Combinatorial quantification of 5mC and 5hmC at individual CpG dyads and the transcriptome in single cells reveals modulators of DNA methylation maintenance fidelity.

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Inheritance of 5-methylcytosine from one cell generation to the next by DNA methyltransferase 1 (DNMT1) plays a key role in regulating cellular identity. While recent work has shown that the activity of DNMT1 is imprecise, it remains unclear how the fidelity of DNMT1 is tuned in different genomic and cell state contexts. Here we describe Dyad-seq, a method to quantify the genome-wide methylation status of cytosines at the resolution of individual CpG dinucleotides to find that the fidelity of DNMT1-mediated maintenance methylation is related to the local density of DNA methylation and the landscape of histone modifications. To gain deeper insights into methylation/demethylation turnover dynamics, we first extended Dyad-seq to quantify all combinations of 5-methylcytosine and 5-hydroxymethylcytosine at individual CpG dyads. Next, to understand how cell state transitions impact maintenance methylation, we scaled the method down to jointly profile genome-wide methylation levels, maintenance methylation fidelity and the transcriptome from single cells (scDyad&T-seq). Using scDyad&T-seq, we demonstrate that, while distinct cell states can substantially impact the activity of the maintenance methylation machinery, locally there exists an intrinsic relationship between DNA methylation density, histone modifications and DNMT1-mediated maintenance methylation fidelity that is independent of cell state.