Structural basis of DNA target recognition by the B3 domain of Arabidopsis epigenome reader VAL1

Abstract Arabidopsis thaliana requires a prolonged period of cold exposure during winter to initiate flowering in a process termed vernalization. Exposure to cold induces epigenetic silencing of the FLOWERING LOCUS C (FLC) gene by Polycomb group (PcG) proteins. A key role in this epigenetic switch is played by transcriptional repressors VAL1 and VAL2, which specifically recognize Sph/RY DNA sequences within FLC via B3 DNA binding domains, and mediate recruitment of PcG silencing machinery. To understand the structural mechanism of site-specific DNA recognition by VAL1, we have solved the crystal structure of VAL1 B3 domain (VAL1-B3) bound to a 12 bp oligoduplex containing the canonical Sph/RY DNA sequence 5′-CATGCA-3′/5′-TGCATG-3′. We find that VAL1-B3 makes H-bonds and van der Waals contacts to DNA bases of all six positions of the canonical Sph/RY element. In agreement with the structure, in vitro DNA binding studies show that VAL1-B3 does not tolerate substitutions at any position of the 5′-TGCATG-3′ sequence. The VAL1-B3–DNA structure presented here provides a structural model for understanding the specificity of plant B3 domains interacting with the Sph/RY and other DNA sequences.

(A) Two DNA-bound VAL1-B3 subunits that form an asymmetric unit are colored yellow and green. Three adjacent asymmetric units are shown. Two 12 bp DNA oligoduplexes present in the asymmetric unit form continuous DNA structures along the a = 40.09 Å edge of the crystal lattice. Blue and red spheres mark Cα atoms of the N-and Cterminal residues resolved in the structure. (B) Gel-filtration of VAL1-B3. The apparent MW of apo-VAL1-B3 determined by gelfiltration (18.25 ml elution volume, equivalent to 10.2 kDa, blue square) is close to the theoretic mass of VAL1-B3 monomer (13.3 kDa). Gel-filtration was carried out at room temperature on an AKTA FPLC system using a Superdex 200 10/300 GL column (GE Healthcare) pre-equilibrated with 10 mM sodium phosphate (pH 7.0 at 25°C), 250 mM NaCl and 5 mM 2-mercaptoethanol. The sample (10 μM VAL1-B3) was prepared in 100 μl of the same buffer. Elution from the column (flow rate 0.5 ml/min) was monitored by measuring absorbance at 280 nm. A calibration line was generated by measuring the elution volumes of a series of standards of known molecular mass (Gel Filtration Calibration Kit from GE Healthcare, black circles). The molecular weight of VAL1-B3 was calculated by interpolating its elution volume onto the calibration line.
Supplementary Figure S2. Overall DNA conformation in complexes with plant B3 domains.
Left: structure of the VAL1-B3-DNA complex (PDB ID 6fas, chains BEF). The N-arm residues are colored green, C-arm residues are orange, other residues are yellow. The DNA strands containing the 5′-TGCATG-3′ and 5′-CATGCA-3′ sequences are blue/gray, the DNA axis calculated by the Curves+ web server (24) is red. The total DNA bend as determined by Curves+ is 5.1° (4.7° for the CD strands). Right: structure of the ARF-B3-DNA complex (PDB ID 4ldx, chain A residues 119-226, chain D residues 1-10, chain C residues 12-21). The N-arm residues are colored pale green, C-arm residues are light orange, other residues are pink. The DNA strands containing the 5′-GAGACA-3′ and 5′-TGTCTC-3′ sequences are green/gray, the DNA axis is magenta. The total DNA bend of the shown DNA fragment as determined by Curves+ is 3.1° (5.8° for an equivalent DNA fragment bound by the ARF1 B chain). Center: superimposition of protein chains of VAL1-B3-DNA and ARF1-B3-DNA complexes generated with Multiprot (18) (orientation as in Figure 1D), showing the slight difference in DNA orientation relative to the proteins.
Supplementary Figure S3. Contacts of VAL1-B3 residues to DNA backbone. In all panels, electrostatic potential surfaces of wt VAL1-B3 (experimental structure) and the respective mutants (models) are shown. Rotamers for R306 and R345 were selected in COOT (34) such that they could make direct contacts to the phosphates. Positions of free DNA, specific complex and non-specific complex in the EMSA gels are marked by blue, red and yellow rectangles, respectively. (A) DNA binding by wt VAL1-B3 and VAL1-B3 mutants Q345R, E328Q and E360Q. DNA concentration (either non-specific 12/12NSP, or cognate 12/12SP) was 1 µM, protein concentrations were 1, 2, 4 and 10 µM (no protein in lanes '0') , EMSA experiments were performed in a pH 8.3 buffer as described in Materials and Methods. Under these experimental conditions only the mutants are capable of specific complex formation.
(B) Mutations of residues that make direct contacts to DNA phosphates impair specific binding. DNA concentration was 1 µM, VAL1-B3 concentrations were 1, 2, 4 and 10 µM, EMSA experiments were performed in a pH 6.0 buffer as described in Materials and Methods.
Supplementary Figure S4. The effect of the C-terminal (His) 6 tag on specific and non-specific DNA binding. In all panels, positions of free DNA, specific complex and non-specific complex in the EMSA gels are marked by blue, red and yellow rectangles, respectively. DNA concentration (either non-specific 12/12NSP, or cognate 12/12SP) was 1 µM, protein concentrations were 1, 2, 4 and 10 µM (no protein in lanes '0') , EMSA experiments were performed either in a pH 6.0 or a pH 8.

Construction of expression vectors and site-directed mutagenesis
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Molecular Replacement
The structure of DNA-bound VAL1-B3 was solved by molecular replacement We note that all attempts to place two copies of any of the individual VAL1-B3 models with PHASER were not successful (PHASER TFZ < 7.5, and no solutions with correct orientation of the subunits among the top-scoring solutions). P+D ⇔PD