After epigenetic reprogramming and coincident with commitment to gametogenesis, mouse (and a subset of human) fetal germ cells acquire a unique genome organization: individual chromosomes separate, centromeres anchor to the nuclear periphery in an H3K9me3-dependent manner, and topologically associating domains (TADs), chromatin loops, and active-active compartment interactions are almost completely dismantled alongside reduced CTCF. How this reorganization is molecularly executed — beyond CTCF loss — and whether the resulting chromosome configuration is required for homologous chromosome pairing and meiotic entry is unknown. In vitro primordial germ cell-like cells (PGCLCs) fail to recapitulate the reorganization and remain meiosis-incompetent, suggesting the two failures are causally linked. The open problem is to identify the molecular cascade that executes the reorganization (including the contribution of cohesin dynamics such as elevated WAPL and altered subunit stoichiometry, and the tether that anchors H3K9me3-enriched pericentromeric heterochromatin to the nuclear periphery where canonical telomere-tethering factors MAJIN and TERB1 are lowly expressed), to test the functional requirement of chromosome separation and perinuclear centromere anchoring for efficient pairing and meiotic entry, and to determine why PGCLC systems fail to undergo this reorganization and whether restoring it confers meiotic competence.

Background

Primordial germ cells (PGCs), the embryonic precursors of oocytes and sperm, undergo a global epigenetic reset after they settle in the genital ridges: genome-wide DNA demethylation, remodeling of repressive histone modifications, and changes in nuclear morphology. Huang et al. (Nat Struct Mol Biol 33, 433–447, 2026; bioRxiv preprint 2025.10.09.681386) used immunofluorescence, DNA-FISH, electron microscopy and low-input Hi-C (a method that maps genome-wide chromatin contact frequencies) to show that premeiotic mouse gonadal germ cells at embryonic day 13.5 (E13.5) acquire a distinctive architecture: conventional chromocenters (clusters of pericentromeric heterochromatin, PCH, marked by histone H3 lysine 9 trimethylation, H3K9me3) dissolve, individual chromosomes separate and disentangle, centromeres anchor at the nuclear periphery, interchromosomal contacts drop, and all canonical levels of 3D genome organization weaken — topologically associating domains (TADs, self-interacting genomic intervals formed by cohesin-mediated loop extrusion and bounded by the architectural protein CTCF), discernible chromatin loops, and active-active compartment interactions. This occurs while CTCF levels fall, but the disruption exceeds what acute CTCF depletion causes in other systems, so additional mechanisms must contribute; the cohesin-release factor WAPL (which unloads cohesin from DNA and thereby dissolves loops) is upregulated in PGCs, and the stoichiometry of cohesin subunits (for example levels of RAD21, the kleisin subunit of the cohesin ring) is altered. Two results implicate H3K9 methylation in peripheral anchoring: the lamin B receptor (LBR, an inner nuclear membrane protein known from Solovei et al., Cell 152, 584–598, 2013 to tether peripheral heterochromatin together with lamins A/C) is strongly upregulated precisely when lamins A/C and B1 are reduced, and inhibiting H3K9 methyltransferases with chaetocin in cultured E13.5 gonads detaches centromeres from the periphery. The identity of the mammalian protein(s) that directly read H3K9me3 and anchor heterochromatin to the nuclear lamina in these cells (analogous to the CEC-4 reader in C. elegans) is not established, and LBR remains an untested candidate. The timing is puzzling: the canonical meiotic telomere-tethering machinery — the MAJIN–TERB1–TERB2 complex that attaches telomeres to the nuclear membrane during prophase I (Shibuya et al., Cell 163, 1252–1266, 2015) — is lowly or not expressed in premeiotic PGCs, so how telomeres and centromeres first attach to the nuclear envelope before meiosis is unclear. Related work at adjacent stages sharpens rather than answers these questions: Nagano et al. (Nat Struct Mol Biol 32, 2203–2218, 2025) showed that a mitotic cohesin complex containing STAG3 (the meiosis-type paralog of the SA1/SA2 cohesin subunit) with short chromatin residence time attenuates TADs in postnatal spermatogonia, and Cheng et al. (Nat Commun 17, 7550, 2026) mapped how meiotic loops anchored by CTCF form as cells pass meiotic entry — neither addresses the embryonic premeiotic PGC transition. Functionally, both sexes acquire this configuration before sex-specific divergence, and male germ cells at this stage are intrinsically poised to enter meiosis (exogenous retinoic acid or loss of the retinoic acid-degrading enzyme CYP26B1 triggers premature meiotic entry), leading Huang et al. to propose that the reorganization primes meiosis — a hypothesis not yet rigorously tested by perturbing the configuration and measuring homolog pairing and meiotic entry. Finally, PGC-like cells (PGCLCs) generated from pluripotent stem cells in vitro — the basis of reconstituted gametogenesis (Hayashi et al., Science 338, 971–975, 2012) — do not undergo the same architectural remodeling as in vivo PGCs (Nagano et al., EMBO J 41, e110600, 2022), retain chromocenters, and show variable centromere positioning; even with BMP2 and vitamin A supplementation or gonadal-tissue reconstitution, which induce synaptonemal complex formation (Hamada et al., PLoS Genet 16, e1008676, 2020), homolog pairing remains inefficient and meiotic defects frequent, pointing to an unknown in vivo prerequisite. A subset of human fetal germ cells shows the same perinuclear centromere anchoring and reduced CTCF/increased RAD21, but the timing, regulation and significance in human development are uncharacterized.

Problem Statement

For mammalian germ cells at the transition to gametogenesis, determine (i) the molecular cascade that executes the premeiotic 3D genome reorganization — in particular the contribution of cohesin dynamics (elevated WAPL, altered cohesin subunit stoichiometry) to the dissolution of TADs and loops beyond what reduced CTCF explains, and the simultaneous weakening of compartment interactions; (ii) the mechanism that attaches centromeres/telomeres to the nuclear envelope in premeiotic germ cells, where the canonical telomere-tethering factors MAJIN and TERB1 are lowly or not expressed, including the identity of the mammalian protein(s) that directly read H3K9me3 and anchor pericentromeric heterochromatin to the nuclear periphery; (iii) whether the resulting configuration — chromosome separation plus perinuclear centromere anchoring — is functionally required for efficient homologous chromosome pairing and entry into meiotic prophase; (iv) why in vitro PGCLC systems fail to recapitulate this reorganization, and whether that failure causally underlies their inability to undergo meiosis efficiently (i.e. whether restoring the in vivo configuration in PGCLCs confers meiotic competence); and (v) the timing, regulation and functional significance of perinuclear centromere anchoring in human fetal germ cell development.

Parts (i)–(iv) form the core of this open problem and are to be addressed together, with part (v) extending the same question to human fetal germ cells.

References

  1. Huang, T.-C., Rigau, M., Malysheva, V., Whildling, C., Siciliani, S., Li, J., Balaguer Balsells, I., Artemov, P., Dion, C., Spivakov, M., Vaquerizas, J.M. & Hajkova, P. Global reorganization of genome architecture at the transition to gametogenesis. Nature Structural & Molecular Biology 33, 433–447 (2026). doi:10.1038/s41594-026-01747-1, https://doi.org/10.1038/s41594-026-01747-1
  2. Huang, T.-C., Rigau, M., Malysheva, V., Whildling, C., Siciliani, S., Li, J., Balaguer Balsells, I., Artemov, P., Dion, C., Spivakov, M., Vaquerizas, J.M. & Hajkova, P. Global re-organisation of genome architecture at the transition to gametogenesis. bioRxiv preprint (2025). doi:10.1101/2025.10.09.681386, https://doi.org/10.1101/2025.10.09.681386
  3. Nagano, M., Hu, B., Yokobayashi, S., Yamamura, A., Umemura, F., Coradin, M., Ohta, H., Yabuta, Y., Ishikura, Y., Okamoto, I., Ikeda, H., Kawahira, N., Nosaka, Y., Shimizu, S., Kojima, Y., Mizuta, K., Kasahara, T., Imoto, Y., Meehan, K., Stocsits, R., Wutz, G., Hiraoka, Y., Murakawa, Y., Yamamoto, T., Tachibana, K., Peters, J.-M., Mirny, L.A., Garcia, B. & Saitou, M. Nucleome programming is required for the foundation of totipotency in mammalian germline development. The EMBO Journal 41, e110600 (2022). doi:10.15252/embj.2022110600, https://doi.org/10.15252/embj.2022110600
  4. Nagano, M., Hu, B., Ogata, K., Umemura, F., Ishikura, Y., Suzuki, S., Katsifis, C.C., Yoshinaga, M., Litos, G., Nagasaka, K., Tang, W., Nosaka, Y., Sasada, H., Wang, H., Kondo, D., Katou, Y., Mizuta, K., Yabuta, Y., Ohta, H., de Luna Vitorino, F.N., Arima, H., Ichikawa, T., Gabriele, M., Majewski, J., Garcia, B., Takeuchi, O., Yoshida, S., Hansen, A.S., Peters, J.-M., Ishihama, Y. & Saitou, M. The mitotic STAG3–cohesin complex shapes male germline nucleome. Nature Structural & Molecular Biology 32, 2203–2218 (2025). doi:10.1038/s41594-025-01647-w, https://doi.org/10.1038/s41594-025-01647-w
  5. Kitamura, Y., Takahashi, K., Maezawa, S., Munakata, Y., Sakashita, A., Katz, S.P., Kaplan, N. & Namekawa, S.H. CTCF-mediated 3D chromatin sets up the gene expression program in the male germline. Nature Structural & Molecular Biology 32, 1227–1240 (2025). doi:10.1038/s41594-025-01482-z, https://doi.org/10.1038/s41594-025-01482-z
  6. Cheng, G., Li, X., Brick, K., Alleva, B., Huang, M., Lam, K.G., Pratto, F. & Camerini-Otero, R.D. High-resolution chromatin mapping reveals that CTCF anchors meiotic loops to the chromosome axis. Nature Communications 17, 7550 (2026). doi:10.1038/s41467-026-73644-6, https://doi.org/10.1038/s41467-026-73644-6
  7. Zickler, D., Rodrigues Neves, A.R. & Kleckner, N. Cohesin release-factor Wapl modulates axis and chromatin structure to promote conjunction and separation of meiotic chromosomes. Cell Reports 45, 117776 (2026). doi:10.1016/j.celrep.2026.117776, https://doi.org/10.1016/j.celrep.2026.117776
  8. Shibuya, H., Hernández-Hernández, A., Morimoto, A., Negishi, L., Höög, C. & Watanabe, Y. MAJIN links telomeric DNA to the nuclear membrane by exchanging telomere cap. Cell 163, 1252–1266 (2015). doi:10.1016/j.cell.2015.10.030, https://doi.org/10.1016/j.cell.2015.10.030
  9. Solovei, I., Wang, A.S., Thanisch, K., Schmidt, C.S., Krebs, S., Zwerger, M., Cohen, T.V., Devys, D., Foisner, R., Peichl, L., Herrmann, H., Blum, H., Engelkamp, D., Stewart, C.L., Leonhardt, H. & Joffe, B. LBR and lamin A/C sequentially tether peripheral heterochromatin and inversely regulate differentiation. Cell 152, 584–598 (2013). doi:10.1016/j.cell.2013.01.009, https://doi.org/10.1016/j.cell.2013.01.009
  10. Hayashi, K., Ogushi, S., Kurimoto, K., Shimamoto, S., Ohta, H. & Saitou, M. Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 338, 971–975 (2012). doi:10.1126/science.1226889, https://doi.org/10.1126/science.1226889
  11. Hamada, N., Hamazaki, N., Shimamoto, S., Hikabe, O., Nagamatsu, G., Takada, Y., Kato, K. & Hayashi, K. Germ cell-intrinsic effects of sex chromosomes on early oocyte differentiation in mice. PLoS Genetics 16, e1008676 (2020). doi:10.1371/journal.pgen.1008676, https://doi.org/10.1371/journal.pgen.1008676
  12. Chang, Y.W., Trimp, M., van der Helm, T., Moustakas, I., Blanch-Asensio, A., Overeem, A.W. & Chuva de Sousa Lopes, S.M. Reconstitution of the cellular niche requirements for primordial germ cell-like cell progression in humans. Stem Cell Reports 21, 102826 (2026). doi:10.1016/j.stemcr.2026.102826, https://doi.org/10.1016/j.stemcr.2026.102826
  13. Sun, Q., Li, W., Meng, J., Zhang, Y., Bu, X., Huang, J., Qian, D., Li, N., Zhi, E., Tian, R., Huang, Y., Zhao, J., Zhao, F., Ru, Y., Liu, G., Li, Z. & Yao, C. Novel variants in LINC and TTM complexes of meiotic chromosome dynamics are associated with meiotic arrest and non-obstructive azoospermia. Journal of Human Genetics (2026). doi:10.1038/s10038-026-01492-9, https://doi.org/10.1038/s10038-026-01492-9