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A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation

Mario Iurlaro1, Gabriella Ficz2*, David Oxley3, Eun-Ang Raiber4, Martin Bachman45, Michael J Booth4, Simon Andrews7, Shankar Balasubramanian456 and Wolf Reik189*

Author affiliations

1 Epigenetics Programme, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK

2 Centre for Haemato-Oncology, Barts Cancer Institute, Charterhouse Square, London EC1M 6BQ, UK

3 Proteomics Research Group, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK

4 Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK

5 Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Robinson way, Cambridge CB2 0RE, UK

6 School of Clinical Medicine, The University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 0SP, UK

7 Bioinformatics Group, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK

8 Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK

9 1Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK

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Citation and License

Genome Biology 2013, 14:R119  doi:10.1186/gb-2013-14-10-r119

Published: 24 October 2013

Abstract

Background

DNA 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.

Results

We 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).

Conclusions

0ur 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.