article published yes James Walker author Jingyi Zhang author Yalin Liu author Shujuan Xu author 9724dd9d-f591-11ee-bd51-e97ed0652286 Yiming Yu author 318e643b-8b61-11ed-b69e-aafa103ec8dd0000-0002-9919-7282 Martin Vickers author Weizhi Ouyang author fec73395-8b60-11ed-b69e-927fda99c743 Judit Tálas author Liam Dolan author Keiji Nakajima author Xiaoqi Feng author e0164712-22ee-11ed-b12a-d80fcdf359580000-0002-4008-1234 XiFe department Establishment, modulation and inheritance of sexual lineage specific DNA methylation in plants project N4-methylcytosine (4mC) is an important DNA modification in prokaryotes, but its relevance and even its presence in eukaryotes have been mysterious. Here we show that spermatogenesis in the liverwort Marchantia polymorpha involves two waves of extensive DNA methylation reprogramming. First, 5-methylcytosine (5mC) expands from transposons to the entire genome. Notably, the second wave installs 4mC throughout genic regions, covering over 50% of CG sites in sperm. 4mC requires a methyltransferase (MpDN4MT1a) that is specifically expressed during late spermiogenesis. Deletion of MpDN4MT1a alters the sperm transcriptome, causes sperm swimming and fertility defects, and impairs post-fertilization development. Our results reveal extensive 4mC in a eukaryote, identify a family of eukaryotic methyltransferases, and elucidate the biological functions of 4mC in reproductive development, thereby expanding the repertoire of functional eukaryotic DNA modifications. https://research-explorer.ista.ac.at/download/19602/20871/2025_Cell_Walker.pdf application/pdfno https://creativecommons.org/licenses/by/4.0/ Elsevier2025 eng 0092-8674 1097-4172 40209706 00150474480000610.1016/j.cell.2025.03.014 188112890-2906.e14 Walker J, Zhang J, Liu Y, et al. Extensive N4 cytosine methylation is essential for Marchantia sperm function. <i>Cell</i>. 2025;188(11):2890-2906.e14. doi:<a href="https://doi.org/10.1016/j.cell.2025.03.014">10.1016/j.cell.2025.03.014</a> Walker, J., Zhang, J., Liu, Y., Xu, S., Yu, Y., Vickers, M., … Feng, X. (2025). Extensive N4 cytosine methylation is essential for Marchantia sperm function. <i>Cell</i>. Elsevier. <a href="https://doi.org/10.1016/j.cell.2025.03.014">https://doi.org/10.1016/j.cell.2025.03.014</a> J. Walker, J. Zhang, Y. Liu, S. Xu, Y. Yu, M. Vickers, W. Ouyang, J. Tálas, L. Dolan, K. Nakajima, X. Feng, Cell 188 (2025) 2890–2906.e14. Walker, James, Jingyi Zhang, Yalin Liu, Shujuan Xu, Yiming Yu, Martin Vickers, Weizhi Ouyang, et al. “Extensive N4 Cytosine Methylation Is Essential for Marchantia Sperm Function.” <i>Cell</i>. Elsevier, 2025. <a href="https://doi.org/10.1016/j.cell.2025.03.014">https://doi.org/10.1016/j.cell.2025.03.014</a>. Walker, James, et al. “Extensive N4 Cytosine Methylation Is Essential for Marchantia Sperm Function.” <i>Cell</i>, vol. 188, no. 11, Elsevier, 2025, p. 2890–2906.e14, doi:<a href="https://doi.org/10.1016/j.cell.2025.03.014">10.1016/j.cell.2025.03.014</a>. Walker J, Zhang J, Liu Y, Xu S, Yu Y, Vickers M, Ouyang W, Tálas J, Dolan L, Nakajima K, Feng X. 2025. Extensive N4 cytosine methylation is essential for Marchantia sperm function. Cell. 188(11), 2890–2906.e14. J. Walker <i>et al.</i>, “Extensive N4 cytosine methylation is essential for Marchantia sperm function,” <i>Cell</i>, vol. 188, no. 11. Elsevier, p. 2890–2906.e14, 2025. 196022025-04-20T22:01:28Z2025-12-29T13:41:13Z