Monitoring Dynamics of DNA Methylation at Single-Cell Resolution during Development and Disease

DNA methylation is an epigenetic modification that is essential for normal mammalian development. Various technologies have been established to address the inherent challenge in reading the "second-dimension" epigenetic code. Development and improvement of next-generation sequencing methodologies, recently enabled genome-wide base-resolution methylomes of multiple tissues and cell types across different species. However, DNA methylation during development, lineage commitment and disease is dynamic and most methods that are currently used to study DNA methylation share three fundamental experimental constraints: (i) They preclude assessment of methylation heterogeneity at the single-cell level, while (ii) providing only a static “snapshot” view of methylation at a given cell state. (iii) Moreover, current methods do not allow the prospective isolation of cells with a given change in methylation, thus impeding mechanistic studies of methylation and gene regulation during cell fate changes. Taken together, a key challenge in the field is to generate tools that allow tracing dynamics of DNA methylation at single-cell resolution. We have recently established a reporter system that now allows monitoring real-time changes of DNA methylation in single cells, while facilitating prospective isolation of cells based on their methylation signature.

The reporter system was utilized to address a long-standing question in the field: are parent-specific methylation marks simply maintained or subjected to regulation during development and in postnatal animals? Our study identified striking regulation of parental imprinting following fertilization, resulting in tissue- and cell type-specific methylation patterns in adults. Interestingly, methylation changes were found to be highly dynamic in the adult brain, thus contributing to cellular epigenetic heterogeneity over time, with potential implications for aging. As prevalent belief posits that following fertilization, imprinted differentially methylated regions (DMRs) are mostly maintained in a passive fashion, our findings challenge the current dogma, supporting a dynamic role of DNA methylation in regulating imprinted DMRs during development. Collectively, these results substantiated the promise of the reporter system to be implemented in studying multiple biological systems, in the context of normal development as well as in disease.