Transient Relaxation of DNA Methylation at the Onset of Meiosis

Meiotic prophase I (MPI) is a unique phase of the cell cycle, specific to germ cells and defining of sexual reproduction. MPI is a period of extensive and specialized homologous chromosome interactions and genetic exchange. Proper progression of MPI requires elaborate epigenetic control, deficiencies in which often lead to infertility. Changes in DNA methylation during MPI can endanger genome integrity by activating transposable elements (TEs) that when mobilized induce DNA breaks and mutations. Therefore, MPI was thought to be under strict surveillance by DNA methylation, whose levels were assumed to be high and stable throughout MPI. Interestingly, expression of LINE retrotransposons, specifically LINE-1 (L1)-encoded protein ORF1p has been observed in MPI germ cells of wild-type male mice. Since tight epigenetic regulation is associated with transposon silencing, we hypothesized that L1 expression in MPI may indicate relaxation of epigenetic silencing in meiotic germ cells. Thus, we investigated the dynamics of CpG DNA methylation during MPI. We enriched and isolated individual MPI stages by Fluorescence Activated Cell Sorting (FACS) and profiled individual MPI germ cells using whole-genome bisulfite sequencing, and RNA-sequencing. Using this approach we uncovered transient and stage-specific changes in DNA methylation dynamics. In contrast to the prevailing view, we show that male germ cells undergo genome-wide transient relaxation of DNA methylation (TRDM) during early MPI. Specifically, we find that a transition from pre-meiotic spermatogonia to meiotic onset in preleptotene spermatocytes is accompanied by genome-wide hypomethylation. Gradual, but uneven remethylation of the genome creates hypomethylated domains throughout meiotic prophase, with pre-meiotic levels of DNA methylation achieved only by late MPI. Our data are most consistent with a DNA replication- coupled mechanism of DNA demethylation in pre-meiotic S-phase. Intriguingly, a TRDM- independent set of hypomethylated domains emerges in mid to late MPI and is enriched in transcriptionally upregulated spermatogenic genes. Using Mael -/- mice defective in piRNA pathway, we show that early MPI offers an opportunity for TE expression and reactivation. We demonstrate that if germ cells enter MPI with insufficient levels of DNA methylation at L1 elements, then during TRDM, meiotic onset can be hijacked to reactivate potentially active L1s. Cumulatively, we demonstrate that early MPI is epigenetically relaxed, exhibits dynamic DNA methylation pattern in MPI and that transient genome-wide DNA hypomethylation at meiotic onset might have implications in gamete quality control

Transient reduction of DNA methylation at the onset of meiosis in male mice

Background: Meiosis is a specialized germ cell cycle that generates haploid gametes. In the initial stage of meiosis, meiotic prophase I (MPI), homologous chromosomes pair and recombine. Extensive changes in chromatin in MPI raise an important question concerning the contribution of epigenetic mechanisms such as DNA methylation to meiosis. Interestingly, previous studies concluded that in male mice, genome-wide DNA methylation patters are set in place prior to meiosis and remain constant subsequently. However, no prior studies examined DNA methylation during MPI in a systematic manner necessitating its further investigation. Results: In this study, we used genome-wide bisulfite sequencing to determine DNA methylation of adult mouse spermatocytes at all MPI substages, spermatogonia and haploid sperm. This analysis uncovered transient reduction of DNA methylation (TRDM) of spermatocyte genomes. The genome-wide scope of TRDM, its onset in the meiotic S phase and presence of hemimethylated DNA in MPI are all consistent with a DNA replication-dependent DNA demethylation. Following DNA replication, spermatocytes regain DNA methylation gradually but unevenly, suggesting that key MPI events occur in the context of hemimethylated genome. TRDM also uncovers the prior deficit of DNA methylation of LINE-1 retrotransposons in spermatogonia resulting in their full demethylation during TRDM and likely contributing to the observed mRNA and protein expression of some LINE-1 elements in early MPI. Conclusions: Our results suggest that contrary to the prevailing view, chromosomes exhibit dynamic changes in DNA methylation in MPI. We propose that TRDM facilitates meiotic prophase processes and gamete quality control

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Additional file 9: Figure S4. CpG DNA methylation levels across chromosome length. DNA methylation was averaged using sliding non-overlapping windows of 100 kb

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Additional file 1: Figure S1. Schematic representation of main events in meiotic prophase I. Following premeiotic DNA replication in preleptonema (PL), parental homologous chromosomes (each containing two sister chromatids) develop chromosome axes (marked by SYCP3 protein), pair and synapse in leptonema (L) and zygonema (Z). Synapse is complete in pachynema (P) indicated by the complete overlap of SYCP3 and SYCP1 proteins. Following the completion of meiotic recombination, the synaptonemal complex disassembles in diplonema (D). Approximate duration of MPI substages are indicated (hrs). Figure adapted from [70]

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Additional file 5: Table S2. The efficiency of bisulfite conversion based on unmethylated lambda DNA

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Additional file 7: Table S4. Pearson correlation between biological replicates

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Additional file 6: Table S3. Methylation evidence results for the uniquely aligned, de-duplicated and M-bias filtered reads

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Additional file 14: Table S7. Characteristics of pairwise comparisons for large DMR block analysis

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Additional file 10: Figure S5. CpG DNA methylation levels across chromosome length. DNA methylation was averaged using sliding non-overlapping windows of 100 kb

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Additional file 4: Table S1. Bisulfite sequencing, alignment and read de-duplication results summary