Histone FLIM-FRET microscopy reveals spatiotemporal regulation of chromatin organization by the DNA damage response
- Department of Biochemistry, University of Melbourne, Australia.
- Biomedical Engineering, University of California, Irvine, USA.
- EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, Australia.
- Children’s Medical Research Institute, University of Sydney, Australia.
Here we describe a biophysical method to measure chromatin organisation in live cells with nucleosome level resolution. The method is based on a localised phasor image correlation analysis (ICS) of FLIM-FRET microscopy data acquired in human cells co-expressing H2B-eGFP and H2B-mCherry. This multiplexed approach produces spatiotemporal maps of nuclear wide chromatin compaction and quantifies the stability, size and spacing between detected chromatin foci. We used this method in cells where double strand breaks (DSBs) were induced by near-infrared laser micro irradiation to assay chromatin dynamics during the DNA damage response (DDR). These experiments revealed that ATM and RNF8 directed rapid local chromatin decompaction at DSBs, coupled with formation of a stable ring of compact chromatin surrounding the repair locus. Based on these data we built a longevity map of sites with high FRET indicating the time scale of large scale compaction events directed by ATM and RNF8. Then by use of a phasor-based ICS analysis we identified the locations where the DDR shapes local and global chromatin dynamics and demonstrate the utility of phasor ICS-FLIM analysis of histone FRET for the study of chromatin biology.