Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase.
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.
Eun-Ang
EA
Raiber
Dario
D
Beraldi
Gabriella
G
Ficz
Heather E
HE
Burgess
Miguel R
MR
Branco
Pierre
P
Murat
David
D
Oxley
Michael J
MJ
Booth
Wolf
W
Reik
Shankar
S
Balasubramanian
D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D002874:Chromosome Mapping; D019295:Computational Biology; D018899:CpG Islands; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D049308:Dioxygenases; D015536:Down-Regulation; D053595:Embryonic Stem Cells / Q000378:metabolism*; D044127:Epigenesis, Genetic; D018507:Gene Expression Regulation, Developmental; D016678:Genome; D059014:High-Throughput Nucleotide Sequencing; D051379:Mice; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D044502:Thymine DNA Glycosylase / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
C560973:5-formylcytosine; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; C540839:TET1 protein, mouse; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D049308:Dioxygenases; C551914:Tet2 protein, mouse; D044502:Thymine DNA Glycosylase