Errors in cell division, the process during which replicated DNA is partitioned between two daughter cells, have been linked to diseases and developmental defects. Improper cell division has also been exploited in therapeutic strategies widely used to treat diseases, such as cancer. Accurate chromosome segregation, proper cell cycle progression and repair of DNA damage rely on key proteins recognizing post-translational modifications on histones, which assemble the basic units of chromatin known as nucleosomes. Comprehensively profiling proteins that `read' specific post-translational modifications (or `marks') on histones to regulate chromosome biology has been difficult using conventional approaches. Our goal is to fill this knowledge gap by devising new chemistry-based strategies to profile direct `readers' of histone `marks' in specific cellular contexts. Central to our approach s the conversion of dynamic and weak (typically micromolar) protein-protein interactions into stable associations, via covalent bonds, using photo-cross-linkers. This strategy is combined with state-of-the-art quantitative mass spectrometry to identify `readers' of specific histone `marks'. In the completed project period, we have developed this `chemical proteomics' approach, named CLASPI (cross-linking-assisted and SILAC-based protein identification), and demonstrated its ability to identify new `readers' of histone `marks', including histone H3 methylation and phosphorylation. We will build on our recent publications and preliminary data and will focus on identifying `readers' of two different histone phosphorylation `marks', one that indicates DNA damage and is observed during prolonged mitotic arrest with cytotoxic drugs, and another that associates with chromosomes in dividing cells and depends on the activity of Aurora kinase, a conserved regulator of cell division and a target of anti-cancer drugs. The functional significance of `reading' these histone `marks' at different stages of the cell cycle wil be examined using high-resolution microscopy assays, mouse embryonic fibroblasts with key proteins knocked out, and chemical inhibitors that act on fast time-scales to block the activity of kinases responsible for generating these `marks' in cells. The proposal has three aims: (i) To identify proteins that `read' a phosphorylation `mark' on histone H2AX, (ii) To characterize the functions of histone H2AX phosphorylation-`readers', and (iii) To profile `readers' of histone post-translational modifications in living cells. The proposed research combines chemistry and biology approaches to unravel how histone `marks' are `interpreted' by proteins to ensure stable genome propagation by regulating chromosome segregation, DNA damage repair and cell cycle progression. These studies should also shed light on how chemical inhibitors of cell division kill cancer cells. In addition, the comprehensive profiling of key post-translational modification-dependent protein-protein interactions should lead to the selection of new targets for therapeutic agents. Finally, the approaches we develop are general and can be broadly applied to dissect complex and dynamic networks of protein-protein interactions in cells.

Public Health Relevance

The goals of the proposed research are to develop and apply chemistry-based approaches to profile proteins that bind (or `read') histones with particular post-translational modifications (or `marks'). By identifying and analyzing these protein-protein interactions, we will gain insight into how chromosome function is regulated to ensure the stable propagation of our genomes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Synthetic and Biological Chemistry B Study Section (SBCB)
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Fabian, Miles
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Rockefeller University
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New York
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Kleiner, Ralph E; Hang, Lisa E; Molloy, Kelly R et al. (2018) A Chemical Proteomics Approach to Reveal Direct Protein-Protein Interactions in Living Cells. Cell Chem Biol 25:110-120.e3
Chen, Zhen; Suzuki, Hiroshi; Kobayashi, Yuki et al. (2018) Structural Insights into Mdn1, an Essential AAA Protein Required for Ribosome Biogenesis. Cell 175:822-834.e18
Kapoor, Tarun M; Miller, Rand M (2017) Leveraging Chemotype-Specific Resistance for Drug Target Identification and Chemical Biology. Trends Pharmacol Sci 38:1100-1109
Steinman, Jonathan B; Santarossa, Cristina C; Miller, Rand M et al. (2017) Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action. Elife 6:
See, Stephanie K; Hoogendoorn, Sascha; Chung, Andrew H et al. (2016) Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity. ACS Chem Biol 11:53-60
Kawashima, Shigehiro A; Chen, Zhen; Aoi, Yuki et al. (2016) Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis. Cell 167:512-524.e14
Kleiner, Ralph E; Verma, Priyanka; Molloy, Kelly R et al. (2015) Chemical proteomics reveals a ?H2AX-53BP1 interaction in the DNA damage response. Nat Chem Biol 11:807-14
Kashyap, Sudhir; Sandler, Joel; Peters, Ulf et al. (2014) Using 'biased-privileged' scaffolds to identify lysine methyltransferase inhibitors. Bioorg Med Chem 22:2253-60
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
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

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