Monitoring global changes in chromatin compaction states upon localized DNA damage with tools of fluorescence anisotropy

Abstract

In the eukaryotic nucleus, DNA, packaged in the form of chromatin, is subject to continuous damage. Chromatin has to be remodeled in order to repair such damage efficiently. But compact chromatin may also be more refractory to damage. Chromatin responses during DNA double-strand break (DSB) repair have been studied with biochemistry or as indirect readouts for the physical state of the chromatin at the site of damage. Direct measures of global chromatin compaction upon damage are lacking. We used fluorescence anisotropy imaging of histone H2B-EGFP to interrogate global chromatin compaction changes in response to localized DSBs directly. Anisotropy maps were preserved in fixation and reported on underlying chromatin compaction states. Laser-induced clustered DSBs led to global compaction of even the undamaged chromatin. Live-cell dynamics could be coupled with fixed-cell assays. Repair factors, PARP1 and PCNA, were immediately recruited to the site of damage, though the local enrichment of PCNA persisted longer than that of PARP1. Subsequently, nodes of PCNA that incorporated deoxynucleotide analogs were observed in regions of low-anisotropy open chromatin, even away from the site of damage. Such fluorescence anisotropy-based readout of chromatin compaction may be used in the investigation of different forms of DNA damage.