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. 2014 Apr;42(8):4859-67.
doi: 10.1093/nar/gku134. Epub 2014 Feb 11.

Structural basis for Klf4 recognition of methylated DNA

Yiwei Liu  1 , Yusuf Olatunde Olanrewaju, Yu Zheng, Hideharu Hashimoto, Robert M Blumenthal, Xing Zhang, Xiaodong Cheng
Affiliations

Structural basis for Klf4 recognition of methylated DNA

Yiwei Liu et al. Nucleic Acids Res. 2014 Apr.

Abstract

Transcription factor Krüppel-like factor 4 (Klf4), one of the factors directing cellular reprogramming, recognizes the CpG dinucleotide (whether methylated or unmodified) within a specific G/C-rich sequence. The binding affinity of the mouse Klf4 DNA-binding domain for methylated DNA is only slightly stronger than that for an unmodified oligonucleotide. The structure of the C-terminal three Krüppel-like zinc fingers (ZnFs) of mouse Klf4, in complex with fully methylated DNA, was determined at 1.85 Å resolution. An arginine and a glutamate interact with the methyl group. By comparison with two other recently characterized structures of ZnF protein complexes with methylated DNA, we propose a common principle of recognition of methylated CpG by C2H2 ZnF proteins, which involves a spatially conserved Arg-Glu pair.

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Figures

Figure 1.
Figure 1.
Klf4 binds methylated CpG. (a) Sequence alignment of the C-terminal ZnF DNA-binding domains of mouse Klf4 (mKlf4) and human KLF4 (hKLF4), which are identical in sequence. The mutations made by Hu et al. (22), R458A and D460A of hKLF4 are located in the last (third) ZnF, which does not directly participate in methyl-CpG binding. (b) Schematic representation of mKlf4 DNA-binding ZnF domain. The sequence and the secondary structure are shown as follows: (arrows) β strands and (ribbons) α helices. The positions highlighted are responsible for Zn ligand binding (C2H2) and DNA base-specific interactions at −1, −4, −5 and −7 positions (relative to the first zinc-binding histidine): solid lines (direct hydrogen bonds) and dashed lines (van der Waals contacts). The DNA sequence used for the study is shown with the majority of base interactions involving the top strand from 3′-to-5′ (left-to-right). The central GCG sequence is colored in magenta and the letter ‘m’ indicates the methyl group in 5mC. Dotted and solid vertical lines indicate specific binding interactions. (c) The mKlf4 ZnF protein binds in the major groove of DNA with ZnF1 (blue), ZnF2 (green) and ZnF3 (pink). (d) Lys413 of ZnF1 at the −7 position interacts with the O6 oxygen atoms of both guanines at G9 (of upper strand) and G10 (of lower strand). (e) His416 of ZnF1 at the −4 position interacts with the TpG dinucleotide. (f) Arg443–Gua6 interaction; a layer of ordered water molecules (marked ‘w’) shields the methyl group of lower strand 5mC. (g) The upper strand 5mCpG interacts with Arg443 and forms a 5mC-Arg-Gua triad. (h) One of the carboxylate oxygen atoms of Glu446 forms a weak C-H…O type of hydrogen bond with the methyl group of the upper strand 5mC. (i) Asp445 of ZnF2 at the −5 position interacts with Arg443 at the −7 position and the N4 atom of 5mC of the upper strand. (j) Arg449–G4 interaction. (k) Arg471–G3 interaction; Asp473 of ZnF3 at the −5 position interacts with Arg471 at the −7 position and the N4 atom of Cyt4 of the lower strand. (l) His474–A2 interaction. (m and n) Structural comparison of mKlf4 Glu446 in the absence (m) and presence of methylation (n).
Figure 2.
Figure 2.
The effects of CpG modifications and DNA sequence on DNA-binding by Klf4. (a) Binding affinities measured by fluorescence polarization assays between Klf4 and DNA with five different modification states on the upper strand (5mC = M, C, 5hmC, 5fC and 5caC). (b) Binding affinities measured between Klf4 and DNA that is fully methylated, unmodified or hemimethylated (on either strand). For these experiments, only M (5mC) and C were used. (c) A GCG-containing DNA sequence partially matching the consensus binding element of Klf4 (underlined) was used as a negative control. Fluorescence polarization (FP) is measured in millipolarization (mP). (d) Binding affinities measured between the E446A variant of Klf4 and DNA having five different modification states on the top strand. In all cases, the lower strand has M (5mC). (e) Distribution of DNA CpG methylation in mouse ES cells and NP cells (33) that is present within the core GGCG Klf4-ChIP sites identified in ES cells (23). The red arrows indicate changes of methylation levels, from hypomethylation in mouse ES cells to hypermethylation in NP cells (see Table 2), during differentiation and/or reprogramming. (f) Distribution of DNA methylation of randomly chosen GGCG sites in the mouse ES genome. (g) Distribution of DNA methylation in human H1 ES cells presented within the human KLF4-ChIP sites (34). GGCGTG sequences (green) have a higher proportion of methylated sites than do GGCGGG sequences.
Figure 3.
Figure 3.
Structural and sequence comparisons of three C2H2 ZnF proteins and their respective DNA interactions. (a and b) The second ZnF of Klf4 or Zfp57 recognizes a methylated GCG sequence. (c) Superimposed GCG-recognition helices of Klf4 (green) and Zfp57 (brown). Arg at −7 position of Klf4 and Arg at −8 position of Zfp57 are spatially aligned. (d) Kaiso uses two neighboring ZnF fingers, an arginine at the −1 position of the N-terminal ZnF and a Glu at the −5 position of the C-terminal ZnF, to recognize 5mCpG or TpG. (e) In Klf4, together with the Arg at the −7 position, the side chain of Glu at the −4 position forms a C-H…O type of hydrogen bond with the 5mC methyl group. (f) In Zfp57, together with the Arg at the -8 position, the side chain of Glu at the -4 position forms a van der Waals contact with the 5mC methyl group and one of its carboxylate oxygen atoms also interacts with the N4 atom of the same 5mC base. (g) Although not aligned at the primary sequence level, Kaiso has spatially conserved Arg and Glu, from two neighboring ZnF fingers (d), forming similar interactions with 5mC as that of Zfp57. (h) Sequence alignment of the second ZnF of three-ZnF DNA-binding domains among the mouse Sp/Klf family members. The negatively charged glutamate, at the −4 position, appears to be critical in discriminating against the negatively charged carboxylate moiety of 5caC, the final oxidative product of 5mC.
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