Abstract

DNA methylation is essential for mammalian embryonic development, yet its functional implications remain debated. Here, we introduce a quantitative model that reconciles conflicting perspectives on the roles of methylation in genome regulation and cell specification. Using single-cell multiome analysis in chimeric and whole embryo mutants during mouse gastrulation and organogenesis, we separate cell-autonomous from indirect effects of methylation and demethylation machinery. We show that while the transcriptional and chromatin programs defining basic lineages can be established independently of methylation, the fidelity of differentiated states is severely impaired in its absence. Specifically, we identify hundreds of genes and thousands of cis-regulatory elements (CREs) dependent on methylation for precise regulation. CRE accessibility alterations in methylation mutants are linked with CpG dinucleotide content, and correlate with multiple transcription factor binding motifs. Our data support a weak-interaction model in which DNA methylation moderates, but does not block or instruct the potency of trans-acting regulatory machineries genome-wide. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, https://ror.org/0472cxd90, ERC_CoG EmbryoCellEnsemble, ERC cells2Tissues Israel Science Foundation, https://ror.org/04sazxf24, 2824/24 Minerva Foundation, https://ror.org/0152xm391 European Molecular Biology Organization, ALTF 268-2018 National Natural Science Foundation of China, 32570691 Council for Higher Education