ChIP analysis of H3K9me3 and H3K4me3 for mouse spermatogonia and leptotene/zygotene spermatocytes defficient in Dnmt3l and Pld6.

In the male germline of mammals, retrotransposon expression is restricted by DNA methylation, trimethylation of histone H3 at lysin-9 (H3K9me3), and PIWI-interacting small RNAs (piRNAs). To elucidate their relative importance in regulating retrotransposons during germ cell development and the relationships between these mechanisms, we performed mRNA, DNA methylation, and histone methylation analyses using mouse spermatogonia from Dnmt3l and Pld6 mutants deficient in de novo DNA methylation and piRNA production, respectively. The results revealed that loss of DNA methylation resulted in decreased H3K9me3 in young L1 subfamilies and increased H3K4me3 in many retrotransposons, suggesting a pivotal role of DNA methylation in maintaining epigenomic integrity in spermatogonia and later stages of spermatogenesis. The transcriptional upregulation of retrotransposons by a loss of DNA methylation was more evident during meiosis (spermatocytes) than before meiosis (spermatogonia). These results are aligned with a global reduction of H3K9me3 at retrotransposons in spermatocytes. The piRNA system also regulated H3K9me3 and H3K4me3 at retrotransposons in spermatogonia likely through the regulation of DNA methylation, since the loss of DNA methylation resulted in decreased H3K9me3 and increased H3K4me3 at the same retrotransposon loci even in the presence of piRNAs. Overall design: Chromatin was prepared from wild-type, Dnmt3l KO, and Pld6 KO spermatonia at P7 (50,000 to 150,000 cells, EPCAM-positive) and spermatocytes at P21 (~200,000 cells, sorted by Hoechst fluorescence) , and fragmantaded by micrococal nuclease treatment. Chromatin immunoprecipitation was conducted against anti-H3K9me3 antibody (MABI0308, Monoclonal Research Institute) or anti-H3K4me3 antibody (07-473, Merck) for 2 hours at 4 C using Dynabeads M280 Sheep anti-mouse or rabbit IgG (ThermoFisher Scientific). Captured DNA was obtained by proteinase K digestion and ethanol precipitation. The sequence library was prepared by using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolab) and sequenced on HiSeq Ten X (Illumina) using 150-bp paired-end mode.