Why deposit 4mC in Marchantia sperm?
We see no evidence of dual-modified 4,5mC, suggesting that 5mC blocks 4mC. This creates a clear division: 4mC marks genes, 5mC marks repeats.
This allows global methylation for compaction while preserving the TE-specific 5mC signature.
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The motility defect is rescued by reintroducing wild-type MpDN4MT1a.
By contrast, sperm from global 5mC mutants show none of these distinctive phenotypes.
9/13

By contrast, transcripts for key sperm function genes—like CENTRIN1 and DYNEIN LIGHT CHAIN 7—are reduced, likely due to a dilution effect, helping explain the sperm motility defect in 4mC mutants.

7/13

The MpDN4MT1 gene appears to have originated via horizontal gene transfer from prokaryotes and has been retained for at least 200 million years— we detect it even in Lunularia cruciata, a liverwort from a distinct lineage to Marchantia.
4/13

We show that MpDN4MT1a, a eukaryotic homolog of bacterial 4mC methyltransferases, is the enzyme required for this modification.
Loss of MpDN4MT1a abolishes 4mC, and reintroduction of wild-type—but not the catalytic mutant—restores it.
3/13

In our new paper, we use six methods to unambiguously validate the presence of extensive 4mC at CG sites across genic regions in mature sperm:
🧪 immunodot blot
🧪 LC-MS
🧬 Bisulfite-seq
🧬 SMRT-seq
🧬 4mC-TAB-seq
🧬 4mC-AMD-seq
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