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. 2017 Jun 1;66(5):711-720.e3.
doi: 10.1016/j.molcel.2017.05.004. Epub 2017 May 18.

Structural Basis for the Versatile and Methylation-Dependent Binding of CTCF to DNA

Hideharu Hashimoto  1 , Dongxue Wang  1 , John R Horton  2 , Xing Zhang  2 , Victor G Corces  3 , Xiaodong Cheng  4
Affiliations

Structural Basis for the Versatile and Methylation-Dependent Binding of CTCF to DNA

Hideharu Hashimoto et al. Mol Cell. .

Abstract

The multidomain CCCTC-binding factor (CTCF), containing a tandem array of 11 zinc fingers (ZFs), modulates the three-dimensional organization of chromatin. We crystallized the human CTCF DNA-binding domain in complex with a known CTCF-binding site. While ZF2 does not make sequence-specific contacts, each finger of ZF3-7 contacts three bases of the 15-bp consensus sequence. Each conserved nucleotide makes base-specific hydrogen bonds with a particular residue. Most of the variable base pairs within the core sequence also engage in interactions with the protein. These interactions compensate for deviations from the consensus sequence, allowing CTCF to adapt to sequence variations. CTCF is sensitive to cytosine methylation at position 2, but insensitive at position 12 of the 15-bp core sequence. These differences can be rationalized structurally. Although included in crystallizations, ZF10 and ZF11 are not visible, while ZF8 and ZF9 span the backbone of the DNA duplex, conferring no sequence specificity but adding to overall binding stability.

Keywords: C2H2 zinc-finger arrays; CTCF; DNA methylation; DNA-binding adaptability to sequence variations; epigenetics; protein-DNA recognition.

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Figures

Figure 1
Figure 1. CTCF ZF3-7 binds the 15-bp CORE sequence
(A) CTCF-binding consensus sequence as determined by ChIP-exo (Rhee and Pugh, 2011). DNA cytosine methylation (indicated by red circles and letters m) occurs at positions 2 and 12 of the consensus sequence in a subset of CTCF-binding sites (Wang et al., 2012). (B) CTCF consensus binding motifs as determined by ChIP-seq (Jothi et al., 2008). (C) Predicted CTCF DNA binding specificity (Persikov and Singh, 2014). The notable differences from the consensus sequence involves a Gua (instead of Ade) at position 3 and a Thy (instead of Cyt) at position 12. We note that both Thy (5-methyluracil) and methylated Cyt (5-methylcytosine) contain a methyl group at ring carbon C5. (D) A model of CTCT ZF2-9 binding DNA, generated by superimposing the common four fingers (4-7) from structures of ZF2-7 and ZF4-9. (E) Three DNA sequences used for binding assays (CORE, H19 and control). (F) Binding affinities of CTCF ZF4-7 against the three oligos defined in panel E. (G-H) ZF3 increases binding affinity against the CORE sequence under two different NaCl concentrations. Because ZF3-7 binds too tightly against CORE (KD being close to probe concentration of 5 nM), we increased NaCl concentration in the binding assays from 150 mM (G) to 250 mM (H). (I) ZF8 increases non-specific binding affinity. (J) The 11-ZF DNA binding domain binds the CORE sequence depending on the ionic strength. DNA binding data represent the mean ± SEM of two independent determinations performed in duplicate.
Figure 2
Figure 2. CTCF ZF3-7 forms base-specific contacts
(A) Structural superimposition of ZF2-7 and ZF3-7. (B) Schematic representation of the ZF3–7 interactions with DNA. The top line indicates the 15-bp consensus sequence. The second line indicates the base pair positions (1–15). The third and the fourth lines are the sequence of the double-stranded oligo used for crystallization, shown with the top strand (orange) matching the consensus sequence. Amino acids of each finger interact specifically with the DNA bases shown below. (C-Q) DNA base specific interactions involve a particular residue of each ZF, colored according to panel A. Atoms are colored dark blue for nitrogen, red for oxygen, and carbon atoms are decorated with finger specific colors. The numerical numbers indicate the inter-atomic distance in angstroms, ‘w’ is water molecules (small red spheres). Note in panel D, hydrogen atoms on the G2:C2 base pair were shown to illustrate the C-H…O type of hydrogen bonds between C2 ring carbon atom C5-H and D451. (R) In the structure of ZF5-8, side chains of Y392 and K393 interact respectively with the cytosine and guanine of the C8:G8 base pair. (S) Side chains of Y392 and K393 in the structure of ZF5-8 (grey) adopted different conformations from that of ZF3-7 (blue; as shown in panel J). The red arrows indicate a concerted movement of the two side chains.
Figure 3
Figure 3. Differential cytosine methylation influences CTCF binding
(A) A cancer-associated mutation (K365T) shows diminished DNA binding. (B) The H19 sequence deviates from the CORE consensus sequence at two locations, a Gua instead of Ade at position 3 and a Thy instead of Ade at position 6. (C) R448 of ZF7 makes bidentate contacts with the Gua at position 3 in the structure of ZF6-8 in complex with the H19 sequence. (D) The Ade-to-Gua change at position 3 of the CORE sequence does not affect DNA binding affinity by ZF4-7. (E) The Ade-to-Thy change at position 6 of the CORE sequence shows much reduced DNA binding by ZF4-7. (F) Methylation at C2 of the CORE sequence abolishes DNA binding, whereas methylation of C12 enhances DNA binding by ZF4-7. (G) Modeling a methyl group onto unmodified C2 potentially results in repulsion (indicated by a star) with D451 of ZF7. (H) Methylation (hemi- or fully) of the CpG dinucleotide at position 2 of the H19 sequence shows reduced DNA binding affinity by ZF4-7. (I) In the structure of ZF3-7 in complex with the methylated DNA, the omit electron density (grey mesh), contoured at 5σ above the mean, is shown for the 5mC methyl group (in yellow sphere). (J) Structural comparison of ZF3-7 in complex with methylated DNA (in orange) and unmethylated DNA (in green). (K) A model of strand-specific interaction associated with differential methylation at C2 by CTCF (open circle: un/de-methylated) and Zfp57 (filled circle: methylated). DNA binding data represent the mean ± SEM of two independent determinations performed in duplicate. See also Figure S3.
Figure 4
Figure 4. (A) CTCF ZF8-9 spans across the DNA phosphate backbone (A)
Two views of ZF4-9, displayed by the crystallographic heat map, from low-to-high thermal B-factor (blue, cyan, green, and yellow). (B-D) Aligned structures of ZF5-8 (B), ZF6-8 (C), and ZF7-8 (D) against a reference DNA molecule. (E) Superimposition of 4-finger structures of ZF4-7 and ZF5-8 indicates that the C-terminal ZF7 lies in the major groove whereas ZF8 spans across the minor groove of DNA. See also Figure S4. (F) Superimposition of four fragments of two-finger structures, ZF4-5, ZF5-6, ZF6-7 and ZF7-8, reveals that the C-terminal ZF8 swings to the right whereas the rest swing to the left. (G) Enlarged linker regions between ZF6-7 and ZF7-8, with the alignment of linker sequences. (H) Superimposition of DNA-bound ZF6-7 and DNA-free ZF6-7 (PDB 2CT1).
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