Repair of DNA double-strand breaks leaves heritable impairment to genome function.

Upon DNA breakage, a genomic locus undergoes alterations in three-dimensional chromatin architecture to facilitate signaling and repair. Although cells possess mechanisms to repair damaged DNA, it is unknown whether the surrounding chromatin is restored to its naïve state. We show that a single DNA double-strand break (DSB) within a topologically associated domain (TAD) harboring conformation-sensitive genes causes lasting chromatin alterations, which persist after completion of DNA repair and feature topological rearrangements and loss of local RNA species. These newly acquired features of postrepair chromatin are transmitted to daughter cells and manifest as heritable impairments of gene expression. These findings uncover a hitherto concealed dimension of DNA breakage, which we term postrepair chromatin fatigue and which confers heritable impairment of gene function beyond DNA repair. Editor's summary: Cells organize their DNA in a three-dimensional structure that helps control which genes are turned on or off. Bantele et al. investigated whether this structure fully recovers after DNA damage is repaired. The researchers introduced precise DNA breaks and tracked changes in genome organization and gene activity. They found that even after the DNA was repaired, the affected regions remained misfolded and showed reduced gene expression, and these changes were passed on to daughter cells. DNA damage thus leaves lasting marks on genome function, a phenomenon called "chromatin fatigue," which has important implications for aging, disease, and gene-editing technologies such as CRISPR. —Di Jiang INTRODUCTION: Eukaryotic genomes are subjected to hierarchical folding that is required to accommodate DNA wrapped around the histone scaffold (collectively called chromatin) within the three-dimensional (3D) nuclear space. Evolution harnessed the 3D arrangement of nuclear chromatin to facilitate interactions among genomic segments such as promoters and enhancers, whose proximity influences gene expression and who thus have an important role in cell fate decisions such as orderly execution of developmental programs, adaptation to a new environment, or transmission of cell identity across successive generations of dividing cells. Although beneficial in these and other physiological contexts, the 3D arrangement of the nuclear genome also enables a distinct vulnerability to environmental or metabolic assaults that can modify chromatin folding and thus derail cellular functions. RATIONALE: A prominent example of such stress assaults is the DNA double-strand break (DSB). Besides disrupting DNA integrity, DSBs are intrinsically coupled to massive chromatin alterations that include changes in 3D arrangement and gene silencing across megabase distances from the primary DNA lesions. Although the DSB-induced chromatin response is initially beneficial to attract genome caretakers and generate structural scaffolds for timely and efficient DNA repair, its fate after restoring the integrity of DNA sequence is unknown. This seems to be a formidable gap in understanding genome maintenance that poses important questions: Do cells restore DSB-induced chromatin folding and the associated gene expression after completion of DNA repair? If yes, is the restoration of postrepair chromatin complete and back to the predamage level? If not, do the lingering chromatin alterations cause physiological impairments that can be inherited by successive cell generations? RESULTS: To answer these questions, we directed Cas9-induced DSBs to genomic loci harboring topologically sensitive protein-coding genes, as well as regulatory RNA species, to interrogate long-term consequences of DNA breakage on chromatin topology and gene activity. By combining quantitative imaging of large cell populations, DNA and RNA fluorescence in situ hybridization (FISH), and Region Capture Micro-C as readouts, we found that DSB-induced chromatin alterations do not recover to predamage level but persist as lasting changes in 3D arrangement and impaired gene expression throughout large chromatin neighborhoods that encounter, and subsequently repair, a single DSB. We show that such impairments persist through several rounds of successive cell divisions and can trigger concrete pathophysiological consequences—manifested, for instance, by reduced responsiveness of the c-MYC gene to upstream signaling even if the primary DSB was generated (and subsequently repaired) in the c-MYC neighborhood but outside its coding region. CONCLUSION: Our results reveal that DNA breakage leaves long-term marks in postrepair genomic loci, which disrupts 3D arrangement of large chromatin neighborhoods, persists through several rounds of successive cell divisions, and primes for heritable alterations in gene expression even if the primary DNA lesions are fully repaired. We term this phenomenon as chromatin fatigue and propose that it represents a hitherto unknown dimension of heritable responses to DNA breakage, with a potential to permanently alter physiology of cells that encounter DSBs through environmental or metabolic stress—but also lineages engineered for various experimental or therapeutic purposes by nuclease-based genome editing. Chromatin fatigue as a heritable consequence of DNA breakage and repair.: Three-dimensional chromatin exposed to a DSB (blue) undergoes conformational changes during DNA repair (green) and maintains a changed 3D structure with corresponding transcriptional alterations once repair has succeeded (tan). Inheritance of such alterations in structure and function can cause chromatin fatigue in the ensuing cell lineages. [ABSTRACT FROM AUTHOR]