DNA methylation-sensitive transcription factors and bivalency orchestrate transposon expression in the absence of DNA methylation [Retrotransposon expression in conditional NRF1 mutant testes]

Silencing of young retrotransposons by Cytosine DNA methylation is essential for spermatogenesis. Failure to methylate retrotransposon promoters leads to their reactivation, meiotic catastrophe and infertility. How transposons become reactivated and why their reactivation follows spermatogenic developmental patterns remains unclear. Here, we show that specific retrotransposon families exhibit distinct expression patterns and chromatin landscapes throughout spermatogenesis when DNA methylation is absent from their promoters. We find a strong correlation between the loss of bivalent H3K4me3-H3K27me3 chromatin modifications and the transition from low retrotransposon expression in spermatogonia to reactivation in meiotic spermatocytes. Using a combination of DNA pulldowns, mass spectrometry and chromatin profiling by CUT&Tag, we identify the DNA methylation-sensitive transcription factor NRF1 as a potential regulator of unmethylated retrotransposons in spermatogenesis. Germline conditional ablation of Nrf1 reduced the upregulation of IAPs in the absence of DNA methylation and rescued the accumulation of IAP-derived Pol protein to wild type germ cell levels. Our study demonstrates that a combination of chromatin modifications and a DNA methylation-sensitive TF regulates young retrotransposons upon loss of repressive DNA methylation in germ cells, suggesting these interactions may be a core strategy used by retrotransposons to proliferate in the germline after evading silencing by DNA methylation. We found that NRF1 binds retrotransposons in the absence of DNA methylation in mouse germ cells, suggesting that NRF1 might activate their transcription in Dnmt3CKO/KO (D3C_KO) mutants (no DNA methylation at retrotransposon promoters). To test this hypothesis, we generated a conditional Nrf1 germ cell-specific knockout (Nrf1_KO) in the Dnmt3CKO/KO (DKO) and wild type backgrounds. For all downstream analysis, we sampled total RNA, total protein, and fixed tissue for immunofluorescence and tested genotypes from the same animal, matching littermate controls whenever possible. To assess the impact of Nrf1 knockout on retrotransposon expression, we performed poly-A-enriched RNA sequencing on whole testes at P5 and analyzed differentially expressed transposon families through pairwise comparisons of the mutants.