An antagonistic epigenetic mechanism regulating gene expression in pollen revealed through single-nucleus multiomics [snmCT-seq]

Arabidopsis MBD5, MBD6, and MBD7 are CG-specific methyl-readers with opposite functions: MBD5 and MBD6 (MBD5/6) repress methylated loci in pollen vegetative nuclei (VN), while MBD7 prevents transgene silencing, possibly by promoting DNA demethylation. Here we show that loss of MBD7 rescues transcriptional defects at a large subset of MBD5/6-bound loci. Using simultaneous profiling of DNA methylation and transcription in single pollen nuclei, we found that MBD5/6-bound loci that are actively demethylated in the early post-mitotic VN lose additional methylation in mbd5/6, prior to transcriptional derepression. A subset of these loci is also bound by MBD7, correlating with demethylation and transcriptional derepression in mbd5/6 that are both reversed by loss of MBD7. Conversely, ectopically recruiting the MBD7 complex to MBD5/6 targets causes partial demethylation and upregulation. We propose that MBD5/6 maintain silencing in VN in part by preventing the MBD7 complex from enhancing the active demethylation that occurs during VN maturation. Overall design: Goal was to examine DNA methylation dynamics in different pollen nuclei types, and how these change in mbd5/6, mbd7, and mbd5/6/7 mutants. Three experiments were performed (jun2022, sep2022, mar2025). In experiments 1 and 2, 8 x 384-well plates containing 1 FACS-sorted pollen nucleus per well were prepared for each of the two genotypes (Col and mbd5/6) using the snmCT-seq protocol (Luo et al. 2022 Cell Genomics). Note that one plate failed in each of the two sep2022 samples; these are excluded. In experiment 3, 6 x 384 well plates were prepared for each of the four genotypes (Col, mbd7, mbd5/6, mbd5/6/7)investigated in this study. Barcodes for each well of the plate are provided in feature_README.txt.