Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells

Abstract

5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals1–5. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC)1,2,4–7. 5hmC and TET proteins have been implicated in stem cell biology and cancer1,4,5,8,9, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS5. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the ‘poised’ chromatin signature found at developmentally-regulated genes in ES cells.

Active DNA demethylation by oxidation and repair

“…npg TET1 plays a role in recruiting PRC2 [17]. Consistent with a role for TET1 in gene regulation, TET1 binding sites are enriched with the repressive chromatin marker H3K27me3, the active marker H3K4me3, or both [16,17,19]. TET1 protein contains a CXXC motif that can bind to unmodified C-or 5mC-and 5hmC-modified CpG-rich DNA [16].…”

“…One prediction is that TET proteins may activate the expression of their targeted genes via their role in active DNA demethylation. However, genome-wide analysis of 5hmC distribution reveals that TET1 has dual functions in transcriptional regulation because it both enhances and inhibits the expression of some genes in mESCs [16][17][18][19]. TET1 is highly expressed in mESCs that accumulate relatively high levels of 5hmC, while 5hmC levels are significantly decreased after mESC differentiation [7].…”

“…These results suggest a dynamic regulation of 5hmC by TET proteins [16][17][18][19]. Using various chromatin immunoprecipitation methods combined with high-throughput DNA sequencing, several laboratories analyzed the genome-wide distribution of 5hmC and TET1 binding sites [16][17][18][19]. Interestingly, TET1 is preferentially bound to CpG-rich regions at transcriptional start sites in promoters of both transcriptionally active and Polycomb-targeted repressive genes and also within exons [16][17][18].…”

“…Downregulation of TET proteins by RNAi decreases the accumulation of 5hmC but increases 5mC. These results suggest a dynamic regulation of 5hmC by TET proteins [16][17][18][19]. Using various chromatin immunoprecipitation methods combined with high-throughput DNA sequencing, several laboratories analyzed the genome-wide distribution of 5hmC and TET1 binding sites [16][17][18][19].…”

Dynamics of 5-hydroxymethylcytosine during mouse spermatogenesis

“…Indeed, two recent studies showed that two methyl CpG binding proteins, MeCP2 and Mbd3, preferentially bind to 5hmC and may have important roles in transcription regulation 20,21 . Previous studies indicate that 5hmC is enriched in the exons of highly expressed genes in brain tissues, and in cis-regulatory elements such as active enhancers, insulatorbinding sites in ESCs [22][23][24][25][26][27][28] . Enrichment of 5hmC is also found in the promoter region of polycomb-repressed 'poised' genes in ESCs [23][24][25]27 , which disappeared in the adult brain 29 .…”