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.