Global chromatin fibre compaction in response to DNA damage.
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.
Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR, UK.
Charlotte
C
Hamilton
Richard L
RL
Hayward
Nick
N
Gilbert
D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D006657:Histones / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D012097:Repressor Proteins / Q000378:metabolism; D000076183:Tripartite Motif-Containing Protein 28
D002843:Chromatin; C468783:H2AX protein, human; D006657:Histones; D012097:Repressor Proteins; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28