BackgroundDNA methylation (5mC) plays important roles in epigenetic regulation of genome function. Recently, TET hydroxylases have been found to oxidise 5mC to hydroxymethylcytosine (5hmC), formylcytosine (5fC) and carboxylcytosine (5caC) in DNA. These derivatives have a role in demethylation of DNA but in addition may have epigenetic signaling functions in their own right. A recent study identified proteins which showed preferential binding to 5-methylcytosine (5mC) and its oxidised forms, where readers for 5mC and 5hmC showed little overlap, and proteins bound to further oxidation forms were enriched for repair proteins and transcription regulators. We extend this study by using promoter sequences as baits and compare protein binding patterns to unmodified or modified cytosine using DNA from mouse embryonic stem cell extracts.ResultsWe compared protein enrichments from two DNA probes with different CpG composition and show that, whereas some of the enriched proteins show specificity to cytosine modifications, others are selective for both modification and target sequences. Only a few proteins were identified with a preference for 5hmC (such as RPL26, PRP8 and the DNA mismatch repair protein MHS6), but proteins with a strong preference for 5fC were more numerous, including transcriptional regulators (FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3), DNA repair factors (TDG and MPG) and chromatin regulators (EHMT1, L3MBTL2 and all components of the NuRD complex).Conclusions0ur screen has identified novel proteins that bind to 5fC in genomic sequences with different CpG composition and suggests they regulate transcription and chromatin, hence opening up functional investigations of 5fC readers.

5-Formylcytosine (5fC) is a rare base found in mammalian DNA and thought to be involved in active DNA demethylation. Here, we show that developmental dynamics of 5fC levels in mouse DNA differ from those of 5-hydroxymethylcytosine (5hmC), and using stable isotope labeling in vivo, we show that 5fC can be a stable DNA modification. These results suggest that 5fC has functional roles in DNA that go beyond being a demethylation intermediate.

Chromatin remodeling complexes evict, slide, insert or replace nucleosomes, which represent an intrinsic barrier for access to DNA. Remodelers function in most aspects of genome utilization including transcription factor (TF) binding, DNA replication and repair1,2. While they are frequently mutated in cancer3, it remains largely unclear how the four mammalian remodeler families (SWI/SNF, ISWI, CHD and INO80) orchestrate the global organization of nucleosomes. To gain insight into this matter, we generated viable embryonic stem cells that lack Snf2h, the ATPase of ISWI complexes, enabling to study its cellular function and contrast it to Brg1, the ATPase of SWI/SNF. Loss of Snf2h decreases nucleosomal phasing and increases linker lengths providing in vivo evidence for ISWI function in ruling nucleosomal spacing in mammals. Systematic analysis of transcription factor binding reveals their selective requirement on either remodeling activity. One group critically depends on Brg1 and contains the transcriptional repressor REST, while a non-overlapping set relies on Snf2h and includes the insulator protein CTCF. This selectivity readily explains why chromosomal folding and insulation of topologically associated domains requires Snf2h, but not Brg1. Collectively, this study shows that mammalian ISWI is critical for nucleosomal periodicity and nuclear organization and that transcription factors rely on specific remodeling pathways for proper genomic binding.

In the original version of the above article, a recent publication and its findings had not been acknowledged. The online and print versions have now been corrected, and the corrected sentence reads ''. but the importance of loss of H3K9me2 is not clear (Walter et al., 2016).'' The full citation to the reference has been added to the reference list: Walter, M., Teissandier, A., Pé rez-Palacios, R., Bourc'his, D. (2016). An epigenetic switch ensures transposon repression upon dynamic loss of DNA methylation in embryonic stem cells. Elife 5.

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