Abstract
The initiation of gene expression during development, known as zygotic genome activation (ZGA), is accompanied by massive changes in chromosome organization. However, the earliest events of chromosome folding and their functional roles remain unclear. Using Hi-C on zebrafish embryos, we discovered that chromosome folding begins early in development with the formation of fountains, distinct elements of chromosome organization. Emerging preferentially at enhancers, fountains show an initial accumulation of cohesin, which later redistributes to CTCF sites at TAD borders. Knockouts of pioneer transcription factors driving ZGA enhancers cause a specific loss of fountains, establishing a causal link between enhancer activation and fountain formation. Polymer simulations demonstrate that fountains may arise as sites of facilitated cohesin loading, requiring two-sided but desynchronized loop extrusion, potentially caused by cohesin collisions with obstacles or internal switching. Moreover, we detected cohesin-dependent fountain patterns at enhancers in mouse cells and found them reemerging with cohesin loading after mitosis. Altogether, fountains represent enhancer-specific elements of chromosome organization and suggest that chromosome folding during development and after cell division starts with facilitated cohesin loading. Observations in multiple systems further support facilitated loading at enhancers as a widespread phenomenon.
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Data availability
Raw and processed Hi-C data for zebrafish embryogenesis is available at GEO at (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE195609). The list of zebrafish fountains is available in Supplementary Dataset 1, Xenopus and medaka fountains in Supplementary Dataset 2, zebrafish TAD boundaries in Supplementary Dataset 3, zebrafish initiation zones at 4.3 hpf in Supplementary Dataset 4, and fountains group assignment based on chromatin accessibility change in mutants is available in Supplementary Dataset 5. The detailed list of the sequencing datasets is available in Supplementary Dataset 6. Additional datasets (including intermediary processed files) are available at OSF at (https://osf.io/mt4vf). Interactive HiGlass views are available at Resgen at (https://resgen.io/galitsyna/Zebrafish_embryogenesis/). Source data are provided with this paper.
Code availability
Fontanka tool for fountain calling (with examples) is available at (https://github.com/agalitsyna/fontanka) (v0.2, version available at https://doi.org/10.5281/zenodo.18018594). Simulations of facilitated loop extrusion are available at (https://github.com/agalitsyna/polychrom_workbench) under the directory targeted_extrusion. TAD calling for zebrafish is available at (https://github.com/encent/danio-2022). Code for reproducing the figures from the paper (main Figures and Supplementary Figs.) is available at (https://github.com/agalitsyna/embryonic-chromatin).
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Acknowledgements
We are grateful to Edward Banigan for proofreading the text and to all members of the Mirny lab for many productive discussions. We thank Damir Baranasic and Chris Sansam for sharing unpublished data. A.G. thanks Max Imakaev, Nezar Abdennur, and Anton Goloborodko for early ideas for the polymer simulations; Henrik Pinholt, Simon Grosse-Holz, and Emily Navarrete for ideas on the model and figures; Antoine Coulon, Mark Pownall, and Christopher Bohrer for the thorough discussions of the results and critical assessment of the hypotheses; students Alexey Shkolikov (FBB MSU) and Eli Rybnikova (PRIMES MIT) for their preliminary analysis of fountains. D.O. is grateful to Prof. Lev Yampolsky for the consultation about statistical analysis. This work was supported by DFG-ON86/5-1, DFG ON86/6-1 and DFG-EXC2189 for D.O., by NIH GM114190 to L.M., by RSF 23-14-00136 to M.S.G., and by RSF 21-64-00001-P to S.R.; A.G. and L.M. were also supported by NIH R01 AI164728 and NIH R01 NS113929.
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S.U.: Hi-C libraries; A.G.: Hi-C and genomic analysis, polymer simulations; M.B.: replication timing data analysis, N.B., K.P., M.S.G.: bioinformatics; S.U., M.V., M.G., D.O.: zebrafish mutants; D.O.: study design; S.R., D.O.: supervision of the wet part, funding acquisition; M.S.G., L.M.: supervision of Hi-C and genomic analysis and simulations. A.G. wrote the first draft, and S.U., S.R., L.M., and D.O. developed and edited the manuscript.
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Galitsyna, A., Ulianov, S.V., Bazarevich, M. et al. Extrusion fountains are hallmarks of chromosome organization emerging upon zygotic genome activation. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69105-9
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DOI: https://doi.org/10.1038/s41467-026-69105-9
