Fig. 6

MMP-9 binding to H3K27me1 nucleosomes. a Schematic depiction of the domain structure of MMP-9. b Peptide pull-down assays with biotinylated H3 1–21 and 21–44 peptides and recombinant His-MMP-9 were analyzed by Western blotting with anti-His antibody. H3 peptides were unmodified, K18ac or K27me1 as indicated. Lane 1 represents 10% of the input MMP-9. c Nucleosomes were reconstituted on a 207-bp 601 nucleosome positioning sequence using unmodified or H3K27me1 histone octamers and immobilized on streptavidin beads. His-MMP-9 was incubated with immobilized nucleosomes, and its binding to nucleosomes was analyzed by Western blotting with anti-His antibody. Lane 1 contains 10% of the input MMP-9. d H3K27me1 nucleosomes were incubated with immobilized MMP-9 N-terminal (amino acids 112–447) and C-terminal (amino acids 448–730) domains. After extensive washing, the binding of H3K27me1 nucleosomes to MMP-9 domains was determined by Western blotting with anti-H3 antibody. Input corresponds to 10% of H3K27me1 nucleosomes used in the binding reactions. e After incubation with H3K27me1 nucleosomes, the binding of MMP-9N-terminal subregions to nucleosomes was determined by Western blotting with anti-His antibody. Input lanes 1–3 represent 10% of MMP-9 fragments used in the binding reactions. f OCP-induced cells were transfected with FLAG-H3 wild type (WT) or K27R mutant (K27R), and mononucleosomes were prepared by micrococcal nuclease digestion as summarized in Figure S3. Mononucleosomes containing ectopic H3 were immunoprecipitated from total mononucleosomes with FLAG antibody and analyzed by Western blotting with anti-MMP-9 antibody. g Model of the MMP-9-H3K27me1 interaction. PDB entries 4h3x (mMMP-9) and 3avr (H3.1) were used in docking simulations using the program Cluspro 2.0 [28,29,30]. Simulations were run with non-methylated H3. For context, H3K27 is shown monomethylated. h Nucleosome binding assays were conducted as in e, except that His-MMP-9 amino acids 384–447 carrying E402A mutation were used