Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response

Abstract

To investigate how chromatin architecture is spatiotemporally organised at a double strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image correlation spectroscopy (ICS) of histone FLIM-FRET microscopy data acquired in live cells co-expressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that when coupled with laser micro-irradiation induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ATM and RNF8 regulate rapid chromatin decompaction at DSBs and formation of a compact chromatin ring surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility. SIGNIFICANCE STATEMENTChromatin dynamics play a central role in the DNA damage response (DDR). A long-standing obstacle in the DDR field was the lack of technology capable of visualising chromatin dynamics at double strand break (DSB) sites. Here we describe novel biophysical methods that quantify spatiotemporal chromatin compaction dynamics in living cells. Using these novel tools, we identify how chromatin architecture is reorganised at a DSB locus to enable repair factor access and demarcate the lesion from the surrounding nuclear environment. Further, we identify novel regulatory roles for key DDR enzymes in this process. Finally, we demonstrate method utility with physical, pharmacological and genetic manipulation of the chromatin environment, identifying method potential for use in future studies of chromatin biology.

Contribution of advanced fluorescence nano microscopy towards revealing mitotic chromosome structure

“…FLIM has been used to study the chromatin compaction state during cell cycle progression in fixed samples where heterogeneous DNA distribution was seen (Murata et al 2000;Murata et al 2001;Görlitz et al 2017). FLIM on live interphase nuclei has shown chromatin decompaction upon chemical or radiation stimulus (Abdollahi et al 2018;Lou et al 2019;Pelicci et al 2019;Sherrard et al 2018). Live cell imaging studies have examined naive embryonic stem cells (ESCs) undergoing differentiation and showed that the presence of NANOG is essential for chromatin decompaction during pluripotency together with Rex1 downregulation (Hockings et al 2020).…”

Chromatin investigation in the nucleus using a phasor approach to structured illumination microscopy

“…It will be of interest to extend this robust SIM-ICCS analysis to data acquired with faster SIM setups (38,39) and study the organization of chromatin, dynamically, in living cells. Information on the organization of chromatin functional motifs at a scale of $100 nm, extracted by SIM-ICCS, would be complementary to other live-cell methods, such as Förster resonance energy transfer, that provide spatial information on a scale below $10 nm (40,41).…”

Cooperation between Caenorhabditis elegans COMPASS and condensin in germline chromatin organization

“…Importantly, FRET is a phenomenon sensitive to fluorophore proximity that occurs efficiently only when 6 the donor and acceptor fluorescent fusion proteins are closely positioned (<10nm) in the 3D nuclear space following chromatin compaction. By measuring histone-histone proximity, this FRET assay can respond to changes in nucleosome spacing, and therefore provides a read-out of nanoscale chromatin compaction (38)(39)(40)(41). FLIM-FRET also provides accurate quantification due to the independence of the fluorescence lifetime from the relative concentrations of the interacting proteins, and is independent of their diffusion rates (42).…”