Extensive N4 cytosine methylation is essential for Marchantia sperm function

James Walker; Jingyi Zhang; Yalin Liu; Shujuan Xu; Yiming Yu; Martin Vickers; Weizhi Ouyang; Judit Tálas; Liam Dolan; Keiji Nakajima; Xiaoqi Feng

doi:10.1016/j.cell.2025.03.014

Extensive N4 cytosine methylation is essential for Marchantia sperm function

Cell. 2025 Apr 2:S0092-8674(25)00287-9. doi: 10.1016/j.cell.2025.03.014. Online ahead of print.

Authors

James Walker¹, Jingyi Zhang², Yalin Liu³, Shujuan Xu⁴, Yiming Yu⁴, Martin Vickers¹, Weizhi Ouyang⁴, Judit Tálas¹, Liam Dolan⁵, Keiji Nakajima⁶, Xiaoqi Feng⁷

Affiliations

¹ Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK.
² Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK; Guangdong Basic Research Center of Excellence for Precise Breeding of Future Crops, College of Agriculture, South China Agricultural University, Guangzhou 510642, China.
³ Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK; Guangdong Provincial Key Laboratory for the Development Biology and Environmental Adaptation of Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China.
⁴ Institute of Science and Technology Austria, Klosterneuburg 3400, Austria.
⁵ Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna 1030, Austria.
⁶ Nara Institute of Science and Technology, Nara 630-0192, Japan.
⁷ Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK; Institute of Science and Technology Austria, Klosterneuburg 3400, Austria. Electronic address: Xiaoqi.Feng@ista.ac.at.

PMID: 40209706
DOI: 10.1016/j.cell.2025.03.014

Abstract

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.

Keywords: 5-methylcytosine; DNA methylation reprogramming; DNA methyltransferases; Marchantia; N4-methylcytosine; gene transcription; sperm; spermatogenesis.