One of the most intensively studied chromatin binding factors is HP1␣. HP1␣ is associated with silenced, heterochromatic regions of the genome and binds to H3K9me3. While H3K9me3 is necessary for HP1␣ recruitment to heterochromatin, it is becoming apparent that it is not sufficient suggesting that additional factors are involved. One candidate proposed as a potential regulator of HP1␣ recruitment is the linker histone H1.4. Changes to the underlying makeup of chromatin, such as the

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... of the histone variant H2A.Z, has also been linked with regulating HP1 binding to chromatin. Here, we rigorously dissected the effects of H1.4, H2A.Z and H3K9me3 on the nucleosome binding activity of HP1␣ in vitro employing arrays, mononucleosomes and nucleosome core particles. Unexpectedly, histone H1.4 impedes the binding of HP1␣ but strikingly, this inhibition is partially relieved by the incorporation of both H2A.Z and H3K9me3 but only in the context of arrays or nucleosome core particles. Our data suggests that there are two modes of interaction of HP1␣ with nucleosomes. The first primary mode is through interactions with linker DNA. However, when linker DNA is missing or occluded by linker histones, HP1␣ directly interacts with the nucleosome core and this interaction is enhanced by H2A.Z with H3K9me3.