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Imaging the Response to DNA Damage in Heterochromatin Domains - PubMed

  • ️Sat Jan 01 2022

Imaging the Response to DNA Damage in Heterochromatin Domains

Audrey Chansard et al. Front Cell Dev Biol. 2022.

Abstract

The eukaryotic genome is assembled in a nucleoprotein complex called chromatin, whose organization markedly influences the repair of DNA lesions. For instance, compact chromatin states, broadly categorized as heterochromatin, present a challenging environment for DNA damage repair. Through transcriptional silencing, heterochromatin also plays a vital role in the maintenance of genomic integrity and cellular homeostasis. It is thus of critical importance to decipher whether and how heterochromatin affects the DNA damage response (DDR) to understand how this chromatin state is preserved after DNA damage. Here, we present two laser micro-irradiation-based methods for imaging the DDR in heterochromatin domains in mammalian cells. These methods allow DNA damage targeting to specific subnuclear compartments, direct visualization of the DDR and image-based quantification of the repair response. We apply them to study DNA double-strand break repair pathways in facultative heterochromatin and the repair of UV photoproducts in constitutive heterochromatin. We discuss the advantages and limitations of these methods compared to other targeted approaches for DNA damage induction.

Keywords: DNA damage; DNA repair; UV; confocal microscopy; heterochromatin; laser micro-irradiation.

Copyright © 2022 Chansard, Pobega, Caron and Polo.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1

Targeting DNA damage to heterochromatin domains in mammalian cells with laser micro-irradiation. Scheme illustrating the main steps for inducing DNA damage in the inactive X-chromosome (Xi) by 405 nm laser micro-irradiation (A) and in pericentric heterochromatin by UVC laser micro-irradiation (B). UVC micro-irradiation requires quartz coverslips and quartz optics since UVC light does not penetrate conventional glass materials.

FIGURE 2
FIGURE 2

Imaging the recruitment of DSB repair factors to the inactive X chromosome. (A) RNA-FISH staining for XIST RNA on the inactive X chromosome in RPE-1 cells using the method described in Chaumeil et al. (2008). The XIST cloud colocalizes with the most intense DAPI region in the nucleus. The microscopy image is a maximum intensity projection of 25 z-planes. (B) Nucleus of an RPE-1 cell stably expressing macroH2A1.2-GFP and stained with Hoechst. A white line depicts how to position the laser line to damage the Xi. (C,E) Live cell imaging showing the recruitment of the indicated GFP-tagged NHEJ and MMEJ factors to the inactive X chromosome (Xi) following 405 nm laser micro-irradiation in human RPE-1 cells. (D,F) Quantifications of the signals in the Xi (red) and outside the Xi (blue) as a function of time after laser damage are shown for each repair factor. Data are presented as mean values ± SD from a total of n nuclei scored in two independent experiments. (G) Hoechst photoconversion control after 405 nm micro-irradiation in RPE-1 cells (parental untransfected cell line vs cells expressing GFP-Ku70). Imaging settings were kept the same for both cell types. (H) Recruitment of the indicated HR factors in the inactive X chromosome (Xi) analyzed by immunofluorescence, 1 h (pRPA) or 4 h (RAD51) after 405 nm laser micro-irradiation in RPE-1 cells stably expressing GFP-tagged macroH2A1.2 (mH2A1.2-GFP). The percentages indicate the proportion of HR-prone cells (S/G2) that display recruitment of the respective HR factor to the damaged Xi. Antibodies used: primary antibodies anti-phospho-RPA32 Ser4/8 (Bethyl Laboratories A300-245A, 1:1,000), anti-RAD51 (Abcam ab176458, 1:1,000), anti-γH2A.X (Merck Millipore 05-636, 1:1,000); secondary antibodies anti-rabbit Alexa Fluor 568 (Invitrogen A11036, 1:1,000), anti-mouse Alexa Fluor 647 (Invitrogen A21236, 1:1,000). The position of the laser track is indicated by arrowheads. All microscopy images are confocal sections unless stated otherwise. Scale bars, 10 μm.

FIGURE 3
FIGURE 3

Pericentric heterochromatin decompaction and recruitment of UV damage repair proteins. (A) Decompaction of pericentric heterochromatin following UVC laser damage analyzed in NIH/3T3 cells expressing GFP-DDB2 (UV damage sensor). (B,C) Quantification of GFP-DDB2 intensity in damaged heterochromatin (B) and chromocenter area (C) relative to before damage. Data are presented as mean values ± SEM from 12–45 cells (depending on the timepoint) scored in at least eleven independent experiments. (D) Recruitment of XPB repair protein analyzed by immunofluorescence 1 h after UVC laser micro-irradiation in NIH/3T3 GFP-DDB2 cells. Antibodies used: primary antibody anti-XPB (Santa cruz Biotechnology sc-293, 1:400), secondary antibody anti-rabbit Alexa Fluor 594 (Invitrogen A11037, 1:1,1000). (E) Hoechst photoconversion control after UVC laser micro-irradiation in NIH/3T3 cells (parental cell line vs cells expressing GFP-DDB2) followed by immunofluorescence for UV lesions (Cyclobutane Pyrimidine Dimers, CPD) 30 min after irradiation. Antibodies used: primary antibody anti-CPD (Cosmo Bio CAC-NM-DND-001, clone TDM2, 1:1,000), secondary antibody anti-mouse Alexa Fluor 647 (Invitrogen A21236, 1:1,000). Imaging settings were kept the same for both cell types. The arrowheads point to damaged chromocenters. All microscopy images are confocal sections. Scale bars, 10 μm. Data from Fortuny et al. (2021).

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