How Does DNA Damage Impact the Transcriptome and Epigenome?

July 2, 2025 By Stuart P. Atkinson

Paired-Damage-seq experimental workflow diagram. — Paired-Damage-seq workflow: DNA damage labeling, antibody-guided tagmentation, reverse transcription (BC1), cell barcoding (BC2/BC3), and separate RNA/DNA library prep; inset details enzymatic repair, nick translation, and ligation of 8-oxoG lesions. From Bai et al.

How Does DNA Damage Impact the Transcriptome and Epigenome?

Maintaining genome integrity supports the ongoing function of the molecular programs required for multicellular organisms to thrive; however, exposure to endogenous and environmental factors over a lifespan can damage DNA. Complexity at the cell-type composition level and the stochasticity of damage formation represent obstacles to a better understanding of the preferred genomic distribution, functional consequences of DNA damage, and how DNA damage impacts gene regulatory programs/cell fate. We possess evidence for the existence of cross-talk between DNA damage and the epigenome (Dabin et al.), with DNA damage linked to chromatin modifier relocalization, the loss of a youthful epigenetic profile (Oberdoerffer et al. and Yang et al.), and chromatin decondensation (Qian et al.).

Chenxu Zhu (New York Genome Center/Weill Cornell Medicine) developed a technique - Paired-Damage-seq that supports the simultaneous high-throughput analysis of DNA damage and gene expression in single cells (Bai et al.). Part 1 of this series of articles from the Epigenome Technologies blog reports on how the authors of this new Nature Methods paper described/validated their novel technique employing HeLa cells. Parts 2 and 3 will explore relationships between DNA damage formation and epigenetic alterations and 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.

Genome tracks of damage, ATAC, CLAPS, CUT&Tag, and single-cell signals. — hg38 chr 9 locus in HeLa cells showing Paired-Damage-seq DNA and RNA, ATAC-seq, CLAPS-seq, Damage CUT&Tag tracks, and aggregated single-cell damage signals (n = 200), with shaded regions of interest. From Bai et al.

Paired-Damage-seq: The Basics

Paired-Damage-seq employs an in situ labeling-by-repair strategy that attaches biotin to DNA damage sites (based on previous studies; Riedl et al. and Shu et al.)

Nucleosome depletion in formaldehyde crosslinked cells minimized labeling bias due to chromatin epigenetic states (Mulqueen et al.)
Treatment with DNA repair proteins removes pre-existing 8-oxoG and cleave apyrimidinic sites to generate single-strand breaks (SSBs) before nick translation with Bst polymerase incorporates biotin-modified deoxyuridine triphosphates at pre-existing and newly generated SSBs and nick sealing occurs
300,000500,000 nuclei are incubated with a biotin antibody before targeted tagmentation with protein A-Tn5 attaches tube-specific DNA barcodes (B1) to the DNA damage sites
Two rounds of ligation-based combinatorial barcoding introduce barcodes BC2 and BC3
DNA fragments and complementary (c)DNA molecules from the same cells receive an identical combination of barcodes (BC1, 2, and 3)
Cell lysis and the purification of barcoded DNA/cDNA molecules allow library amplification steps similar to Paired-Tag (Zhu et al.)
Barcodes simultaneously attached to DNA and cDNA assign each read to individual cells

UMAP of damage-seq RNA time course and violin plots of library yields. — UMAP embedding of single-cell Paired-Damage-seq RNA in HeLa cells across H₂O₂ treatment times (0–48 h); violin plots of transcript and fragment counts for Paired-Damage-seq, ATAC-seq, CUT&Tag, and control assays in multiple sample types. From Bai et al.

Paired-Damage-seq: Validation in Bulk and Single-cell Samples

Validation steps employing bulk cells indicated that Paired-Damage-seq faithfully detects DNA damage

Paired-Damage-seq detected DNA damage levels in HeLa nuclei as a mode of differing levels of artificially generated SSBs and varying concentrations of H O
Pre-repair of DNA damage reduced the signal detected by Paired-Damage-seq, suggesting that this approach can detect endogenous DNA damage

Analyses confirmed the ability of Paired-Damage-seq robustly to simultaneously measure DNA damage and gene expression in single HeLa cells

Paired-Damage-seq displayed similar sensitivity in RNA detection when compared to alternative single-cell multiomics sequencing methods for joint RNA and epigenome analyses
Bulk and single-cell results compared well

A final validation step revealed the power of multiplexing different samples when applying Paired-Damage-seq to reduce batch effects

The team barcoded HeLa cells exposed to sub-toxic H2O2 concentrations, collected at varying time points with BC1, and multiplexed them into a single experiment
Transcriptomic analyses revealed ongoing alterations to gene expression, although DNA damage displayed a weak gradient distribution across different treatments

Chromosome 1 damage and ATAC-seq tracks under control and H₂O₂. — Genome browser tracks on chromosome 1 comparing DNA damage (control vs. H₂O₂), ATAC-seq signal and fold-change, and A/B compartment profiles in 250 kb bins. From Bai et al.

Paired-Damage-seq: A Validated Means of Evaluating DNA Damage and Gene Expression in Single Cells

Xie et al.) and to additional DNA damage types by adopting different labeling strategies (Mingard et al. and Amente et al.). This 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.

Nature Methods, March 2025.