Post-translational modifications of conserved histone residues regulate many aspects of chromosome biology. Clusters of phosphorylated, acetylated, methylated and deiminated amino acids produce a high degree of combinatorial complexity, the so-called """"""""histone code,"""""""" that has the potential to specify a vast number of different downstream events. Full interpretation of the histone code requires approaches that can unscramble the contribution of multiple post-translational modifications to the binding and activity of histone-associated proteins. Particularly unclear is the molecular basis for the regulatory effect of histone phosphorylation, especially during mitosis. As a model system, we will study haspin, a kinase required for normal chromosome alignment at metaphase. Haspin phosphorylates a novel residue, Thr-3, of the core histone H3 during mitosis in vivo. We hypothesize that this type of phosphorylation, in combination with other modifications, generates binding sites on histones for regulatory proteins during mitosis.
In Aim 1, we propose new and broadly applicable methodology to screen the human proteome for proteins that bind to clustered post-translational modifications. As a specific example, we will isolate proteins that bind to phospho-histone H3 (Thr-3) peptides with or without combinations of acetylated, methylated, deiminated and phosphorylated surrounding residues.
In Aim 2, we propose a general means to determine the patterns of post-translational modification recognized by modification-specific enzymes and binding proteins. As a test case, we will use innovative technology to screen an array of modified peptide libraries representing histone H3 to determine the post-translational requirements for haspin activity. Together, these motif-based approaches will allow us to define pathways of signaling through histone modification. Recent studies have revealed, in addition to phosphorylation, the methylation and acetylation of many cellular proteins and demonstrated their importance in diverse processes including transcription factor activity, cell motility, intracellular signaling and trafficking, immune synapse formation and apoptosis. While histones are striking examples, it is likely that the existence of protein modification codes is a more general phenomenon. The development of methodology to determine the functional impact of these marks is therefore a critical requirement for future work in all areas of biomedicine. ? ?
