The DNA double-strand “breakome” of mouse spermatids

Abstract

De novo germline mutations arise preferentially in male owing to fundamental differences between spermatogenesis and oogenesis. Post-meiotic chromatin remodeling in spermatids results in the elimination of most of the nucleosomal supercoiling and is characterized by transient DNA fragmentation. Using three alternative methods, DNA from sorted populations of mouse spermatids was used to confirm that double-strand breaks (DSB) are created in elongating spermatids and repaired at later steps. Specific capture of DSB was used for whole-genome mapping of DSB hotspots (breakome) for each population of differentiating spermatids. Hotspots are observed preferentially within introns and repeated sequences hence are more prevalent in the Y chromosome. When hotspots arise within genes, those involved in neurodevelopmental pathways become preferentially targeted reaching a high level of significance. Given the non-templated DNA repair in haploid spermatids, transient DSBs formation may, therefore, represent an important component of the male mutation bias and the etiology of neurological disorders, adding to the genetic variation provided by meiosis.

Endogenous DNA Double-Strand Breaks during DNA Transactions: Emerging Insights and Methods for Genome-Wide Profiling

“…Initially, DBrIC was used to demonstrate locus-specific and genome-wide DSBs in human cancer cells. More recently, the approach was harnessed to profile DSB hotspots during the transient genome fragmentation that accompanies post-mitotic chromatin remodeling in mouse spermatids [215,216].…”

Genetic Instability and Chromatin Remodeling in Spermatids

“…Several methods were used to confirm that the transient DNA strand breakage in spermatids also included a significant proportion of DNA double-strand breaks (DSBs). These included neutral comet assay, pulse-field gel electrophoresis [12], γH2AX labelling [13] and qTUNEL assay, whereby double-strand breaks were specifically labelled in solution following a prior step involving DNA nicks and gap filling [14]. Indirect evidence of a DSB repair response based on γH2AX expression must however be taken with caution, as the latter has also been associated with chromatin alteration [15].…”

“…Whereas meiosis may have evolved mechanisms to prevent these error-prone end-joining processes [18,46], haploid spermatids likely cannot avoid such mutagenic repair mechanisms. However, our initial mapping data indicated that the transient post-meiotic DSBs arise preferentially within repeated elements of the genome, which should minimize the genetic threat associated with the DSB formation [12]. It is important that coding sequences be protected from the global DNA fragmentation process, especially because of the general loss in DNA repair capacity that has been observed as the chromatin remodeling in spermatids proceeds to the final steps [47,48,49].…”

“…Second, our initial screening in mice indicated that a large part of the transient DSBs in spermatids map to repeated elements of the genome, in accordance with the relative abundance of these intergenic regions. Interestingly, however, DSBs arise at a greater frequency in LINEs and microsatellites relative to their normal representation in the genome [12]. Coincidentally, a higher frequency of de novo mutations was found to arise within repetitive DNA sequences [67], and a strong paternal bias has been reported for mutations within microsatellite repeats [71].…”

“…For instance, 88% of de novo indels arise on the paternal chromosome and are associated with autism spectrum disorders [65]. When considering only the gene subset, our preliminary gene ontology term analysis showed that DSBs in spermatids arise preferentially within synaptic genes, and with high significance [12]. Hence, the transient DSB formation in spermatids deserves more attention as a potentially significant mechanism by which paternal variation may be transmitted with implications for neurodevelopment.…”