Defossez, P. | Search bioRxiv

bioRxiv2026

DNA methylation links lagging strand replication to transposable element control

DNA methylation is an essential epigenetic mark that silences transposable elements (TEs) in mammalian genomes1,2. Following DNA replication, methylation patterns must be faithfully restored3,4, yet how the two processes are coordinated remains unclear. Here, using strand-specific, genome-wide analyses5-7 in mouse embryonic stem cells, we show that DNA methylation maintenance is coupled to the lagging-strand synthesis in TE-rich regions. Paradoxically, despite this targeting, the lagging strand is more permissive for TE integration than the leading strand. Notably, insertions of full-length LINE-1s, SINEs, and satellite repeats are all enriched on the lagging strand over evolutionary time. Consequently, most TEs, particularly young elements, are oriented head-on relative to replication forks in the mouse genome, creating an unfavorable genomic configuration8 that is preferentially targeted by DNA methylation maintenance. Mechanistically, DNA methylation maintenance is coupled to the lagging-strand replication via UHRF1-LIG19 and PCNA-PAF1510 interactions, and the interference in this mechanism slows Okazaki fragment maturation, and thereby potentially may facilitate TE retention. Together, we show a mechanism of TE control during DNA replication with an unexpected evolutionary interplay in which DNA methylation may facilitate, rather than solely prevent, TE expansion.

Molecular Biology↗

bioRxiv2026

Dual profiling of DNA modifications with enhancer features during the exit of naive pluripotency

Cis-regulatory elements, such as enhancers, play an essential role in coordinating gene expression programs during cellular transitions. As such, substantial efforts have been made to characterize enhancer elements, e.g., chromatin accessibility, transcription factor (TF) binding sites, histone post-translational modifications (PTMs), and 5-cytosine DNA methylation and hydroxymethylation (5mC and 5hmC). Elevated 5mC levels are typically correlated with the inactive enhancer state. However, whether 5mC precludes TF binding or is simply a downstream consequence of enhancer decommissioning is difficult to determine. Bulk genomics assays from cell populations fail to fully capture cellular heterogeneity, and single-cell assays often suffer from limited read counts per cell. Here, we leveraged tagmentation-based technologies to assess chromatin features with 5mC simultaneously: Methyl-ATAC and Methyl-CUT&Tag. In addition, we modified the technique to interrogate 5hmC dynamics (hM-ATAC). We employed these techniques during the exit of naive pluripotency in mouse embryonic stem cells (mESCs), which recapitulates the embryonic DNA methylation establishment program. This system withstands the complete absence of DNA methylation and demethylation machinery, allowing us to further dissect the temporal contributions of 5mC and 5hmC. Given the affordability of these techniques, we were able to obtain robust, dynamic single-molecule 5mC/5hmC information during this cellular transition for chromatin accessibility and histone marks in wild-type and mutant conditions. The sum of these data allowed for unprecedented insight into the role that 5mC turnover plays at enhancers during a key window of mammalian embryonic development.

Molecular Biology↗