Does the Repair of DNA Damage Restore the Altered Chromatin Landscape?

By Stuart P. Atkinson

CRISPR-induced DNA damage results in locally divergent transcriptional changes across an entire topologically associated domain (TAD) persisting beyond DNA repair. From figure 1 of Bantele et al.).

Does the Repair of DNA Damage Restore the Altered Chromatin Landscape?

The 3D arrangement of the chromatin landscape has a widely recognized impact on gene expression, which, in turn, can significantly influence cell-fate decisions; therefore, any insult that disrupts the topology of the normal chromatin landscape may lead to significant cellular dysfunction. While DNA double-strand breaks disrupt DNA integrity, they also induce significant alterations in the chromatin landscape (including at distances far from the primary DNA lesion), which facilitate signaling and DNA damage repair (Sanders et al.). The presence of DNA double-strand breaks in active chromatin regions also inhibits local transcription, thereby preventing harmful collisions between DNA- and RNA-associated factors (Vtor et al., Shanbhag et al., and Pankotai et al.). Cells generally possess the intricate machinery required to repair DNA double-strand breaks, but whether this approach restores the normal chromatin landscape and associated gene expression profiles remains somewhat underexplored.

Researchers from the laboratories of Susanne Bantele and Jiri Lukas (University of Copenhagen) sought to explore the long-term consequences of DNA damage and repair on the chromatin landscape and gene expression by directing CRISPR/Cas9-induced DNA double-strand breaks to genomic loci harboring topologically sensitive protein-coding genes. Now, their new Science study reveals that DNA double-strand break-induced alterations to chromatin conformation do not recover to pre-damage levels and instead persist (even through cell division), altering the expression of genes distant from the initial insult and triggering pathophysiological consequences (Bantele et al.). Overall, this exciting new study reports on a new phenomenon that the authors call chromatin fatigue, which may have an enormous impact on cells engineered for experimental or therapeutic purposes via nuclease-based genome editing approaches.

This study focused on 3D chromatin conformation and gene expression alterations in response to an initial DNA damage insult and subsequent repair; however, subsequent research linking these alterations to the histone modification profiles and transcriptomes of affected regions in single cells may provide even more profound insight. Paired-Tag technology from Epigenome Technologies, which generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays, could represent an efficient means of exploring this exciting concept.

DNA damage induces alterations in nuclear DNA localization (top) and chromatin accessibility (MNAse-seq; bottom) that persist long after DNA repair. From Figure 4 of Bantele et al.).

Chromatin Fatigue: Alterations to the Chromatin Landscape that Persist after DNA Damage Repair

Bantele et al. combined quantitative imaging of large cell populations, DNA/RNA fluorescence in situ hybridization, and Region Capture Micro-C to demonstrate that DNA double-strand break-induced chromatin alterations do not recover to pre-damage level after the repair of precise DNA breaks within a topologically associated domain harboring protein-coding genes and regulatory RNA species. Instead, these changes persisted as lasting alterations in the 3D chromatin architecture (misfolding) and impaired gene expression across large chromatin neighborhoods. Importantly, these impairments, or chromatin fatigue, persisted through cell division and can trigger concrete pathophysiological consequences; for example, the authors describe the reduced responsiveness of c-MYC gene expression to upstream signaling even when the primary DNA double-strand break generated (and subsequently repaired) lay outside the coding region. Importantly, the authors note the distinct nature of the chromatin fatigue concept when compared to the recently described micronuclei-associated transcriptional repression of genes after reintegration into the nuclear genome (Papathanasiou et al. and Agustinus et al.). Of note, this study also detected the reduced retention of at least two RNA species known to locally cluster at the c-MYC topologically associated domain, which adds another layer of epigenetic intrigue, given that RNA can contribute to genome structure in multiple ways (Quinodoz et al., Creamer et al., Zhang et al., and Isoda et al.).

DNA damage induces epigenetic alternations that change c-MYC production response to EGF stimulation, regardless of damage location in the TAD. From figure 6 of Bantele et al.).

The Significant Implications of Chromatin Fatigue

This never-tiring study describes chromatin fatigue as a new phenomenon that may permanently alter cells that undergo DNA double-strand breaks induced via external stressors. Notably, this finding has significant implications for research into aging, disease, and gene-editing technologies such as CRISPR/Cas9. Do alterations to histone modification profiles also underlie chromatin fatigue? Could the implementation of Paired-Tag technology from Epigenome Technologies help to answer this question? Epigenome Technologies Blog to find out!