Supplementary Figure 3: CHD1 interacts with KDM1A K114me2.

(a) Protein-domain microarray. Samples are GST fusion proteins listed in (b) that were arrayed onto nitrocellulose. M contains GST alone. (c) The microarrays were probed with either anti-GST antibody and visualized with a FITC-conjugated secondary antibody or Cy3-labelled KDM1A and KDM1A K114me2 peptides. (d) Coomassie blue staining and table. Samples are extracts of LNCaP cells incubated with column bound KDM1A peptides that were eluted and analyzed as indicated. The table depicts the proteins enriched with the methylated KDM1A peptide. (e) Representative ITC experiment displaying titration of H3 and H3K4me3 peptides to CHD1. (f) Two-dimensional error surface projections of CHD1 (A) and H3K4me3 (B) ITC fit. (g) View of the intermolecular interactions between the KDM1A peptide and CHD1 residues. (h) Superimposition of KDM1A and H3 in complex with CHD1. The surface that could be exploited for the design of inhibitors specifically interfering with binding of KDM1A K114me2 but not H3K4me3 is shown in red. (i,j) Representative ITC experiments displaying titration of KDM1A R113A-K114me2 mutant peptide to CHD1 (i) and KDM1A K114me2 to CHD1 D425A (j). (k) Anti-CHD1, anti-EHMT2, anti-KDM1A and anti-KDM1A K114A Western blots. Samples are extracts from LNCaP cells cultured with or without DHT and Bix-01294 that were immunoprecipitated with anti-EHMT2 or rIgG. Results are representatives of 1 (c,d), 2 (k, i,j) and 3 (e) independent experiments.