Single-Cell 5-Formylcytosine Landscapes of Mammalian Early Embryos and ESCs at Single-Base Resolution

Abstract

Active DNA demethylation in mammals involves ten-eleven translocation (TET) family protein-mediated oxidation of 5-methylcytosine (5mC). However, base-resolution landscapes of 5-formylcytosine (5fC) (an oxidized derivative of 5mC) at the single-cell level remain unexplored. Here, we present "CLEVER-seq" (chemical-labeling-enabled C-to-T conversion sequencing), which is a single-cell, single-base resolution 5fC-sequencing technology, based on biocompatible, selective chemical labeling of 5fC and subsequent C-to-T conversion during amplification and sequencing. CLEVER-seq shows intrinsic 5fC heterogeneity in mouse early embryos, Epi stem cells (EpiSCs), and embryonic stem cells (ESCs). CLEVER-seq of mouse early embryos also reveals the highly patterned genomic distribution and parental-specific dynamics of 5fC during mouse early pre-implantation development. Integrated analysis demonstrates that promoter 5fC production precedes the expression upregulation of a clear set of developmentally and metabolically critical genes. Collectively, our work reveals the dynamics of active DNA demethylation during mouse pre-implantation development and provides an important resource for further functional studies of epigenetic reprogramming in single cells.

Two are better than one: HPoxBS - hairpin oxidative bisulfite sequencing

“…to understand and model the dynamic loss and gain of DNA methylation in non dividing aging cells, to characterise the heterogeneity of epihaplotypes (epigenetic chromosomal patterns) and most importantly to understand changes occurring during development and differentiation with and without DNA replication. Ultimately, in combination with new detection methods, our pipeline could easily be adjusted to likewise, describe the distribution and the behavior 5fC or 5caC (38–40,50,51). …”

Chemoselective labeling and site‐specific mapping of 5‐formylcytosine as a cellular nucleic acid modification

“…A similar bisulfite-free approach for single-cell and single-base resolution sequencing of 5fC, likewise based on the principle of the Friedl€ ander reaction, has recently been reported by Zhu et al [90]. In this approach, named CLEVER-Seq (Fig.…”

DNA methylation in epigenetic inheritance of metabolic diseases through the male germ line

“…It is now clear, however, that the paternal genome demethylation employs both a rapidly acting active demethylation mechanism dependent on TET3 activity and a passive replication-dependent demethylation mechanism , Oswald et al 2000, Guo et al 2014, Shen et al 2014. Notably, the decline in 5mC in the paternally derived genome is accompanied by a prominent increase in 5hmC and less prominent increases in 5fC and 5caC (Gu et al 2011, Inoue & Zhang 2011, Iqbal et al 2011, Wossidlo et al 2011, Zhu et al 2017. TET3 is also present in the maternal pronucleus, but 5mC is largely protected from active demethylation (Guo et al 2014, Peat et al 2014, Shen et al 2014, Wang et al 2014, Zhu et al 2017.…”

“…Notably, the decline in 5mC in the paternally derived genome is accompanied by a prominent increase in 5hmC and less prominent increases in 5fC and 5caC (Gu et al 2011, Inoue & Zhang 2011, Iqbal et al 2011, Wossidlo et al 2011, Zhu et al 2017. TET3 is also present in the maternal pronucleus, but 5mC is largely protected from active demethylation (Guo et al 2014, Peat et al 2014, Shen et al 2014, Wang et al 2014, Zhu et al 2017. The PGC7 (also called STELLA or DPPA3) protein, which is genomically recruited through H3K9me2, is a candidate to mediate protection of 5mC in the maternal genome against active demethylation (Nakamura et al 2007, 2012, Wossidlo et al 2011, Bian & Yu 2014.…”