Z-DNA is Remodeled by ZBTB43 in Prospermatogonia to Safeguard the Germ Line Genome and Epigenome (Affinity-seq)

Mutagenic purine-pyrimidine repeats can adopt the left-handed Z-DNA conformation. DNA breaks at potential Z-DNA sites can lead to somatic mutations in cancer or to germ line mutations which are transmitted to the next generation. It is not known whether any mechanism exists in the germ line to control Z-DNA structure and DNA breaks at purine-pyrimidine repeats. Here, we provide genetic, epigenomic, and biochemical evidence for the existence of a biological process that erases Z-DNA specifically in germ cells of the mouse male fetus. We show that a previously uncharacterized zinc finger protein, ZBTB43, binds to and removes Z-DNA, preventing the formation of DNA double-strand breaks. By removing Z-DNA, ZBTB43 also promotes de novo DNA methylation at CG-containing purine-pyrimidine repeats in prospermatogonia. Therefore, the genomic and epigenomic integrity of the species is safeguarded by remodeling DNA structure in the mammalian germ line during a critical window of germline epigenome reprogramming. ZBTB43 in vitro affinity-sequencing was done using purified MBP-ZBTB43 full length protein and fully unmethylated DNA isolated from Dnmt1/Dnmt3a/Dnmt3b triple knockout (TKO) ES cells (Tsumura, A. et al. Genes Cells 11, 805-814 2006) and also fully methylated lung DNA prepared with SssI bacterial CpG DNA methyltransferase, in duplicates. Background affinity was detected using MBP (vector). ZBTB43 affinity -seq was also caried out on DNA from Zbtb43-/- and wild type spermatozoa.