Nucleosome context regulates chromatin reader preference

Chromatin is more than a simple genome packaging system, and locally distinguished by histone post-translational modifications (PTMs) that can change nucleosome structure and / or be “read” by chromatin-associated proteins to mediate downstream events. An accurate understanding of histone PTM binding preference is vital to understanding chromatin function and disease development, and has led to the identification of multiple therapeutic targets. Such studies most often use histone peptides, even though this cannot explore the full regulatory potential of nucleosome context. Here we apply a range of complementary and easily adoptable biochemical and genomic approaches to interrogate fully defined peptide and nucleosome targets containing acyl, methyl, methyl analog and phospho PTMs with a diversity of mono- or multi- valent chromatin readers. Nucleosome context consistently refined reader binding, and was particularly informative when analyzing multivalent engagement. This included abrogating the binding of L3LBTL1 MBT to histone tails with lower methyl states (me1 or me2 at H3K4, H3K9, H3K27, H3K36 or H4K20); and demonstrating that specificity of the CBX7 chromodomain for H3K27me3 is enabled by an adjacent DNA-binding AT-hook like (ATL[MK1] ) motif. Further, the in vitro binding preferences of readers to nucleosomes was confirmed by in vivo reader-CUT&RUN genomic mapping. Such data confirms that interrogating with more representative substrates provides greater insight to chromatin reader capability and biological mechanism.