Mapping an undulating, dynamic, and partially hidden landscape represents a vexing problem to say the least, especially when the landmarks of interest are rare DNA demethylation by-products in a single cell! This is the task faced by those intrepid explorers hoping to map the genome-wide location of 5-formylcytosine (5fC), a fleetingly rare oxidized form of 5-methylcytosine (5mC) whose excision completes the active DNA demethylation process.
In response, researchers from the laboratories of Fuchou Tang and Chengqi Yi (Peking University, Beijing, PRC) have described a CLEVER new technique to map the 5-formylcytosine landscape at single-base resolution in single cells: chemical-labeling-enabled C-to-T conversion sequencing.
CLEVER sequencing combines biocompatible chemical labeling of 5-formylcytosine, C-to-T conversion during amplification, and sequencing to map the genome-wide 5-formylcytosine landscape. As 5fC is indistinguishable from 5caC and regular cytosines during bisulfite treatment (normally used in associated detection techniques), the authors instead employed malononitrile for 5fC labeling and mapping.
So what did this CLEVER new technique tell the team about the 5-formylcytosine landscape in single pluripotent stem cells and early embryos?
- Mouse gametes, pre-implantation embryos, epiblast stem cells (mEpiSCs), and embryonic stem cells (mESCs) all displayed intrinsic heterogeneity in 5fC levels
- However, 5fC levels are highly dynamic and enriched in functionally important genomic regions
- In mEpiSCs and mESCs, 5fC displayed enrichment at exons, introns, 3’ untranslated regions, and transcriptional termination sites
- 5fC presence correlated with poised enhancer elements marked by H3K27ac in mESCs and two-cell embryos
- Analysis of mouse gametes and early embryos also suggested parent-specific 5fC dynamics during pre-implantation development
- Provides strong evidence of active DNA demethylation of both paternal and maternal genomes
- The production of 5fC correlated with 5mC DNA demethylation
- Integrated analysis demonstrated that promoter-specific 5fC production preceded the upregulation of critical developmental and metabolic gene expression
Single cells maps of the 5-formylcytosine landscape will help us to understand the epigenetics of pre-implantation development and will provide an important resource for functional studies of epigenetic reprogramming. Furthermore, the authors suggest that the combination of CLEVER sequencing with single-cell techniques for genome, transcriptome, and epigenome sequencing may move us a step forward in the quest for integrated profiling of the same single cell.
See how this CLEVER new technique helps to map the 5-formylcytosine landscape over at Cell Stem Cell, March 2017.