Effect of DNA damage and chromatin damage on transcription in HT1080 cells with wild type p53 and p53 inhibited by dominant negative mutant

DNA-targeting drugs may damage DNA or chromatin. Many anti-cancer drugs do both, making difficult understanding their mechanism of actions. Using molecules causing DNA breaks without altering nucleosome structure (bleomycin) or destabilizing nucleosomes without damaging DNA (curaxin), we investigated consequences of DNA or chromatin damage in normal and tumor cells. As expected, DNA damage caused p53-dependent growth arrest followed by senescence. Chromatin damage caused higher p53 accumulation than DNA damage, but growth arrest was p53-independent and did not result in senescence. Chromatin damage activated transcription of multiple genes, including classical p53 targets, in a p53-independent manner. These activated genes, though they were not highly expressed, have chromatin organization around transcription start sites (TSS) characteristic for the most highly expressed genes and the highest level of paused RNA polymerase. We proposed that nucleosomes around TSS of these genes are the most sensitive to chromatin damage, and therefore nucleosome loss upon curaxin treatment enables transcription without assistance of sequence-specific transcriptional factors. We confirmed this by showing greater loss of nucleosomes around TSS of these genes upon curaxin treatment and activation of specific p53 reporter in p53-null cells by chromatin damaging agents, but not DNA damaging agents. Overall design: HT1080 cells, parental or expressing p53 dominant negative mutant GSE56 were treated with 400ug/ml of bleomycin or 0.5 uM of CBL0137 for 24 hours. After 24 hr, cells were incubated with 1 mM EU for 20 min. RNA was isolated using the Monarch Total RNA Miniprep Kit (New England BioLabs, cat #T2010) following the manufacturer's protocol. Nascent RNA was labeled with biotinylated azide and captured on magnetic beads using the Click-iT Nascent RNA Capture Kit (Molecular Probes, cat # C10365). cDNA was synthesized from RNA bound to the beads using the SuperScript VILO cDNA Synthesis Kit (ThermoFisher Scientific, cat #11754-050). cDNA was sequenced with the Illumina NextSeq 500/550 at the Roswell Park Comprehensive Cancer Center Genomics Shared Resource using Illumina NextSeq 500/550.