Extensive N4 cytosine methylation is essential for Marchantia sperm function
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| English
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Author
Walker, James;
Zhang, Jingyi;
Liu, Yalin;
Xu, ShujuanISTA;
Yu, YimingISTA;
Vickers, Martin;
Ouyang, WeizhiISTA;
Tálas, Judit;
Dolan, Liam;
Nakajima, Keiji;
Feng, XiaoqiISTA 
Corresponding author has ISTA affiliation
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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.
Publishing Year
Date Published
2025-04-09
Journal Title
Cell
Publisher
Elsevier
Acknowledgement
We thank Sir Richard Roberts (NEB) for the kind gift of anti-4mC antibodies. We are also grateful to the JIC Small Molecule Mass Spectrometry (Lionel Hill) and Chemistry (Martin Rejzek) platforms as well as the High Resolution Metabolomics Laboratory (Manfred Beckmann, Aberystwyth University) for their assistance with LC-MS. Additionally, we acknowledge the assistance of the JIC Bioimaging Facility and ISTA Imaging and Optics Facility for microscopy. Finally, we appreciate the High Performance Computing resources provided by the ISTA Scientific Computing Facility and Norwich BioScience Institute Partnership Computing Infrastructure. This work was funded by a Sainsbury Charitable Foundation studentship (J.W.), a UKRI-BBSRC Doctoral Training Partnerships studentship (BBT0087171 to J.T.), a European Research Council Starting Grant (“SexMeth” 804981 to J.W., S.X., and X.F.), two Biotechnology and Biological Sciences Research Council (BBSRC) grants (BBS0096201 and BBP0135111 to J.Z., M.V., and X.F.), an EMBO Long Term Fellowship (Y.L.), an ISTA Bridge Fellowship (S.X.), and ISTA core funding (Y.Y. and X.F.).
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