Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase

Abstract

Background: Methylation of cytosine in DNA (5mC) is an important epigenetic mark that is involved in the regulation of genome function. During early embryonic development in mammals, the methylation landscape is dynamically reprogrammed in part through active demethylation. Recent advances have identified key players involved in active demethylation pathways, including oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) by the TET enzymes, and excision of 5fC by the base excision repair enzyme thymine DNA glycosylase (TDG). Here, we provide the first genome-wide map of 5fC in mouse embryonic stem (ES) cells and evaluate potential roles for 5fC in differentiation.

Results: Our method exploits the unique reactivity of 5fC for pulldown and high-throughput sequencing. Genome-wide mapping revealed 5fC enrichment in CpG islands (CGIs) of promoters and exons. CGI promoters in which 5fC was relatively more enriched than 5mC or 5hmC corresponded to transcriptionally active genes. Accordingly, 5fC-rich promoters had elevated H3K4me3 levels, associated with active transcription, and were frequently bound by RNA polymerase II. TDG down-regulation led to 5fC accumulation in CGIs in ES cells, which correlates with increased methylation in these genomic regions during differentiation of ES cells in wild-type and TDG knockout contexts.

Conclusions: Collectively, our data suggest that 5fC plays a role in epigenetic reprogramming within specific genomic regions, which is controlled in part by TDG-mediated excision. Notably, 5fC excision in ES cells is necessary for the correct establishment of CGI methylation patterns during differentiation and hence for appropriate patterns of gene expression during development.

Figure 1

Comparison between the 5fC-antibody immunoprecipitation and chemical pulldown method. (a) For the 5fC DNA immunoprecipitation we used 1 pg of 5fC-103mer and 10 pg C-103mer in the presence of 5 µg salmon sperm DNA. The immunoprecipitation resulted in an enrichment of 1.6-fold of the 5fC-103mer over the C-103mer. Error bars represent the standard error of the mean. (b) Conditions for the biotinylation reaction of a 9-mer containing a single 5fC. The oligonucleotide was incubated at room temperature with an ARP in the presence of anisidine at pH 5 for 24 h and resulted in the formation of a single product. (c) Pulldown of 1 pg 5fC-biotin-103mer in the presence of 10 pg C-103mer and 5 µg salmon sperm DNA using streptavidin-coated magnetic beads resulted in an enrichment of the biotinylated DNA of around 1,000-fold. Error bars represent the standard error of the mean.

Figure 2

Genomic distribution of 5fC in comparison to 5hmC and 5mC. (a) Average enrichment (log2 of read count in pull down/read count in input) of 5fC, 5hmC, and 5mC in different genomic regions. 5fC and 5hmC followed similar distributions with high enrichments in CGIs, promoters and exons. Error bars are the mean of each replicate. (b) Distribution of 5fC, 5hmC and 5mC in the 5' UTR of retrotransposon elements LINE1 and in IAP LTR. Error bars are the mean of each replicate. (c) Genes were classified into CGI- and non-CGI-containing promoters and the normalized read count (reads per kilobase per million mapped reads (RPKM)) averaged across all genes. The profile of genes with CGI promoters showed high levels of 5hmC and 5fC close to the transcription start site (TSS), while an opposite trend was observed for 5mC. In contrast, genes with non-CGI promoters showed low levels of all three cytosine modifications in the promoter. The levels increased slightly near transcription initiation sites and then stayed constant throughout the gene body.

Figure 3

5fC is associated with active gene transcription. (a) Relationship between gene expression levels and cytosine modifications. The first three green bars show the gene expression levels of all genes (All), genes with CGI promoter (+ CpG) and genes with non-CGI promoters (- CpG). The subsequent bars are labeled with the notation 'x > y' (for example, mC > hmC) to indicate genes whose promoter CGI is relatively more enriched in one modification (for example, mC) than in the other (for example, hmC). The number below each label is the number of genes belonging to each category. See Table s5 in Additional file 1 for significance of difference between groups. The × symbols show the group means. In all the boxplots in this figure the whiskers extend up to 1.5 times the interquartile range and data points beyond this range have been omitted for clarity of presentation. (b) Genes are categorized into low (0 to 25%, white), medium (25 to 75%, grey) and high (75 to 100%, blue) expressed genes and correlated with 5fC levels of two biological replicates (normalized read counts). (c) Relationship between 5fC, 5hmC, 5mC and H3K4me3 and H3K27me3 at promoters. (d) Enrichments of cytosine modifications in the transcription factor binding sites of CTCF, p300 and Pol II over the input. RPKM, reads per kilobase per million mapped reads.

Figure 4

Effect of TDG knockdown on 5fC distribution. (a) Percentage change of annotated regions between TDG and siRNA control. Regions affected in the TDG KD (enriched in 5fC in TDG KD relative to the siRNA control) were mostly assigned to CGIs and exons. (b) CGI found in regions enriched in TDG knockout relative to siRNA control classified by genomic annotation.

Figure 5

Effect of TDG knockdown on 5fC distribution. (a) The ratio of methylation was plotted for TDG wild-type (WT) MEFs versus TDG± ES cells for the group of CGIs that show the highest 5fC enrichment in ES cells and a control group of all CGIs. The 5fC-enriched CGIs have higher methylation in the MEFs than the ES cells. (b) The ratio of methylation for WT MEFs (in red), or TDG KO MEFs (in blue), versus the ES cells. The 43 CGIs with the biggest increase in 5fC upon TDG KD have higher methylation in MEFs than ES cells and this increase is bigger in the absence of TDG. (c) Two example profiles (Hic1 and Csf2ra) show the overlap between 5fC CGI peaks in TDG KD ES cells and 5mC peaks at CGIs in TDG KO MEFs. The dotted grey line marks the transcription start site whereas the solid black lines represent CGIs. The shaded grey areas are the regions that are enriched for 5fC over control. RPKM, reads per kilobase per million mapped reads.