Transcriptional Stress as a Cause of DNA Double-Stranded Breaks

ABSTRACT Aims: Double-stranded breaks (DSBs) cause genomic instability, a hallmark of aging, and activate the DNA damage response (DDR) and the cytosolic DNA-sensing proteins (CDSP) pathways. The activated pathways induce cell cycle arrest, inflammation, senescence, cell death, fibrosis, and organ dysfunction. The purpose of the study was to identify and characterize genome-wide DSBs at the nucleotide resolution in adult cardiac myocytes. Methods and Results: We identified the genome-wide DSBs in the genomes of ~ 6 million adult cardiac myocytes (1 million per sample) in the 3 wild-type and 3 myocyte-specific LMNA-deficient mice (Myh6-Cre:LmnaF/F) mice by END-Sequencing. We identified 6,774 differential DSBs between the wild-type and Myh6-Cre:LmnaF/F cardiac myocytes, which, except for 8, were exclusive to the Myh6-Cre:LmnaF/F genotype. The differential DSBs were enriched in the gene regions, transcription initiation sites, cardiac transcription factor motifs, and at the G quadruplex forming structures, suggesting the involvement of the transcriptional stress. Because LMNA regulates transcription, we performed a CUT&RUN assay with an anti-LMNA antibody (N=5) to test the role of transcriptional stress in an increased prevalence of DSBs. We identified an average of 818 genome-wide lamin-associated domains (LADs), which encompassed 30% of the mouse genome. LADs were associated with a 16-fold reduction in the transcript levels of protein-coding genes located at the LAD regions (N=3,975) as opposed to genes located at the non-LAD regions (N=~ 17,778). Consistent with increased transcription at the non-LAD regions DSBs were ~ 6-fold more prevalent in the non-LAD than LAD regions. Likewise, the loss of LAD was associated with a 2.3-fold higher prevalence of differential DSBs in the Myh6-Cre:LmnaF/F cardiac myocyte genomes, which were predominantly localized to the transcription start sites. Conclusions: To our knowledge, this is the first identification of the DSBs, a hallmark of aging, at the nucleotide resolution in the cardiovascular system. The findings implicate transcriptional stress in the pathogenesis of DSB and the protective role of LMNA against DSBs likely through transcriptional suppression. Given the ubiquitous nature of transcriptional stress, DSBs are expected to be pervasive and pathogenic through activation of the DDR and CDSP pathways not only in cardiovascular diseases but also in other conditions, including aging.