Histone Methylation Regulates Gene Expression in the Round Spermatids to Set the RNA Payloads of Sperm

Gene expression during spermatogenesis undergoes significant changes due to a demanding sequence of mitosis, meiosis, and differentiation. We investigated the contribution of H3 histone modifications to gene regulation in the round spermatids. Round spermatids were purified from rat testes using centrifugal elutriation and Percoll density-gradient centrifugation. After enzymatic chromatin shearing, immuno-precipitation using antibodies against histone marks H3k4me3 and H3K9me3 was undertaken. The immunoprecipitated DNA fragments were subjected to massive parallel sequencing. Gene expression in round spermatids and sperm was analyzed by transcriptome sequencing using next-generation sequencing methods. ChIP-seq analysis showed significant peak enrichment in H3K4me3 marks in active chromatin regions and H3K9me3 peak enrichment in repressive regions. We found 53 genes which showed overlapping peak enrichment in both H3K4me3 and H3K9me3 marks. Some of the top H3K4me3-enriched genes were involved in sperm tail formation (Odf1, Odf3, Odf4, Oaz3, Ccdc42, Ccdc63, and Ccdc181), chromatin condensation (Dync1h1, Dynll1, and Kdm3a), and sperm functions such as acrosome reaction (Acrbp and Fabp9), energy generation (Gapdhs), and signaling for motility (Tssk1b, Tssk2, and Tssk4). Transcriptome sequencing in round spermatids found 64% transcripts of the H3K4me3-enriched genes at high levels and of about 25% of H3K9me3-enriched genes at very low levels. Transcriptome sequencing in sperm found that more than 99% of the ChIP-seq corresponding transcripts were also present in sperm. H3K4me3 enrichment in the round spermatids correlates significantly with gene expression and H3K9me3 correlates with gene silencing that contribute to sperm differentiation and setting the RNA payloads of sperm.