Genome Replication As Origin of Regulatory Variation and Epigenetic Drift

Molecular heterogeneity is emerging as a critical feature of multicellular life. While single-cell analyses have revealed the existence of cell-to-cell variation in the levels and activities of the molecules responsible for gene regulation, the source of such variation is still poorly understood. Cytosine methylation is a highly conserved epigenetic modification that plays an important role in mammalian development and its occurrence within phenotypically uniform cell populations is often variable even at the same genomic location. We recently developed a new sequencing method (Repli-BS) that enables analysis of methylation heterogeneity across cytosine residues within newly replicated strands of DNA over time. Using this method, we discovered that much of the methylation heterogeneity observed within HUES64 human embryonic stem cells (hESCs) is temporal in nature and associated with DNA replication (Charlton et al., Nat. Struc. Mol. Bio. 2018). More recently, we used our hESC Repli-BS dataset to establish kinetic rate parameters that numerically reflect the “speed” at which individual cytosines achieve steady-state methylation levels after being replicated (Busto-Moner et al., Plos Comp. Bio. 2020). Here, we employ bioinformatic analyses to explore how properties of post-replication DNA methylation dynamics relate to well-established features of the genome and the broader chromatin landscape. Our preliminary findings reveal that unique patterns of methylome replication associate with distal regulatory regions throughout the genome, enrich for cytosine residues dynamically methylated between stem cells and germ layers, and coincide with the location of stem cell-specific transcription factor binding and chromatin architectures. We also find correlations between sub-cell cycle kinetics in DNA methylation and the divergence of bulk methylation patterns observed during multiple cell generations and natural aging. Taken together, our studies suggest that (epi)genome replication may act as an important source of (temporal) regulatory variation in hESCs while, simultaneously, conferring susceptibility to epigenetic drift throughout the human lifespan.