Fig. 8: ZBTB43 is required for de novo methylation of PPRs in prospermatogonia.
From: Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome
a, Z-DNA structure inhibits DNMT3A catalytic activity in vitro. The CC, CL and LL substrates were prepared from the Rps6kl1 affinity binding sequence and were subjected to methylation by DNMT3A at 30× or 60× molar excess. The level of DNA methylation at each CpG is plotted after multiplexed bisulfite sequencing in two biologically independent replicates. b, DNA methylation is aberrant in sperm of Zbtb43−/− mice at the sites of ZBTB43 binding. IGV browser images display selected in vivo hypomethylated regions detected using MIRA–seq assays in Zbtb43−/− versus wild-type spermatozoa (navy). The ZBTB43 affinity binding peaks (green), and ChIP–seq peaks mapped in 15.5 dpc prospermatogonia (light green) are also shown together with the MBP and IgG backgrounds. Independent replicate samples are displayed. c, DMRs originate at the time of de novo methylation in prospermatogonia. Bisulfite sequencing results show methylated and unmethylated CpGs (black and white circle, respectively) along individual chromosomes at specific DMR sequences and at one control region in adult spermatozoa (top) and 18.5 dpc prospermatogonia (bottom). The genotypes are marked on the left. d, Heatmap displaying the MIRA–seq intensities in navy at the hypomethylated and hypermethylated DMRs identified between Zbtb43−/− and Zbtb43+/+ spermatozoa. Heatmaps to the right in green show the affinity binding of MBP-ZBTB43-FL in unmethylated and methylated genomic DNA (TKO and SssI) centred at the sites of sperm DMRs. The sequencing results shown represent two independent biological replicates in b and d. e, ZBTB43 ChIP–seq peaks in 15.5 dpc prospermatogonia overlap with hypo-DMRs detected in Zbtb43−/− sperm DNA. Venn diagrams. f, Model. Top: ZBTB43 binds Z-DNA in normal prospermatogonia. By removing Z-DNA, ZBTB43 creates an accessible substrate for DNMT3A and facilitates DNA methylation establishment at PPR-rich DNA regions. Bottom: Z-DNA is not removed in the Zbtb43−/− prospermatogonia in the absence of ZBTB43, DNA methylation is not established at PPRs, and hypomethylated DMRs are found in mutant sperm. One prominent ZBTB43 region can induce large re-arrangements in mammalian mutation assays, and ZBTB43 protects from DSBs by directly binding to PPRs and removing mutagenic Z-DNA structures in the foetal germ cells.
