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Response to Mackay et al.
Letters
Trends in Biochemical Sciences September 2013, Vol. 38, No. 9
Response to Mackay et al. Xiaodong Cheng1 and Robert M. Blumenthal2 1
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA Department of Medical Microbiology & Immunology and Program in Bioinformatics and Proteomics/Genomics, The University of Toledo College of Medicine & Life Sciences, Toledo, OH 43614, USA
2
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) [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 [2]), XpG in the CpG-methylated genomes of Homo, Pan, Mus, Danio, and Takifugu genera is 15.7 0.9% of all dinucleotides, whereas TpGjCpA 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. [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 [3,4]), four bind GpG (one of them, KLF4, binds methylated DNA, as recently shown [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 (50 ) pyrimidine. The presCorresponding author: Cheng, X. ([email protected]); Blumenthal, R.M. ([email protected]).
ence 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 [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 [7]). In addition, we provided evidence that a single spatially conserved glutamate in Zfp57 and Kaiso also contribute to the preferred binding of 5mC [7,8]. 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 [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 50 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. References 1 Mackay, J.P. et al. (2013) Is there a telltale RH fingerprint in zinc fingers that recognize methylated CpG dinucleotides? Trends Biochem. Sci. 38, 421–422 2 Simmen, M.W. (2008) Genome-scale relationships between cytosine methylation and dinucleotide abundances in animals. Genomics 92, 33–40 3 Omichinski, J.G. et al. (1997) The solution structure of a specific GAGA factor–DNA complex reveals a modular binding mode. Nat. Struct. Biol. 4, 122–132 4 Stoll, R. et al. (2007) Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA. J. Mol. Biol. 372, 1227–1245 5 Spruijt, C.G. et al. (2013) Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159 6 Zou, X. et al. (2012) Recognition of methylated DNA through methylCpG binding domain proteins. Nucleic Acids Res. 40, 2747–2758 7 Liu, Y. et al. (2012) An atomic model of Zfp57 recognition of CpG methylation within a specific DNA sequence. Genes Dev. 26, 2374–2379 8 Liu, Y. et al. (2013) A common mode of recognition for methylated CpG. Trends Biochem. Sci. 38, 177–183 9 Buck-Koehntop, B.A. et al. (2012) Molecular basis for recognition of methylated and specific DNA sequences by the zinc finger protein Kaiso. Proc. Natl. Acad. Sci. U.S.A. 109, 15229–15234 0968-0004/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibs.2013.06.010 Trends in Biochemical Sciences, September 2013, Vol. 38, No. 9
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Trends in Biochemical Sciences September 2013, Vol. 38, No. 9
Response to Mackay et al. Xiaodong Cheng1 and Robert M. Blumenthal2 1
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA Department of Medical Microbiology & Immunology and Program in Bioinformatics and Proteomics/Genomics, The University of Toledo College of Medicine & Life Sciences, Toledo, OH 43614, USA
2
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) [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 [2]), XpG in the CpG-methylated genomes of Homo, Pan, Mus, Danio, and Takifugu genera is 15.7 0.9% of all dinucleotides, whereas TpGjCpA 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. [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 [3,4]), four bind GpG (one of them, KLF4, binds methylated DNA, as recently shown [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 (50 ) pyrimidine. The presCorresponding author: Cheng, X. ([email protected]); Blumenthal, R.M. ([email protected]).
ence 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 [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 [7]). In addition, we provided evidence that a single spatially conserved glutamate in Zfp57 and Kaiso also contribute to the preferred binding of 5mC [7,8]. 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 [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 50 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. References 1 Mackay, J.P. et al. (2013) Is there a telltale RH fingerprint in zinc fingers that recognize methylated CpG dinucleotides? Trends Biochem. Sci. 38, 421–422 2 Simmen, M.W. (2008) Genome-scale relationships between cytosine methylation and dinucleotide abundances in animals. Genomics 92, 33–40 3 Omichinski, J.G. et al. (1997) The solution structure of a specific GAGA factor–DNA complex reveals a modular binding mode. Nat. Struct. Biol. 4, 122–132 4 Stoll, R. et al. (2007) Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA. J. Mol. Biol. 372, 1227–1245 5 Spruijt, C.G. et al. (2013) Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159 6 Zou, X. et al. (2012) Recognition of methylated DNA through methylCpG binding domain proteins. Nucleic Acids Res. 40, 2747–2758 7 Liu, Y. et al. (2012) An atomic model of Zfp57 recognition of CpG methylation within a specific DNA sequence. Genes Dev. 26, 2374–2379 8 Liu, Y. et al. (2013) A common mode of recognition for methylated CpG. Trends Biochem. Sci. 38, 177–183 9 Buck-Koehntop, B.A. et al. (2012) Molecular basis for recognition of methylated and specific DNA sequences by the zinc finger protein Kaiso. Proc. Natl. Acad. Sci. U.S.A. 109, 15229–15234 0968-0004/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibs.2013.06.010 Trends in Biochemical Sciences, September 2013, Vol. 38, No. 9
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