Orchestrating transposon regulation in the male germline – the role of DNA methylation, histone marks and transcription factors
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Abstract
Transposable elements (TEs) are mobile DNA sequences, which can proliferate and change their position in the genome. They are part of all eukaryotic genomes and make up a significant portion of mammalian genomes. While they drive evolutionary innovation, their ability to replicate poses a major threat to genome stability. In somatic tissues, transposons are effectively silenced by DNA methylation. However, during mammalian development, DNA methylation is dynamic and undergoes reprogramming. The germline reprogramming wave erases somatic methylation patterns, allowing the activation of germline genes that are essential for development. However, this also creates a window of opportunity for TEs to become active.
In male germ cells, this is counteracted by the piRNA pathway, which processes TE-derived mRNA into piRNAs that guide de novo DNA methylation to the promoters of young, active TEs. This process is mediated by the male rodent-specific methyltransferase DNMT3C and its cofactor DNMT3L. The piRNA pathway and TEs are engaged in a continuous evolutionary arms race: while the silencing machinery evolves to suppress newly active TEs, TEs must evade this suppression to proliferate. Failure of the silencing machinery leads to TE upregulation, resulting in defective chromosome pairing, meiotic arrest, and ultimately male sterility. Notably, these spermatogenic defects arise several days after the failure of de novo methylation in fetal germ cells, suggesting that additional regulatory mechanisms govern TEs in the absence of DNA methylation.
To investigate TE regulation in the absence of DNA methylation, we first analyzed TE expression in Dnmt3CKO/KO mutants across sorted germ cell stages. Contrary to previous reports, we found that the young, active TEs, LINE1s and IAPs, become already active before meiosis in the absence of DNA methylation, while LINE1 expression further increases at meiosis. Next, we examined histone modifications associated with TE regulation in the absence of DNA methylation, revealing dynamic chromatin signatures during spermatogenesis. Specifically, we identified bivalent chromatin marks at unmethylated TE promoters: H3K4me3-H3K9me3 at LINE1 elements in fetal germ cells before DNMT3C-mediated methylation, and H3K4me3-H3K27me3 at TEs before meiosis in Dnmt3CKO/KO mutants. The bivalent H3K4me3-H3K27me3 mark may poise TE expression before meiosis while protecting them from DNA methylation. At the onset of meiosis, this mark transitioned to a more open chromatin state, correlating with high TE expression when DNA methylation is not in place.
To further explore TE regulation, we conducted an unbiased proteomic pulldown screen to identify factors binding TE promoters in a DNA methylation-dependent manner. Among other interesting candidates, we found NRF1, a DNA methylation-sensitive transcription factor, known to regulate germline genes, to specifically bind unmethylated TE promoters in male germ cells. To characterize NRF1 and other factors binding at TEs, we explored and optimized low-input chromatin profiling methods. This expertise enabled collaborations with other laboratories, where we demonstrated that the chromatin reader SPIN1 binds the bivalent H3K4me3-H3K9me3 signature at LINE1 elements in fetal germ cells, facilitating SPOCD1 recruitment and DNMT3C-mediated silencing. Furthermore, we showed that NRF1 binds LINE1s and IAPs in postnatal germ cells when DNA methylation is absent at their promoters, with increased binding at meiosis.
Finally, we tested a causative correlation, using a conditional NRF1 knockout in Dnmt3CKO/KO mice, and demonstrated that NRF1 activates unmethylated IAP elements before meiosis, while our data suggest a potential additional role in LINE1 regulation at meiosis.
Overall, we show that a bivalent chromatin signature and NRF1 as a DNA methylation-sensitive transcription factor orchestrate TE regulation in male germ cells when DNA methylation is absent at TE promoters. Our findings shed light on how TEs expand in genomes threatening germline genomic integrity and provide broader insights into how epigenetic mechanisms and transcription factors interact to regulate gene expression.