Methylation-independent binding to histone H3 and cell cycle-dependent incorporation of HP1β into heterochromatin

We have examined HP1 beta-chromatin interactions in different molecular contexts in vitro and in vivo. Employing purified components we show that HP1 beta exhibits selective, stoichiometric, and salt-resistant binding to recombinant histone H3, associating primarily with the helical "histone fold" domain. Furthermore, using "bulk" nucleosomes released by MNase digestion, S-phase extracts, and fragments of peripheral heterochromatin, we demonstrate that HP1 beta associates more tightly with destabilized or disrupted nucleosomes (H3/H4 subcomplexes) than with intact particles. Western blotting and mass spectrometry data indicate that HP1 beta-selected H3/H4 particles and subparticles possess a complex pattern of post-translational modifications but are not particularly enriched in me(3)K9-H3. Consistent with these results, mapping of HP1 beta and me(3)K9-H3 sites in vivo reveals overlapping, yet spatially distinct patterns, while transient transfection assays with synchronized cells show that stable incorporation of HP1 beta-gfp into heterochromatin requires passage through the S-phase. The data amassed challenge the dogma that me(3)K9H3 is necessary and sufficient for HP1 binding and unveil a new mode of HP1-chromatin interactions.