Global chromatin fibre compaction in response to DNA damage

Charlotte Hamilton,Richard L Hayward,Nick Gilbert

doi:10.1016/j.bbrc.2011.10.021

Charlotte Hamilton, Richard L Hayward + Show 1 more

Open Access

https://doi.org/10.1016/j.bbrc.2011.10.021

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Abstract

DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation (γH2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and γH2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by linker histones. We suggest that following DSB formation, although there is localised chromatin unfolding to facilitate repair, the bulk genome becomes rapidly compacted protecting cells from further damage.

Highlights

In mammalian cells DNA is packaged with histone proteins into nucleosomes which fold to form a 30-nm diameter chromatin fibre that are further packaged into large scale chromatin structure [1]
Heterochromatin provides a barrier for DNA repair and KAP1 phosphorylation is required for repairing damage in heterochromatin, but depletion of HP1 proteins alleviates the need for pKAP1 at heterochromatin [9,10] suggesting they are both involved in modulating chromatin structure
We have investigated the global effects of DNA double strand break (DSB) in U2OS and HCT116 cells

Summary

Introduction

In mammalian cells DNA is packaged with histone proteins into nucleosomes which fold to form a 30-nm diameter chromatin fibre that are further packaged into large scale chromatin structure [1]. Chromatin both protects the DNA from damage and provides a regulated environment for nuclear processes such as transcription, replication and DNA repair. We find that in response to DNA DSBs the chromatin fibre adopts a more compact structure, consistent with fibres being more regularly folded or fibre deformations being stabilized This global compaction of chromatin fibres could protect the genome from subsequent damage

Cell lines and reagents

Western blotting

Immunofluorescence

Chromatin modifications in response to DNA DSBs

Chromatin structure changes in response to DNA DSBs

Phosphorylated