Response to mackay et Al.

Mackay et al. have raised some important questions regarding the value of RH-containing Zn finger (ZF) proteins in predicting preferential interaction with methylated DNA bases (5mC or T) [1xIs there a telltale RH ﬁngerprint in zinc ﬁngers that recognize methylated CpG dinucleotides?. Mackay, J.P. et al. Trends Biochem. Sci. 2013; 38: 421–422Abstract | Full Text | Full Text PDF | PubMed | Scopus (1)See all References[1].With respect to the presence of RH-containing ZF proteins in organisms lacking DNA methylation, given the established role of these proteins in recognizing TpG motifs, we would only expect to find them at diminished levels in organisms such as Caenorhabditis elegans, Drosophila melanogaster, and Ciona intestinalis if the missing 5mCpG motifs represent a large fraction of the total XpG motifs (where X is either T or 5mC). According to relative dinucleotide frequencies (adapted from Table 1 in [2xGenome-scale relationships between cytosine methylation and dinucleotide abundances in animals. Simmen, M.W. Genomics. 2008; 92: 33–40Crossref | PubMed | Scopus (37)See all References[2]), XpG in the CpG-methylated genomes of Homo, Pan, Mus, Danio, and Takifugu genera is 15.7 ± 0.9% of all dinucleotides, whereas TpG|CpA alone in the CpG-nonmethylated genomes of Ciona, Drosophila, Anopheles, and Caenorhabditis genera is 11.3 ± 0.2% (mean ± SD). For the sake of argument, if XpG-binding ZF proteins varied in direct proportion to the number of potential targets, we would expect nonmethylators to have a decrease of just 28% in such proteins. Neither we nor, we assume, Mackay et al. [1xIs there a telltale RH ﬁngerprint in zinc ﬁngers that recognize methylated CpG dinucleotides?. Mackay, J.P. et al. Trends Biochem. Sci. 2013; 38: 421–422Abstract | Full Text | Full Text PDF | PubMed | Scopus (1)See all References[1] would predict such direct proportionality, and the data they present in Figure 1C indicate that such comparisons may have limited value. This is a case in which positive data (a significant relationship between DNA methylation and composition at the ZF +6 position) would suggest something, but the lack of a relationship does not.Of greater relevance is the evidence that ZF proteins containing an RH motif in positions 6–7 bind to sequences lacking a methylated base. They refer to ten Zn fingers with RH motifs, complexed with DNA, for which structural data are available, and note that three bind TpG (consistent with our model), two (GAGA and WT1 ZF4) bind unmethylated CpG (although the binding of methylated CpG was not tested [3xThe solution structure of a specific GAGA factor–DNA complex reveals a modular binding mode. Omichinski, J.G. et al. Nat. Struct. Biol. 1997; 4: 122–132Crossref | PubMed | Scopus (146)See all References, 4xStructure of the Wilms tumor suppressor protein zinc finger domain bound to DNA. Stoll, R. et al. J. Mol. Biol. 2007; 372: 1227–1245Crossref | PubMed | Scopus (51)See all References]), four bind GpG (one of them, KLF4, binds methylated DNA, as recently shown [5xDynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Spruijt, C.G. et al. Cell. 2013; 152: 1146–1159Abstract | Full Text | Full Text PDF | PubMed | Scopus (282)See all References[5]), and one binds ApG. For the ApG binder (Zif268 ZF3), the Ade is outside of the DNA recognition site and thus it is unknown whether Zif268 will bind (5mC/T)pG better than ApG. They did not include any of the 5mCpG-binding complexes to which we referred, but the substantial issue is the binding of these RH-containing ZFs to GpG. They argue that R+6 specifies binding to NpG (where N can be any DNA base), whereas we presented data indicating that the bifurcated interaction between R and Gua anchors an additional hydrophobic/van der Waals interaction with methyl groups on the 5-carbon of an adjacent (5′) pyrimidine. The presence of such Arg–methyl interactions seems clear from the data we summarized; the question is the extent to which the additional interaction provides specificity.We noted that for each triad of 5mC-Arg-Gua in MBD1 (a non-ZF protein), the binding energy difference calculated between methylated and non-methylated CpG is approximately 0.8 ± 0.4 kcal/mol [6xRecognition of methylated DNA through methyl-CpG binding domain proteins. Zou, X. et al. Nucleic Acids Res. 2012; 40: 2747–2758Crossref | PubMed | Scopus (36)See all References[6], within the general range of van der Waals contributions (∼0.1–1.0 kcal/mol). Thus, the discriminatory effect from this interaction would not be expected to exclude binding to non-methylated bases (which occurs with a KD value of 150 nM in the case of Zfp57), but would favor binding to methylated bases (by a factor of 10 in the case of Zfp57 [7xAn atomic model of Zfp57 recognition of CpG methylation within a specific DNA sequence. Liu, Y. et al. Genes Dev. 2012; 26: 2374–2379Crossref | PubMed | Scopus (38)See all References[7]). In addition, we provided evidence that a single spatially conserved glutamate in Zfp57 and Kaiso also contribute to the preferred binding of 5mC [7xAn atomic model of Zfp57 recognition of CpG methylation within a specific DNA sequence. Liu, Y. et al. Genes Dev. 2012; 26: 2374–2379Crossref | PubMed | Scopus (38)See all References, 8xA common mode of recognition for methylated CpG. Liu, Y. et al. Trends Biochem. Sci. 2013; 38: 177–183Abstract | Full Text | Full Text PDF | PubMed | Scopus (22)See all References]. On top of this, multi-finger proteins are highly cooperative, so the suboptimal interaction of one finger could be compensated by other fingers (in the case of Kaiso, the RH motif and the glutamate are from two neighboring ZFs [9xMolecular basis for recognition of methylated and specific DNA sequences by the zinc finger protein Kaiso. Buck-Koehntop, B.A. et al. Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 15229–15234Crossref | PubMed | Scopus (36)See all References[9]), and the overall effect would vary with the protein and target sequence combinations.We therefore remain comfortable with the hypothesis, as a promising basis for specific testing, that RH motifs in ZF proteins confer a preference for methylated bases (5mC or T) at the 5′ position in NpG dinucleotides. Having noted this, Mackay et al. are correct in their warning that the presence of the RH motif alone does not mean that the ZF proteins will bind exclusively to sequences containing methylated bases.