Abstract Body: Background: An intrinsic feature of gene transcription is the formation of DNA superhelices near the transcription bubble, which are resolved upon induction of transient double-stranded breaks (DSBs) by topoisomerases. Unrepaired DSBs are pathogenic as they lead to cell cycle arrest, senescence, inflammation, and organ dysfunction.
Hypothesis and Aims: We posit that DSBs would be more prevalent at the genomic sites associated with gene expression. As Lamin-A (LMNA) regulates active transcription through Lamin associated domains (LADS) we predict for DSBs to be more common in non-LAD regions. The objectives were to identify and characterize genome-wide DSBs at the nucleotide resolution and determine the association of DSBs with transcription in cardiac myocytes.
Methods: We identified the genome-wide DSBs in ~ 1 million cardiac myocytes per heart in 3 wild-type and 3 myocyte-specific LMNA-deficient (Myh6-Cre:Lmna^F/F) mice by END-Sequencing, the LADs were defined in cardiac myocytes via LMNA CUT&RUN assay (N=5).
Results: The prevalence of DSBs was 0.8% and 2.2% in the wild-type and Myh6-Cre:Lmna^F/F myocytes, respectively. The END-Seq signals for 8 DSBs were enriched in the wild-type and 6,764 in Myh6-Cre:Lmna^F/F myocytes, respectively (q<0.05). The DSBs were preferentially localized to the gene regions, transcription initiation sites, cardiac transcription factor motifs, and the G quadruplex forming structures. Because LMNA regulates transcription through the LADs, we defined the LADs in cardiac myocytes and found an average of 818 LADs per myocyte. LADs that were shared by at least three mice (N= 2,572) were considered constitutive LADs (cLADs), which comprised about a third of the mouse cardiac myocyte genomes. Transcript levels of the protein-coding genes located at the cLADs (N=3,975) were ~ 16-fold lower than those at the non-LAD regions (N=~17,778). The prevalence of DSBs was higher in the non-LAD as compared to the cLAD regions. Likewise, DSBs were more common in the loss-of LAD regions, defined as the juxtaposed genomic regions in the Myh6-Cre:Lmna^F/F that corresponded to the LAD regions in the wild-type myocytes.
Conclusions: To our knowledge, this is the first identification of DSBs, at the nucleotide resolution in the cardiovascular system. The prevalence of DSBs was higher in the genomic regions associated with transcription. Because transcription is pervasive, DSBs are expected to be common and pathogenic in various disease states and aging.