. Lysine acetylation in proteins contributes significantly to cellular differentiation and development. At the molecular level, effector modules called bromodomains recognize acetylated lysine to facilitate downstream signaling through binding of macromolecular complexes to specific genomic loci. Despite their fundamental role in biology and disease, an unbiased cataloguing of acetylated interacting partners of a specific bromodomain is lacking possibly due to the weak, highly dynamic and context-dependent nature of such interactions. However, such molecular information is essential in delineating precise mechanism by which bromodomains partake in the intricate process of transcriptional regulation in biology and disease. The current proposal outlines a unique strategy termed `Interaction-Based Protein and Promoter Profiling' (IBPP) by introducing a photo-crosslinkable amino acid into the hydrophobic cage of bromodomains to capture transiently interacting partners followed by their proteomic and genomic characterizations. We will apply IBPP to BET bromodomains (BRD2, BRD3, BRD4 and BRDT) to identify their interacting partners from intact cells. Subsequent biochemical validation and functional studies of the newly identified interactome will lead to improved mechanistic understanding of acetylation and bromodomain-mediated gene regulation in normal cells as well as in human malignancies. Salient points of our approach include the ability to identify weak and transient binding partners (because of crosslinking) with high temporal control (because of light as the perturbing agent) of closely related members of BET family (via protein engineering with unnatural mutagenesis) in cell-type specific manner. Since crosslinking occurs in the deeply embedded `aromatic cage' in bromodomains, selectivity in identifying authentic interacting partners is expected to be much higher compared to chromatin immunoprecipitation (ChIP) that relies on formaldehyde-based non-selective crosslinking method. Successful implication of IBPP will be invaluable in providing both molecular as well as system level understanding of the dynamic functions of more than sixty bromodomain-containing human proteins.

Public Health Relevance

Relevancy Statement. Point mutation, translocation and deletion in bromodomain-containing proteins are common to multiple human conditions such as cancer, autoimmune diseases and cognitive disorders. This proposal outlines a unique strategy called 'Interaction-based protein and promoter profiling (IBPP)' for identifying and characterizing the missing molecular links between the biochemical misregulations of bromodomains and these diseases. The successful implication of the proposed work is expected to advance our understanding of such aberrant processes, and also identify new biomarkers for diagnosis and novel targets for drug discovery.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Synthetic and Biological Chemistry B Study Section (SBCB)
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Fabian, Miles
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University of Pittsburgh
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Islam, Kabirul (2018) The Bump-and-Hole Tactic: Expanding the Scope of Chemical Genetics. Cell Chem Biol 25:1171-1184
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Sudhamalla, Babu; Dey, Debasis; Breski, Megan et al. (2017) Site-specific azide-acetyllysine photochemistry on epigenetic readers for interactome profiling. Chem Sci 8:4250-4256
Perell, Gabriella T; Mishra, Neeraj K; Sudhamalla, Babu et al. (2017) Specific Acetylation Patterns of H2A.Z Form Transient Interactions with the BPTF Bromodomain. Biochemistry 56:4607-4615