The fact that DNA is wrapped around a spool, comprised of histones, is believed to influence essentially all aspects of chromosome biology, including DNA replication, repair after damage and segregation. Diverse post-translational modifications (e.g. acetylation, methylation, and phosphorylation) of histones are known and are believed to play key roles in regulating a wide swath of biology linked to our genomes. Based on observed antagonisms and synergies between different histone post-translational modifications (or 'marks') in recruiting proteins to chromosomes, it has been proposed that these 'marks'form a 'code'for regulating chromosome function. It has also been suggested that this 'code'may provide a basis of epigenetic inheritance, which is the transmission of cellular traits that are not encoded at the level of DNA sequence. Many of the proteins that post-translationally modify histones (i.e. 'write'or 'erase'the 'code') have been characterized. In contrast, our knowledge of the proteins that recognize (or 'read') histone post-translational modifications remains incomplete. The difficulty in identifying these effector-proteins (or 'readers') is, in large part, due to the histone modifications being sub-stoichiometric, dynamic, and mediators of weak interactions. With the goal to fill this knowledge gap, we have recently reported an approach, which combines photo-chemical crosslinking with bio-orthogonal chemistry, to 'capture'proteins that bind histone H3 trimethylated at Lys-4. We now combine this method with state-of-the-art mass spectrometry to develop a robust chemical proteomics approach to profile 'readers'of histone methylation 'marks.'Our ongoing work suggests that our approach is general and can be used to analyze these post-translational modification-dependent protein-protein interactions in any human cell type (e.g. normal or cancer), cell state (e.g. mitosis) or context (e.g. drug-treated). Based on these and other unpublished preliminary data, we propose to: (i) comprehensively profile proteins that recognize methylation 'marks'on histones, (ii) characterize how proteins that recognize methylation 'marks'control down-stream biology, and (iii) examine how interplay between histone phosphorylation and methylation ensures error-free chromosome segregation during cell division. We combine chemistry, biochemistry, high-resolution microscopy and cell biological approaches to gain insight into fundamental cellular processes. Our findings may reveal how improper 'reading'of histone post-translational modifications can result in disease. In the long-term, our findings may also provide a basis for developing new therapeutic strategies that target 'readers'of histone methylation 'marks'.
The goals of the proposed research are to develop and apply a chemical proteomics approach to comprehensively profile proteins that recognize histones with particular post-translational modifications (or 'marks'). By characterizing these proteins, we will gain insight into how chromosome function is regulated in normal cells and how diseases may arise when the 'reading'of these histone 'marks'goes awry.
|Kasap, Corynn; Elemento, Olivier; Kapoor, Tarun M (2014) DrugTargetSeqR: a genomics- and CRISPR-Cas9-based method to analyze drug targets. Nat Chem Biol 10:626-8|
|Kashyap, Sudhir; Sandler, Joel; Peters, Ulf et al. (2014) Using 'biased-privileged' scaffolds to identify lysine methyltransferase inhibitors. Bioorg Med Chem 22:2253-60|
|Kawashima, Shigehiro A; Takemoto, Ai; Nurse, Paul et al. (2013) A chemical biology strategy to analyze rheostat-like protein kinase-dependent regulation. Chem Biol 20:262-71|
|Foley, Emily A; Kapoor, Tarun M (2013) Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore. Nat Rev Mol Cell Biol 14:25-37|
|Li, Xiang; Foley, Emily A; Kawashima, Shigehiro A et al. (2013) Examining post-translational modification-mediated protein-protein interactions using a chemical proteomics approach. Protein Sci 22:287-95|
|Kleiner, Ralph E; Ti, Shih-Chieh; Kapoor, Tarun M (2013) Site-specific chemistry on the microtubule polymer. J Am Chem Soc 135:12520-3|
|Wacker, Sarah A; Houghtaling, Benjamin R; Elemento, Olivier et al. (2012) Using transcriptome sequencing to identify mechanisms of drug action and resistance. Nat Chem Biol 8:235-7|
|Li, Xiang; Foley, Emily A; Molloy, Kelly R et al. (2012) Quantitative chemical proteomics approach to identify post-translational modification-mediated protein-protein interactions. J Am Chem Soc 134:1982-5|