FRET-FISH probes chromatin compaction at individual genomic loci in single cells

Abstract

The density or compaction of chromatin throughout the cell nucleus is a key biophysical property that influences DNA replication, transcription, and repair. Chromatin accessibility is often used as a proxy for chromatin compaction or density, however it is not clear how these two properties relate to each other, given the lack of tools for directly probing compaction at defined genomic loci. To fill in this gap, here we developed FRET-FISH, a microscopy-based method combining fluorescence resonance energy transference (FRET) with DNA fluorescence in situ hybridization (FISH) to probe chromatin compaction at selected loci in single cells. We optimized FRET-FISH by testing different probe designs in situ in fixed cells, readily detecting FRET generated by DNA FISH probes. To validate FRET-FISH, we compared it with ATAC-seq and Hi-C, demonstrating that local chromatin compaction and accessibility are strongly correlated and that the frequency of intra-genic contacts measured by Hi-C may be an even better proxy for local chromatin density. To further validate FRET-FISH, we showed that it can detect expected differences in chromatin compaction along the nuclear radius, with peripheral loci being more compacted and central ones less compacted. Lastly, we assessed the sensitivity of FRET-FISH, demonstrating its ability to reproducibly detect differences in chromatin density (i) upon treatment of cells with drugs that perturb global chromatin condensation; (ii) during prolonged cell culture; and (iii) in different phases of the cell cycle. We conclude that FRET-FISH is a robust tool for probing chromatin compaction at selected loci in single cells and for studying inter-allelic and cell-to-cell variability in chromatin density.