Combinatorial profiling of chromatin-binding modules reveals multi-site discrimination

Specific interactions between post-translational modifications (PTMs) and chromatin-binding proteins are central to the idea of a ‘histone code’. Here, a 5000-member, PTM-randomized, combinatorial peptide library based on the N-terminus of histone H3 was utilized to interrogate multi-site specificity of six chromatin-binding modules, which read the methylation status of K4. We found that T3 phosphorylation, R2 methylation, and T6 phosphorylation are critical additional PTMs that modulate the ability to recognize and bind histone H3. Notably, phosphorylation of T6 yielded the most varied effect on protein binding, suggesting an important regulatory mechanism for readers of the H3 tail. Mass spectrometry and antibody-based evidence indicate that this previously uncharacterized modification exists on native H3, and NMR analysis of ING2 revealed the structural basis for discrimination. These investigations reveal a continuum of binding affinities in which multi-site PTM recognition involves both switch- and rheostat-like properties, yielding graded effects that depend on the inherent ‘reader’ specificity.