Figure 3: ERαK235me2 is recognized by the PHF20 Tudor domain.

(a) Identification of ERαK235me2-specific readers by the chromatin-associated domain array (CADOR). The array probed with unmodified ERαK235 peptide was used as a negative control. Positive interactions of the ERαK235me2 peptide are highlighted in coloured boxes. Red: 53BP1 Tudor; Blue: S. pombe SPF30 Tudor; Yellow: PHF20/20L Tudors. The list of all the domains on the CADOR is shown in Supplementary Fig. 5. (b) The PHF20 Tudor domain binds to ERαK235me peptides in vitro. Western blot analysis of peptide pulldowns of PHF20 Tudor using the indicated ERα and histone peptides. GelCode staining shows the peptide input for the assay. (c) Overlays of ¹H,¹⁵N HSQC spectra of PHF20 Tudor collected as the ERαK235me2 peptide (left) or unmodified ER peptide (right) were titrated in. The spectra are colour coded according to the peptide concentration (inset). (d) A ribbon diagram of the structure of PHF20 Tudor (ID: 3P8D). The residues that exhibit large ERαK235me2-induced resonance changes (red) or resonance broadening (brown) are highlighted. (e) PHF20 Tudor binding to the ERαK235me2 peptide is methylation dependent. Western blot analysis of ERαK235me peptide pulldowns of the WT PHF20 Tudor domain or two methyl-binding-deficient mutants (W97A and Y103A).