Dynamic three-dimensional epigenomic reorganization for the development of undifferentiated spermatogonia in mice

Germ cells are the origin of totipotency, giving rise to new individuals. To fulfill this role, while faithfully replicating their genetic information [1], they undergo dynamic epigenomic programming during their development 2, 3, eventually transmitting genetic and epigenetic information from one generation to the next. Abnormalities in germ cell development can lead to severe conditions, including infertility and genetic or epigenetic disorders in offspring. Consequently, the mechanisms governing germ cell development and the acquisition of totipotency have been the focus of intensive investigation, as they are crucial in both biology and medicine.

The mechanisms of germ cell development in mammals have been primarily studied using the mouse as a model organism, with more recent studies extending to humans and nonhuman primates 4, 5. In mice, primordial germ cells (PGCs), the origin of germ cells, are specified in the extraembryonic mesoderm at embryonic day (E) E6.5 6, 7 (Figure 1). Following specification, PGCs undergo migration and colonize the embryonic gonads, in which they initiate their sex-specific development in response to the cues from gonadal somatic cells [8] (Figure 1). During the migration and colonization of embryonic gonads, PGCs show extensive propagation and undergo epigenetic reprogramming, characterized by genome-wide DNA demethylation and histone modification reorganization, differentiating into mitotic prospermatogonia in males or oogonia in females 2, 3, 9. Epigenetic reprogramming is considered a crucial event for acquiring totipotency in the next generation 2, 10, 11. Thereafter in males, mitotic prospermatogonia enter into mitotic arrest (mitotically arrested prospermatogonia) and differentiate into undifferentiated spermatogonia/spermatogonial stem cells (SSCs), and this process involves epigenetic programming, leading to the acquisition of androgenetic epigenome. However, the mechanisms by which epigenetic reprogramming and following epigenetic programming in the male germline establish the basis for gametogenesis and totipotency remain poorly understood.

Epigenetic regulation has been regarded as a key mechanism ensuring the generation of a wide variety of cell types in the body from the same DNA. In this context, the role of 3D genome organization has recently become a topic of intense investigation 12, 13. The 2-m-long DNA is highly folded and compacted within a micrometer-scale nucleus, where spatial proximity plays a crucial role in regulating gene expression via mechanisms such as enhancer–promoter (E-P) interactions and repression mediated by Polycomb repressive complexes 1 and 2 (PRC1/2). The former is essential for cell type–specific gene activation, while the latter is critical for the silencing of developmental genes during early development 13, 14. The development of Hi-C — a technique that measures 3D genomic distances through proximity ligation followed by sequencing — and subsequent higher-resolution 3D genomic techniques over the past decade has revealed that 3D chromatin organization entails two types of interactions: (1) affinity-based interactions creating canonical megabase-scale compartments and finer-scale microcompartments and (2) loop extrusion–based chromatin interaction represented as topologically associating domains (TADs) and loops (typically spanning tens to hundreds of kilobases) 12, 13, 15 (Figure 2). While A and B compartments, which correspond to euchromatin and heterochromatin, are thought to be established by affinity-based interactions potentially driven by active and repressive chromatin modifications, TADs and chromatin loops are formed by cohesin-mediated loop extrusion that stalls at CCCTC-binding factor (CTCF) boundaries 12, 15 (Figure 2). In particular, TADs have been considered primary boundaries that constrain E-P communication, although they exhibit dynamic variation across cells and over time [16] (Figure 2).

Although 2D epigenomic and 3D genomic techniques — such as chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and genome-wide chromatin conformation capture (Hi-C), respectively — often require millions of cells, recent advancements in low-input epigenomic and 3D genomic techniques — reducing the input to fewer than 1000 cells 19, 20, 21•, 22, 23• — along with the opportunities to characterize scalable in vitro germ cells 24, 17•, have facilitated our understanding of how the epigenome and 3D genome organization are dynamically programmed during mammalian germ cell development. We hereby refer to this integrated regulatory layer — comprising both the epigenome and 3D genome organization — as the 3D epigenome. Here, we provide a concise review of our current understanding of the 3D epigenome reorganization for the development of undifferentiated spermatogonia/SSCs, the lifelong source of the male gametes, the spermatozoa. For the 3D epigenome reorganization in both male and female gametogenesis, refer to other recent reviews 21•, 25, 26, 27, 28, 29.

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