Figure 1. hHP1β–chromatin interaction is dependent on H3K9me3.

(a) Domain structure of hHP1β; boundaries of domains are indicated by respective amino acid positions. For more details see Supplementary Fig. 2. (b) Scheme of pull-down experiments in c using different H3K9me templates immobilized on streptavidin-coated magnetic beads via C-terminal incorporation of a biotinylated lysine (peptide) or ligation of 5′-biotinylated oligonucleotides to DNA templates used in chromatin reconstitution (mono- and oligonucleosomes). (c) Immobilized H3K9me templates according to the pull-down experimental schemes in b were incubated with recombinant hHP1β WT. Material recovered after washing was analysed by western blotting. The indicated salt concentrations were used throughout the experiment. (d) Scheme of chromatin coprecipitation assay; factors bound to oligonucleosomes are precipitated with the template when clustering is induced by addition of Mg²⁺ ions. (e,f) Chromatin coprecipitation of hHP1β (e) and hHP1α (f) proteins with oligonucleosomes. Precipitated material was run on SDS-PAGE and stained with Coomassie blue. Input, 10%. (g) The indicated recombinant proteins were incubated with a DNA fragment of 150 bp at 500: 1 and 1,000: 1 molar ratio. Complexes were separated by PAGE. DNA was stained with SYBR Gold.