# p53 status determines the epigenetic response to demethylating agents azacitidine and decitabine

## TL;DR

This study shows that the p53 protein controls how cells respond to demethylating drugs azacitidine and decitabine, with p53 status acting as a potential biomarker for treatment choice.

## Contribution

The study identifies p53 as a key determinant of drug-specific epigenetic responses and introduces p53 status as a novel biomarker for demethylating agent selection.

## Key findings

- Azacitidine activates p53, leading to R-loop accumulation in CpG islands of p53 target genes.
- Decitabine induces DNA damage in the absence of p53, while p53 removal effects on the epigenome are reversible.
- p53 status determines divergent epigenetic responses to azacitidine and decitabine.

## Abstract

5’-Azacitidine (Aza) and 5-Aza-2’-deoxycytidine (Dac) are widely used demethylating drugs that directly integrate into nucleic acids. They are frequently used interchangeably, surprisingly as their selectivity is unique from the other, with no predictors of response or clinical biomarkers to indicate drug preference. Using these drugs to induce demethylation, we combine DRIPc-Seq, Immunostaining, RNA-Seq and Mass spectrometry to uncover unique cellular responses. Activation of p53, exclusively by Aza, sustains accumulation of R-loops in CpG islands of p53 target genes. This effect is abolished by the removal of p53, compounded by destabilisation of heterochromatin marks. Dac treatment induces global chromatin modification, sustaining DNA damage, which is heightened in the absence of p53. Rescue experiments reverse the changes observed in the epigenome, demonstrating a direct role for p53 in preserving H3K9me3 and H3K27me3. These insights further our knowledge of how cells recognize and respond to methylation changes and uncover novel roles for p53 in modulation of the epigenome. Further to this, we determine a first in kind biomarker in p53 status that may be relevant for clinical settings.

Azacitidine and Decitabine induce divergent changes to R-loops and the wider epigenome, subject to p53 mediated control. Removal of p53 ablates these effects, while the damaging impact of p53 removal on the epigenome is reversible.

Azacitidine and Decitabine modulate the wider epigenome in divergent manners, in part through changes to R-loop patterns.Azacitidine activates p53 and requires its activation for an appropriate epigenetic response.Decitabine does not activate p53 but induces substantial DNA damage in its absence.The epigenetic consequences of p53 removal are reversible.

Azacitidine and Decitabine modulate the wider epigenome in divergent manners, in part through changes to R-loop patterns.

Azacitidine activates p53 and requires its activation for an appropriate epigenetic response.

Decitabine does not activate p53 but induces substantial DNA damage in its absence.

The epigenetic consequences of p53 removal are reversible.

Azacitidine and Decitabine induce divergent changes to R-loops and the wider epigenome, subject to p53 mediated control. Removal of p53 ablates these effects, while the damaging impact of p53 removal on the epigenome is reversible.