: 99%

In situ optical sequencing and structure analysis of a trinucleotide repeat genome region by localization microscopy after specific COMBO-FISH nano-probing

et al. 2015

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Trinucleotide repeat expansions (like (CGG)n) of chromatin in the genome of cell nuclei can cause neurological disorders such as for example the Fragile-X syndrome. Until now the mechanisms are not clearly understood as to how these expansions develop during cell proliferation. Therefore in situ investigations of chromatin structures on the nanoscale are required to better understand supra-molecular mechanisms on the single cell level. By super-resolution localization microscopy (Spectral Position Determination Microscopy; SPDM) in combination with nano-probing using COMBO-FISH (COMBinatorial Oligonucleotide FISH), novel insights into the nano-architecture of the genome will become possible. The native spatial structure of trinucleotide repeat expansion genome regions was analysed and optical sequencing of repetitive units was performed within 3D-conserved nuclei using SPDM after COMBO-FISH. We analysed a (CGG)n-expansion region inside the 5' untranslated region of the FMR1 gene. The number of CGG repeats for a full mutation causing the Fragile-X syndrome was found and also verified by Southern blot. The FMR1 promotor region was similarly condensed like a centromeric region whereas the arrangement of the probes labelling the expansion region seemed to indicate a loop-like nano-structure. These results for the first time demonstrate that in situ chromatin structure measurements on the nanoscale are feasible. Due to further methodological progress it will become possible to estimate the state of trinucleotide repeat mutations in detail and to determine the associated chromatin strand structural changes on the single cell level. In general, the application of the described approach to any genome region will lead to new insights into genome nano-architecture and open new avenues for understanding mechanisms and their relevance in the development of heredity diseases.

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“…Localization microscopy of fluorescent proteins (28,29) was performed on a microscopy setup described by Szczurek et al (30). Fluorescent proteins are known to faintly photoswitch and are susceptible to rapid photobleaching.…”

Section: Single-molecule Localization Microscopymentioning

confidence: 99%

PML‐like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication‐based single‐strand breaks

et al. 2018

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DNA lesions induce recruitment and accumulation of various repair factors, resulting in formation of discrete nuclear foci. Using superresolution fluorescence microscopy as well as live cell and quantitative imaging, we demonstrate that X‐ray repair cross‐complementing protein 1 (XRCC1), a key factor in single‐strand break and base excision repair, is recruited into nuclear bodies formed in response to replication‐related single‐strand breaks. Intriguingly, these bodies are assembled immediately in the vicinity of these breaks and never fully colocalize with replication foci. They are structurally organized, containing canonical promyelocytic leukemia (PML) nuclear body protein SP100 concentrated in a peripheral layer, and XRCC1 in the center. They also contain other factors, including PML, poly(ADP‐ribose) polymerase 1 (PARP1), ligase IIIα, and origin recognition complex subunit 5. The breast cancer 1 and ‐2 C terminus domains of XRCC1 are essential for formation of these repair foci. These results reveal that XRCC1‐contaning foci constitute newly recognized PML‐like nuclear bodies that accrete and locally deliver essential factors for repair of single‐strand DNA breaks in replication regions.—Kordon, M. M., Szczurek, A., Berniak, K., Szelest, O., Solarczyk, K., Tworzydlo, M., Wachsmann‐Hogiu, S., Vaahtokari, A., Cremer, C., Pederson, T., Dobrucki, J. W. PML‐like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication‐based single‐strand breaks. FASEB J. 33, 2301–2313 (2019). http://www.fasebj.org

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

Cited by 47 publications

References 70 publications

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

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

In situ optical sequencing and structure analysis of a trinucleotide repeat genome region by localization microscopy after specific COMBO-FISH nano-probing

PML‐like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication‐based single‐strand breaks

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