How Does DNA Damage Impact the Epigenome?

June 9, 2025 By Stuart P. Atkinson

Stepwise Paired-Damage-seq experimental workflow. — Paired-Damage-seq workflow: lesion labeling, antibody-guided tagmentation, RT (BC1), cell barcoding (BC2/BC3), and separate RNA/DNA library prep with enzymatic nick repair inset. From Bai et al.

How Does DNA Damage Impact the Epigenome?

Part 1 of this series of articles from the Epigenome Technologies blog summarized the description and validation of Paired-Damage-seq, an exciting new technique reported in a recent Nature Methods paper from researchers led by Chenxu Zhu (New York Genome Center/Weill Cornell Medicine) (Bai et al.) . This technique hoped to support the study of how DNA damage and epigenomes interact by overcoming the obstacles faced by previous related techniques. Part 2 now explores how Paired-Damage-seq can help to describe relationships between DNA damage formation and epigenetic alterations when employing HeLa cells. Part 3 will describe the application of Paired-Damage-seq to the mouse cerebral cortex and the exploration of cell-type-specific genome vulnerabilities. Importantly, the range of products and services that Epigenome Technologies provides can empower your research aims with flexible, high-resolution technologies that turn hidden regulatory layers into actionable discoveries.

Line chart of damage enrichment by chromatin state over time. — DNA damage levels over H₂O₂ time course (0–48 h) plotted across 16 ENCODE chromatin states (Enhancer, TSS, transcribed, repressive, quiescent, ZNF repeats).

The Intertwining of Chromatin States and Genome Susceptibility

Compartment B (inactive heterochromatic regions) displayed higher DNA damage levels than Compartment A (active/open chromatin regions) in HeLa cells

This finding agrees with the preferential accumulation of cancer-associated single-nucleotide variants in heterochromatin due to inhibited DNA repair (Wang et al. and Schuster-Bockler and Lehner)

Stress-exposed cells possessed more DNA damage in gene bodies of highly transcribed genes compared to less transcribed/repressed genes

Open chromatin/R-loop conformations during transcription may render DNA more susceptible even though average gene body DNA damage levels remain lower when compared to Compartment B
The DNA repair machinery may selectively protect genic regions from accessible chromatin-coupled genome susceptibility

Genic enhancers and transcribed regions represented hotspots for DNA damage

Distal enhancers displayed comparable damage levels to transcribed regions, perhaps due to enhancer RNA transcription or the greater sensitivity of open chromatin regions to stress (Milano et al.)

Cis-regulatory elements (CREs) associated with H3K27me3 (e.g., bivalent enhancers and/or promoters and polycomb-associated heterochromatin) contained the lowest DNA damage levels

The non-coding elements responsible for cell/tissue identity may suffer from selective protection irrespective of activity; however, prioritized protection does not include constitutive heterochromatin

DNA damage, RNA, and ATAC tracks at marker genes by cell type. — Single-cell Paired-Damage-seq DNA (top) and RNA (middle) along with snATAC-seq (bottom) tracks at eight marker gene loci across major brain cell types. From Bai et al.

Identifying Inducible DNA Damage Hotspots

Analysis of single-cell DNA damage in HeLa cells revealed that short tandem repeats (STRs), long interspersed nuclear elements (LINEs), and CpG islands (not promoters and exonic regions) represented DNA damage hotspots

This distribution suggests that conserved factors drive DNA damage formation

Gene expression levels positively associated with local DNA damage levels
DNA damage increased at endogenous retroviruses in both compartments A and B, but chromatin accessibility only reduced at compartment A-residing endogenous retroviruses, which suggests that repeat elements can sense stress and alter gene activity at the epigenetic level (Fleming and Burrows)

Scatter plots of damage vs ATAC and damage vs H3K27me3 fold-change. — Scatter of log₂-fold change in DNA damage (48 h vs control) versus (c) snATAC-seq (PCC = –0.356) and (d) H3K27me3 CUT&Tag. From Bai et al.

DNA Damage Prompts the Loss of Epigenetic Memory

The authors evaluated how DNA damage-epigenome crosstalk may impact epigenetic memory in HeLa cells

Enhancers and H3K9me3-associated heterochromatin displayed high levels of DNA damage after stress
Changes to DNA damage levels over time negatively correlated with changes in chromatin accessibility and H3K9me3 levels over large genome areas
Epigenetic alterations occurred immediately after stress and endured for one or two cell divisions
The data suggest that the association between DNA damage formation and epigenetic alterations occurred at STRs and regulatory regions

Multiomic DNA damage and gene expression analysis revealed that DNA damage preferentially disrupts repressive regulatory states due to the chromatin decondensation needed for repair machinery loading (Oberdoerffer et al.)

DNA damage sites that strongly influence gene expression display enrichment at CREs (associated strongly with sites modified with H3K4me1, H3K4me3, and H3K27ac)

Damage signal violins for compartments A and B across cell types. — Violin plots of DNA damage signal in A (pink) versus B (blue) compartments across eight brain cell types. From Bai et al.

Paired-Damage-seq: Analysis of the Cerebral Cortex and Cell-Specific Genome Vulnerabilities

These applications of Paired-Damage-seq reveal how accumulated DNA damage can prompt the loss of epigenetic information; furthermore, this exciting technique revealed the existence of DNA damage hotspots. The subsequent applications of Paired-Damage-seq will aim to dissect the dynamics of genome and/or epigenome erosion and investigate any functional impacts on the molecular programs of cells in diseases and during aging. The Paired-Damage-seq technique represents an evolution of Paired-Tag from Epigenome Technologies, which generates joint epigenetic and gene expression profiles at the single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays.

For more on how Paired-Damage-seq supports the simultaneous analysis of DNA damage and gene expression in single cells, see Nature Methods, March 2025.