A Sensitive Approach to Map Genome-wide 5-Hydroxymethylcytosine and 5-Formylcytosine at Single-Base Resolution

Abstract

Mapping genome-wide 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) at single-base resolution is important to understand their biological functions. We present a cost-efficient mapping method that combines 5hmC-specific restriction enzyme PvuRts1I with a 5hmC chemical labeling enrichment method. The sensitive method enables detection of low-abundance 5hmC sites, providing a more complete 5hmC landscape than available bisulfite-based methods. This method generated a genome-wide 5fC map at single-base resolution. Parallel analyses revealed that 5hmC and 5fC in non-CpG context exhibit lower abundance, more dynamically, than those in CpG context. In the genic region, distribution of 5hmCpG and 5fCpG differed from 5hmCH and 5fCH (H = A, T, C). 5hmC and 5fC were distributed distinctly at regulatory protein-DNA binding sites, depleted in permissive transcription factor binding sites, and enriched at active and poised enhancers. This sensitive bisulfite conversion-free method can be applied to biological samples with limited starting material or low-abundance cytosine modifications.

Simultaneous mapping of active DNA demethylation and sister chromatid exchange in single cells

“…Consistent with previous reports, 5fC/5caC-modified regions called in Tdg-depleted ES cells are enriched at enhancers, H3K4me1 ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) peaks, and 5hmC/5fC/5caC DIP-seq (DNA immunoprecipitation combined with sequencing) peaks (Supplemental Fig. S1G; Shen et al 2013;Song et al 2013;Lu et al 2015;Sun et al 2015;Xia et al 2015). Among the regions covered by RRBS strategy and overlapping with 5fC/5caC DIP-seq peaks, 27.1% were called as modified in Tdg-depleted cells, while only 1.0% were wrongly called as modified in the Tet triple-knockout-negative control, further confirming the effectiveness of our approach in distinguishing real 5fC/5caC signals from background.…”

“…To understand the mechanism and function of active DNA demethylation, various methods have been developed to map the genomic distribution of 5fC and 5caC (Raiber et al 2012;Shen et al 2013;Song et al 2013;Booth et al 2014;Lu et al 2015;Sun et al 2015;Xia et al 2015). However, the majority of these methods requires a large amount of input DNA (typically hundreds of nanograms or more, corresponding to several hundred thousand cells) and thus cannot be readily applied to biological processes with limited cell availability, such as mammalian preimplantation development and PGC development.…”

5-hydroxymethylcytosine accumulation in postmitotic neurons results in functional demethylation of expressed genes

“…Our data demonstrate that 5hmCH (0.57%) occurs at reduced but substantial levels in granule cell genomes relative to 5mCH (0.94%). Although these values are higher than the levels of 5hmCA determined using Tet-assisted bisulfite sequencing to study 5hmC in neurons in the prefrontal cortex (10), the levels of 5hmC detection by oxBS-Seq are consistent with recent studies that used Pvu-Seal-Seq to determine that ∼24% of 5hmC in mouse embryonic stem cells occurs in the non-CpG context (23,24).…”

Glucosylation Mediated Rolling Circle Amplification Combined with a qPCR Assay for the Detection of 5-Hydroxymethylcytosine

“…Several methods have been developed to detect the abundance and distribution of 5hmC within the genome, such as affinitybased high-throughput sequencing, 11,12 restriction endonucleasemediated detection methods 13,14 and chemical single base conversion. [15][16][17] Among these methods, the selective glucosylation of 5hmC residues by T4 phage β-glucosyltransferase (T4 βGT) is widely used as a pretreatment step to detect 5hmC.…”

“…What is more, the proposed method is not limited to specific sites, like the endonuclease restriction method. 13,14 This RCA-qPCR assay could potentially be used in the fast and sensitive detection of 5hmC in targeted genes and DNA fragments isolated from cells or tissues.…”