Base-Resolution and Single-Cell Analysis of Active DNA Demethylation Using Methylase-Assisted Bisulfite Sequencing
Abstract
In mammals, DNA methylation in the form of 5-methylcytosine (5mC) can be actively reversed to unmodified cytosine through ten-eleven translocation (TET) dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), followed by replication-dependent dilution or base excision repair. This process, known as active DNA demethylation, is present in many biological contexts including development and diseases. To investigate the mechanism and function of active DNA demethylation, various methods have been developed to analyze the genomic distribution of the demethylation intermediates 5hmC, 5fC and 5caC. However, previous methods suffer from limitations including low resolution, non-specificity and incompatibility with single-cell studies. To overcome these limitations, I developed methylase-assisted bisulfite sequencing (MAB-seq), a method capable of profiling 5fC and 5caC (5fC/5caC) at single-base resolution and to the single-cell level.In my dissertation, I present the development of MAB-seq method, its applications in different biological contexts, and the insights revealed by the studies. I first established MAB-seq and applied it to mouse embryonic stem cells, uncovering unique features of active DNA demethylation including the processivity of TET-mediated oxidation and its correlation with chromatin accessibility. I then further modified the protocol to make it compatible with low-input and single-cell studies, termed liMAB-seq and scMAB-seq, respectively. To demonstrate the application of liMAB-seq, I analyzed paternal genome demethylation in mouse zygotes, revealing the dynamics of 5mC/5hmC and 5fC/5caC at individual genomic regions. To demonstrate the utility of scMAB-seq, I applied the method to perform cell type classification, genomic mapping of sister chromatid exchange and reconstruction of cellular lineage during preimplantation development.
Taken together, this work not only establishes novel methods for studying active DNA demethylation at base-resolution and single-cell level, but also demonstrates the utilities of these methods in different biological contexts and provides insights into the demethylation process.
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