Abstract
Chromatin is more than a simple genome packaging system but rather locally distinguished by histone post-translational modifications (PTMs) that can directly 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 explain normal function and pathogenesis and has revealed multiple therapeutic opportunities. Such studies most often use histone peptides, though these cannot represent 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 with a diversity of mono- or multivalent chromatin readers. In the resulting data, nucleosome context consistently refined reader binding, and multivalent engagement was more often regulatory than simply additive. This included abrogating binding of the Polycomb group malignant brain tumor (MBT) protein L3MBTL1 to lysine methylated histone tails and confirmation that the CBX7 chromodomain and AT-hook-like motif (CD-ATL) tandem act as a functional unit to confer specificity for H3K27me3. These in vitro nucleosome preferences were confirmed by in vivo reader-CUT&RUN genomic mapping. Such data confirms that more representative chromatin substrates provide greater insight into biological mechanism and human disease.