Abstract
Chromatin architecture plays major roles in gene regulation as well as in the repair of DNA damaged by endogenous or exogenous factors, such as after radiation. Opening up the chromatin might provide the necessary accessibility for the recruitment and binding of repair factors, thus facilitating timely and correct repair. The observed formation of ionizing radiation-induced foci (IRIF) of factors, such as 53BP1, upon induction of DNA double-strand breaks have been recently linked to local chromatin decompaction. Using correlative light and electron microscopy (CLEM) in combination with DNA-specific contrasting for transmission electron microscopy or tomography, we are able to show that at the ultrastructural level, these DNA damage domains reveal a chromatin compaction and organization not distinguishable from regular euchromatin upon irradiation with carbon or iron ions. Low Density Areas (LDAs) at sites of particle-induced DNA damage, as observed after unspecific uranyl acetate (UA)-staining, are thus unlikely to represent pure chromatin decompaction. RNA-specific terbium-citrate (Tb) staining suggests rather a reduced RNA density contributing to the LDA phenotype. Our observations are discussed in the view of liquid-like phase separation as one of the mechanisms of regulating DNA repair.
Highlights
Genomic DNA is packed in a highly regulated manner within an interphase nucleus to allow gene transcription or duplication of DNA during the S-Phase
We show that the DNA/chromatin compaction state within DNA repair regions generally is lower than in HC, but comparable with euchromatic areas, DNA rearrangement cannot be solely responsible for the Low Density Areas (LDAs) pattern
The LDA phenotype was initially reported after formaldehyde fixation and embedding into acrylic resin (LR White) [21], we show that the LDA phenotype occurs after glutaraldehyde fixation and Epon embedding (Figure 2), the sample preparation protocol used for DNA selective staining ChromEMT and osmium ammine B (OA-B)
Summary
Genomic DNA is packed in a highly regulated manner within an interphase nucleus to allow gene transcription or duplication of DNA during the S-Phase. In contrast to UA staining, this DNA-specific stain revealed non-homogeneous distribution of DNA within the cell nuclei (Figure 3) It consists of dense heterochromatic patches located at the nuclear and nucleolar periphery, and in part within the nucleoplasm as well as lower density areas indicating euchromatic regions. The results of OA-B stain on irradiated U2OS cells were quite similar to ones obtained in ChromEMT stained samples: IRIFs showed a low DNA density, which was again comparable to non-irradiated euchromatin areas (EC-like (EC-l) structures, Figure 4). These observations could be reproduced again using WT NIH/3T3 cells (Supplemental Figure S2C), corroborating our findings.
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