Inside the nucleus of a living cell, DNA is folded around histone proteins into nucleosomes and compacted into a multi-layered three-dimensional (3D) structure called chromatin (Luger et al., 2012; Bickmore, 2013; Bonev and Cavalli, 2016). At any moment in time, a DNA double-strand break (DSB) can occur anywhere within this dynamic structural framework, and somehow, a cellular surveillance system termed the “DNA damage response” (DDR) (Jackson and Bartek, 2009) has the capacity to instantaneously detect DSB induction and recruit repair machinery to this type of genetic damage (Kalousi and Soutoglou, 2016; Hauer and Gasser, 2017; Marnef and Legube, 2017). Initially, chromatin was viewed as an obstacle to DSB repair that the DDR must first “open” and then restore upon DSB resolution. More recently, however, it has become apparent that the chromatin compaction status of a DSB plays a more active role in DNA damage signaling and DSB repair pathway choice (Soria et al., 2012; Lemaître et al., 2014; Clouaire and Legube, 2015; Polo and Almouzni, 2015). Local reorganization in chromatin network architecture has been shown to spatiotemporally modulate the arrival and retention of different DNA repair factors at DSB sites (Hinde et al., 2014; Smith et al., 2019). Thus, in order to understand how genome integrity is maintained at a cellular level, there is an emerging need to study DSB repair within the context of chromatin and the 3D nuclear landscape of a living cell.

The chromatin “opening” and “compacting” events that follow DSB induction (Klement et al., 2014; Kalousi et al., 2015; Thorslund et al., 2015; Luijsterburg et al., 2016) are underpinned by nanoscale changes in the spacing between nucleosomes (Hauer and Gasser, 2017), and these dynamics occur on a spatial scale that is well below the diffraction limit of optical microscopy (Luger and Hansen, 2005; Ou et al., 2017; Ohno et al., 2018; Ochs et al., 2019; Whelan and Rothenberg, 2021). Thus, with the aim of rendering any DDR-induced changes to a local chromatin structure visible in a living cell, we recently demonstrated that Förster resonance energy transfer (FRET) between fluorescently labeled histones is a sensitive real-time readout of nucleosome proximity during DSB repair (Lou et al., 2019) that can be spatially mapped throughout the nucleoplasm by the phasor approach to fluorescence lifetime imaging microscopy (FLIM) (Liang et al., 2020). From coupling FLIM detection of FRET between histone H2B tagged to eGFP (H2B-eGFP) and mCherry (H2B-mCh) with DSB induction via near-infrared (NIR) laser micro-irradiation, we quantified a rapid chromatin decompaction event central to a DNA repair locus that was surrounded by a border of compact chromatin foci and found this chromatin structure to be critical for the timely accumulation of DNA repair factors at a DSB site (Lou et al., 2019). Thus, this phasor histone FLIM-FRET assay has the potential to be an invaluable tool for biologists studying DSB repair, since it has sufficient spatiotemporal resolution to reveal what is normally an invisible layer of regulation to a cellular DDR.

Here in this study, we demonstrate the capacity of the phasor histone FLIM-FRET assay to spatially map chromatin architecture with respect to DNA damage in the DSB inducible via AsiSI cell system (DIvA) (Iacovoni et al., 2010). DIvA cells harbor a 4-hydroxytamoxifen (4OHT)-inducible AsiSI restriction enzyme that allows for induction of approximately 100 site-specific DSBs throughout the genome upon 4OHT treatment (Iacovoni et al., 2010; Massip et al., 2010; Aymard et al., 2014). Thus, by multiplexing phasor histone FLIM-FRET with immunofluorescence (IF) against phosphorylated serine 139 of histone variant 2AX (γH2AX), we are able to spatially map nuclear-wide chromatin compaction at the level of nucleosome proximity with respect to DIvA DSB locations. From image analysis of this three-color experiment across multiple DIvA nuclei, we find in agreement with our previous study employing NIR laser micro-irradiation (Lou et al., 2019) that DSB induction induces a global chromatin compaction event that surrounds sites of DNA damage, which statistically represent nuclear locations that are in a more “open” chromatin state. While a benefit of NIR laser micro-irradiation as a method for DSB induction was temporal resolution, an important advantage of the DIvA cell system is having access to the spatial heterogeneity that underlies this quantified chromatin response. Thus in a final experiment, to demonstrate this utility, we perform a four-color experiment that enables the chromatin structure reported by histone FRET to be studied as a function of the DSB repair pathway. We anticipate that this unique capacity of the phasor histone FLIM-FRET assay in DIvA alongside IF has the potential to facilitate discovery into how exactly chromatin structure regulates a DSB DNA damage response.

Phasor histone FLIM-FRET microscopy coupled with IF maps nuclear-wide changes in chromatin compaction with respect to DSB induction in the DIvA cell system. To quantify the local versus global chromatin compaction status of nucleus architecture with respect to multiple site-specific DSBs, here we combine phasor histone FLIM-FRET analysis with IF of γH2AX in the DIvA cell system. FRET is an optical phenomenon that reports fluorescent protein–protein interaction on a scale of 1–10 nm, and in the context of chromatin labeled with donor–acceptor fluorescent histones (Llères et al., 2009), FRET reports nucleosome proximity with nanoscale resolution. Thus, to implement histone FRET in the DIvA cell system, we first transfected DIvA cells with H2B tagged to eGFP (H2B-eGFP) in the absence (donor control) versus presence of mCherry (H2B-mCh) (Figures 1A,B). Then in fixed and washed DIvA nuclei expressing the donor control versus donor–acceptor FRET pair, we acquired FLIM data in the H2B-eGFP (donor) channel where quenching of the donor lifetime in the presence of H2B-mCh (acceptor) reports histone FRET (Figures 1C,D). Quantification of this donor control versus histone FRET experiment in the DIvA cell system by the phasor approach to lifetime analysis enabled the FRET efficiency of compact chromatin to be characterized as 16% (i.e., donor lifetime shift from 2.5 to 2.1 ns) (Figure 1C) and definition of a cursor-based palette to spatially map compact (red pixels) versus open chromatin (teal pixels) throughout DIvA nuclei (Figure 1D).

To next employ histone FRET as a readout of chromatin network architecture with respect to sites of DSB induction in the DIvA cell system, we first confirmed via IF for γH2AX Alexa Fluorophore 647 (γH2AX-AF647) in DIvA cells fixed 2 h after 4-hydroxytamoxifen (4OHT) treatment that multiple DSBs do form across the genome (Figure 1E). Then from careful design of a multi-colored imaging experiment that aimed to measure histone FRET between H2B-eGFP and H2B-mCh (Figures 1F,G) in the presence of γH2AX-AF647 IF (Figure 1H), we spatially mapped compact versus open chromatin in the presence versus absence of multiple DSB foci (Figure 1I) without artifact from 4OHT addition (Supplementary Figure S1). Quantification of this multiplexed imaging experiment via calculation of the fraction of pixels exhibiting histone FRET (our readout of a compact chromatin state) (Figure 1J) revealed genetic DSB induction to initiate significant nuclear-wide chromatin compaction when applied across multiple cells (Figure 1K). This result alongside a qualitative comparison of γH2AX-AF647 localization with histone FRET after 4OHT treatment (Figures 1H,I, right) suggested DSB sites to occupy the few “open” chromatin regions that exist within the detected nuclear-wide chromatin compaction event. Thus, to further investigate this observation, we next performed a γH2AX-AF647–based mask analysis of the histone FRET maps derived after 4OHT treatment, to enable quantification of the local (inside the DSB site) versus global (outside the DSB site) chromatin response to DSB induction.

To generate a mask that enables histone FRET analysis of chromatin compaction inside versus outside of DSB foci (Figures 2A–D), a threshold based on γH2AX-AF647 IF was employed (Figure 2E). This binary mask allowed for selection of pixels within the FLIM map that occupy DSB sites versus the surrounding nucleoplasm (Figure 2F), and quantitation of the fraction of pixels exhibiting histone FRET in either environment (Figure 2G). From application of this analysis to multiple cells after 4OHT treatment (Figure 2H), we confirmed DSB sites to statistically be in a more “open” chromatin state than the surrounding chromatin environment, which was compacted upon DSB induction (Figure 1). Interestingly, this differentially regulated reorganization in local versus global chromatin structure that was induced by multiple DSBs being genetically cut at distinct nuclear locations is in direct agreement with our previous study, which coupled histone FRET with NIR laser micro-irradiation to cut multiple DSBs at a single nuclear location (Lou et al., 2019). Thus, while NIR laser micro-irradiation was advantageous in terms of temporal resolution and enabling observation of early changes in DSB chromatin structure, a clear advantage of the DIvA cell system for histone FRET assessment of DSB chromatin during repair is the potential for this assay to explore any spatial heterogeneity that underlies this response.

IF-guided image analysis of phasor histone FLIM-FRET microscopy data acquired in DIvA cells quantifies chromatin network organization and enables exploration of DSB foci heterogeneity. To demonstrate the potential of phasor histone FLIM-FRET microscopy and IF in DIvA cells to enable both 1) a quantitative insight into the nuclear-wide spatial organization of compact chromatin with respect to DSBs and 2) exploration of heterogeneity in the local chromatin response at DSBs, here we performed two types of image analysis to acquired FLIM maps of histone FRET. The first type of analysis extracts the nuclear-wide localization of high FRET compact chromatin foci within a FLIM map, treats them as particles, and then quantifies their spatial distribution in terms of particle size. From application of this analysis to DIvA nuclei that were untreated versus treated with 4OHT (Figures 3A,B), we find the extracted network of high FRET compact chromatin foci (Figure 3C), to undergo a spatial reorganization in response to DSB induction that results in an increase in foci area (Figures 3D,E). This result, alongside the finding that DSB induction initiates a nuclear-wide chromatin compaction event at the level of nucleosome proximity (Figures 1, 2), suggests that, in addition to this global but nanoscale reorganization in chromatin structure, which occurs outside of DSB sites, a DSB DNA damage response also initiates sub-micron changes to higher order chromatin network organization (Figure 3).

To next investigate heterogeneity in the local chromatin response reported by histone FRET at DIvA DSB sites, we performed IF against not only γH2AX, which is expected to highlight the total population of DSBs present, but also different DNA repair proteins that highlight the DSB sub-population set to undergo repair by one of two dominant DSB repair pathways. In particular, we performed an IF-guided mask analysis of histone FRET maps acquired in DIvA nuclei co-expressing H2B-eGFP and H2B-mCherry, which were treated with 4OHT for 2 h (Figures 4A–C) and fixed with IF against 1) tumor suppressor p53 binding protein 1 (53BP1-AF405) that highlights DSBs marked for non-homologous end joining (NHEJ) (Figures 4D–F) and 2) breast cancer type 1 susceptibility protein (BRCA1-AF647) that highlights DSBs marked for homologous recombination (HR) (Figures 4G–I). Collectively, these experiments enabled quantification of chromatin compaction inside versus outside of DSB foci marked for NHEJ (Figure 4J) and HR (Figure 4K), as well as investigation into whether NHEJ versus HR DSB repair takes place in different chromatin environments (Figure 4L).

We find from this analysis that both NHEJ and HR DSB sites are statistically in a more “open” chromatin state than their surrounding undamaged chromatin environment (Figures 4J, K), which is in keeping with our γH2AX-guided analysis (Figure 2H). Also, intriguingly, if we take into account the baseline chromatin compaction status of each DIvA nucleus analyzed (i.e., normalized with respect to FRET fraction in pixels outside DSB sites), we find that while 53BP1 DSB foci marked for NHEJ are not significantly different from γH2AX DSB foci, BRCA1 DSB foci marked for HR are statistically more “open” than γH2AX DSB foci (Figure 4L). The molecular mechanism and physiological function of why HR DSB foci are more “open” needs to be further investigated; however, it is in keeping with previous studies that link BRCA1 with roles in chromatin de-condensation (Bochar et al., 2000; Ye et al., 2001), and it does suggest that heterogeneity in terms of chromatin structure does exist as a function of the DSB repair pathway.

In this study, we applied phasor histone FLIM-FRET microscopy to the measurement of nuclear-wide chromatin compaction at the level of nucleosome proximity and demonstrated that this assay can quantify sub-micron changes in the spatial organization of this nanoscale feature upon DSB induction in the DIvA cell system. From coupling this technology with immunofluorescence against histone modifications that highlight DSB sites (e.g., γH2AX) and DNA repair proteins involved in DSB resolution (e.g., 53BP1 and BRCA1), we also highlight the capacity of phasor histone FLIM-FRET to explore spatial heterogeneity in the local DSB chromatin structure as a function of DSB repair pathway choice—NHEJ versus HR. In doing so, we found that DIvA DSBs induce a global chromatin network compaction event that reduces the average spacing between nucleosomes and reorganizes them into larger clusters, in parallel with the local opening of chromatin at DSB sites—especially those marked for repair via HR. Interestingly, this result, which stems from multiple site-specific DSBs being induced at distinct locations throughout the DIvA nucleoplasm, is in strong agreement with our previous study that implemented phasor histone FLIM-FRET in HeLa cells exposed to NIR laser micro-irradiation, which induces multiple DSBs at a single nuclear location (Lou et al., 2019). Thus, chromatin “opening” at a DSB site alongside chromatin compacting of the surrounding DNA appears to be a universal mechanism for efficient repair of DSBs whether they be induced genetically or by a source of radiation.

The next question is the following: What biological function do these detected changes in chromatin structure serve for DSB resolution? In the context of DNA repair, there is already evidence obtained via super-resolution microscopy that a nanoscale reorganization in the chromatin structure regulates DNA repair protein access and retention at DSB sites (Ochs et al., 2019; Whelan and Rothenberg, 2021). Along this line, in our previous study that employed NIR laser micro-irradiation, we found that the compacted chromatin boundary of a DSB repair locus serves to modulate the mobility and access of the NHEJ repair factor tumor suppressor 53BP1 to the central “opened” region of this type of genomic lesion (Lou et al., 2019). Thus, given the demonstrated potential of the histone FRET assay to explore DSB chromatin structure, here as a function of DNA repair pathway choice when coupled with IF in DIvA, future experiments will be dedicated toward bettering understanding what is the role of DSB chromatin structure in controlling 53BP1 versus BRCA1 access and identifying whether chromatin plays a role in the decision to proceed toward DSB resolution via NHEJ versus HR.