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. 2013;14(10):R119.
doi: 10.1186/gb-2013-14-10-r119.

V体育官网入口 - A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation

Mario Iurlaro, Gabriella Ficz, David Oxley, Eun-Ang Raiber, Martin Bachman, Michael J Booth, Simon Andrews, Shankar Balasubramanian, Wolf Reik

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

Mario Iurlaro et al. Genome Biol. 2013.

Abstract (VSports在线直播)

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 VSports手机版. 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 V体育安卓版. 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: Our 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. V体育ios版.

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Figures

Figure 1
Figure 1
A mass spectrometry-based method for detection of 5-formylcytosine binding proteins. (a) Schematic representation of the pull-down method. DNA oligonucleotides corresponding to the promoter regions of the Pax6 (280 bp) and Fgf15 (248 bp) genes were obtained by PCR with biotinylated primers and using dATP, dGTP. dTTP and either dCTP, dmCTP, dhmCTP or dfCTP. DNA was then incubated with Streptavidin-linked beads and with nuclear extract from mouse ES cells. Bound fraction was then eluted and analysed by mass spectrometry. (b) Western blot showing presence of UHRF1 in the protein fraction captured by methylated and hydroxymethylated probes (both Fgf15 and Pax6) compared to umodified DNA. (c, d) Venn diagrams and histograms showing distribution of significantly enriched proteins binding to differentially modified Fgf15 probe (CpG: 14; non-CpG: 69, %CpGs: 11.3%) and Pax6 probe (CpG: 8; non-CpG: 44; %CpG: 5.7%) with schematic representation of their genomic position.
Figure 2
Figure 2
5-formylcytosine specific binders to Fgf15 probe are enriched for transcription factors and chromatin regulators. (a) Heatmap representation of the relative protein enrichment on the Fgf15 probe. Spectral count values for each replicate were analysed by testing the sample groups using a non-parametric Kruskal-Wallis t-test with a P value cutoff of 0.1. For heatmap display, additional filters for the size of absolute change between group means were applied, and the data for each gene were normalised by subtracting the median value for that gene across all experiments from the individual values. A cartoon highlights presence of all the component of the main core of the NuRD complex among the 5fC binders. (b) Functional annotation enrichment analysis performed on 5fC binders using DAVID shows enrichment for transcription (mainly zinc-binding factors) and chromatin regulators. Results are expressed with their corresponding Benjamini-corrected P value.
Figure 3
Figure 3
Relative protein enrichment in pull-downs with the Pax6 probe. Heatmap representation of the relative protein enrichment on the Pax6 probe. Spectral count values for each replicate were analysed by testing the sample groups using a non-parametric Kruskal-Wallis t-test with a P value cutoff of 0.1. For Heatmap display, additional filters for the size of absolute change between group means were applied, and the data for each gene were normalised by subtracting the median value for that gene across all experiments from the individual values.
Figure 4
Figure 4
Validation and functional analysis of 5fC binding proteins. (a) Venn diagram illustrating overlap between 5fC specific binders identified by the two different probes used. (b) ELISA assays performed with purified recombinant MPG, L3MBTL2 and ZSCAN21 proteins and differentially modified Fgf15 probe (blue = unmodified DNA; yellow = methylated DNA; green = hydroxymethylated DNA; red = formylated DNA). MPG (specifically bound to 5fC on both probes) shows strong selective binding for formylated DNA (Kd = 13.4 ±1.4 nM). L3MBTL2 (Kd = 37.1 ±5.6 nM for 5fC and Kd = 81.2 ±18.8 nM for C) and ZSCAN21 show preference of binding. This could reflect the difference in DNA interaction between an enzyme and transcriptional regulators. (c) Mass spectrometry analysis of global 5-formylcytosine (red bars) and 5-carboxycytosine (grey bars) levels in J1 ES cells after three rounds of knockdown of potential 5fC binders, compared to cells transfected with non-targeting siRNA. Bars show average of four biological replicates with corresponding standard deviation, expressed as the number of modified cytosines per million of all cytosines. Dotted line indicates the limit of accurate quantification.
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References

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