Spermatogenesis is a highly coordinated developmental process during which precise temporal control of gene expression is crucial. In a recent study published in Cell Research, Lin et al. systematically profiled the epigenomic landscapes of male germ cells across developmental stages and discovered that a histone methyltransferase SETD1B establishes thousands of spermatid-specific broad H3K4me3 domains to ensure temporal transcriptional control during sperm development.

Male germ cells undergo a highly complex and tightly regulated unidirectional differentiation process, ensuring continuous sperm production. This involves mitotic maintenance of spermatogonia, meiotic division of spermatocytes, and morphological transformation of spermatids into mature spermatozoa. All of these processes require precise stage-specific gene expression orchestrated by transcription factors, chromatin modifiers, and histone modifications.1,2 It is not sufficient that the right genes are activated — they must be activated at exactly the right time. The inherent complexity of spermatogenesis poses a challenge to the isolation of homogeneous populations of each substage, hindering efforts to construct a comprehensive epigenomic landscape of male germ cells. To address these challenges, Lin et al.3 used a germ cell synchronization and isolation method to establish one of the most comprehensive epigenomic atlases of germ cell development during spermatogenesis to date. This was achieved by adding new data sets to their previous data sets.4 They profiled histone modifications, DNA methylation as well as chromatin accessibility across 11 distinct developmental stages, covering the major mitotic, meiotic, and postmeiotic stages of mouse spermatogenesis. This study provides a valuable resource for studying gene regulation during spermatogenesis.

Leveraging this detailed epigenomic profile of spermatogenesis, Lin et al. observed two major waves of chromatin reorganization — one prior to the mitosis-to-meiosis transition and the other upon completion of meiosis. These transitions involve genome-wide redistribution of repressive histone modifications, specifically histone H3 lysine 9 dimethylation (H3K9me2), H3K9 trimethylation (H3K9me3), and Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3. The first rearrangement occurs in type B spermatogonia prior to the mitotic-to-meiotic transition. At this point, a global loss of H3K27me3 has previously been reported upon spermatogonial differentiation.5 Intriguingly, Lin et al. now identify a concurrent genome-wide surge in H3K9me2 when H3K27me3 levels decrease. These shifts correlate with global transcriptional repression at meiotic entry, suggesting that H3K9me2, rather than H3K27me3, plays a role in gene silencing at this stage. The second rearrangement occurs in mid-pachytene spermatocytes. It is marked by a global reduction of H3K9me2 and H3K9me3 on autosomes and coincides with a transcriptional burst essential for meiotic progression.6 In addition, H3K27me3 levels are restored, which leads to the formation of H3K4me3-H3K27me3 bivalent domains. These domains serve as epigenetic placeholders — they maintain temporary repression of genes related to spermatogonial function or embryonic development while keeping these genes poised for future activation.2 This observation aligns well with previous observations that SCML2, a germline-specific Polycomb protein, interacts with PRC2 to establish bivalent chromatin in late spermatogenesis.7

Moreover, Lin et al. identify unexpectedly broad H3K4me3 domains (> 5 kb) in postmeiotic round spermatids. H3K4me3 is a well-established mark of active transcription that typically forms sharp peaks (~1–2 kb) at gene promoters. The broad H3K4me3 domains observed by Lin et al. coincide with promoters and enhancers and serve as hubs for RNA polymerase II (Pol II) recruitment. They are also commonly enriched in H3K27ac, an active promoter and enhancer mark, and overlap with super-enhancers established in late spermatogenesis.6 Thus, the broad H3K4me3 domains are associated with robust, stage-specific gene activation essential for sperm development. Lin et al. identified SETD1B (KMT2G), a relatively unexplored histone methyltransferase, as the key regulator of these broad H3K4me3 domains. Remarkably, germ cell-specific deletion of Setd1b leads to a complete loss of broad H3K4me3 domains and mislocalization of Pol II to conventional sharp H3K4me3 peaks (Fig. 1). This mislocalization disrupts the finely-tuned transcriptional timing of spermiogenesis, leading to a delay in early-stage gene expression and premature activation of late-stage genes. Ultimately, this compromises the precise gene expression cascade required for sperm development.

Fig. 1: Broad H3K4me3 domains orchestrate temporal control of gene expression during sperm development.
figure 1

In spermatids, SETD1B is recruited by RFX2 and other transcription factors to stage-specific promoter and super-enhancer loci and mediates the formation of broad H3K4me3 domains. These broad H3K4me3 domains sequester more Pol II than sharp H3K4me3 peaks, thereby contributing to the transcriptional upregulation of stage-specific genes.

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Epigenetic modifications rarely act in isolation — transcription factors often serve as guides for chromatin-modifying enzymes. RFX2, a transcription factor essential for spermiogenesis, is highly enriched at broad H3K4me3 domains and physically interacts with SETD1B. Loss of Rfx2 results in a dramatic reduction of broad H3K4me3 domains, suggesting that RFX2 acts in concert with SETD1B to establish these regulatory sites. RFX2 may also coordinate additional H3K4me3 methyltransferases because Rfx2 deletion leads to more severe transcriptional and developmental defects compared to Setd1b loss.

The current study challenges the conventional view of H3K4me3 as a static chromatin feature. Instead, H3K4me3 acts as a dynamic transcriptional timer that ensures that early-expressed genes do not remain active for too long and that late-expressed genes are not prematurely activated. Thus, this work establishes SETD1B as an epigenetic timekeeper in spermiogenesis. Interestingly, broad H3K4me3 domains were also detected in human spermatids, suggesting a conserved mechanism for regulating gene expression during spermiogenesis across species.

Broad H3K4me3 domains have also been found in other cell types, including somatic cells, oocytes, and early embryos, but their genomic distribution and function are different for each cell type.8,9,10 In oocytes, broad H3K4me3 domains span > 10 kb and cover ~22% of the genome.8,9,11 They are predominantly localized to intergenic regions and anti-correlated with DNA methylation. Here, another H3K4 methyltransferase, KMT2B (MLL2), is responsible for the formation of broad H3K4me3 domains. However, while loss of Kmt2b leads to the erasure of broad H3K4me3 domains in oocytes, transcription remains largely unchanged. This suggests that the broad H3K4me3 domains in oocytes are not a major regulator of transcription.11 By contrast, the broad H3K4me3 domains in spermatids are shorter (5–10 kb), localized at promoters and enhancers, and function as activators of gene expression. Moreover, as spermatids mature into sperm, broad H3K4me3 domains largely disappear. Because paternal H3K4me3 is largely reprogrammed in zygotes,8 the role of broad H3K4me3 domains is primarily restricted to sperm differentiation, with minimal influence on post-fertilization development. Despite these functional differences, broad H3K4me3 domains in oocytes and spermatids do share one mechanistic feature: they sequester Pol II from regular H3K4me3 peaks by acting as molecular “sponges” (Fig. 1). In this way, they compete with conventional H3K4me3 regions to regulate transcriptional timing, gene dosage, and developmental transitions. Interestingly, SETD1B is also required during oogenesis, and loss of SETD1B in oocytes leads to redistribution of H3K4me3 from gene promoters.12 Thus, oocytes have two layers of H3K4me3 regulation — one is SETD1B-mediated H3K4me3 at active gene promoters, and the other is MLL2-mediated H3K4me3 at broad H3K4me3 domains. These findings highlight a fundamental sex-specific difference in the function of broad H3K4me3 domains, which warrants further investigation.

In summary, by defining stage-specific chromatin transitions during spermatogenesis and identifying broad H3K4me3 domains as a crucial regulator of gene expression timing, Lin et al. provide fundamental insights into how epigenetic remodeling fine-tunes gene expression during spermatogenesis and ensures male fertility. They also uncover a new layer of epigenetic control, demonstrating how a chromatin-based mechanism governs developmental timing.