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Epigenetics Chromatin. 2014 Jul 13;7:14. doi: 10.1186/1756-8935-7-14. eCollection 2014.

Assessing cellular efficacy of bromodomain inhibitors using fluorescence recovery after photobleaching.

Author information

1
Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK ; Target Discovery Institute, Nuffield Department of Clinical Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK ; Current address: Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Windmill Road, Oxford OX3 7LD, UK.
2
Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK ; Target Discovery Institute, Nuffield Department of Clinical Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
3
Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK ; Current address: Nanotether Discovery Sciences Ltd, Museum Avenue, Cardiff University, Cardiff CF10 3AX, UK.
4
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada.
5
Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK.
6
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada ; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.

Abstract

BACKGROUND:

Acetylation of lysine residues in histone tails plays an important role in the regulation of gene transcription. Bromdomains are the readers of acetylated histone marks, and, consequently, bromodomain-containing proteins have a variety of chromatin-related functions. Moreover, they are increasingly being recognised as important mediators of a wide range of diseases. The first potent and selective bromodomain inhibitors are beginning to be described, but the diverse or unknown functions of bromodomain-containing proteins present challenges to systematically demonstrating cellular efficacy and selectivity for these inhibitors. Here we assess the viability of fluorescence recovery after photobleaching (FRAP) assays as a target agnostic method for the direct visualisation of an on-target effect of bromodomain inhibitors in living cells.

RESULTS:

Mutation of a conserved asparagine crucial for binding to acetylated lysines in the bromodomains of BRD3, BRD4 and TRIM24 all resulted in reduction of FRAP recovery times, indicating loss of or significantly reduced binding to acetylated chromatin, as did the addition of known inhibitors. Significant differences between wild type and bromodomain mutants for ATAD2, BAZ2A, BRD1, BRD7, GCN5L2, SMARCA2 and ZMYND11 required the addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) to amplify the binding contribution of the bromodomain. Under these conditions, known inhibitors decreased FRAP recovery times back to mutant control levels. Mutation of the bromodomain did not alter FRAP recovery times for full-length CREBBP, even in the presence of SAHA, indicating that other domains are primarily responsible for anchoring CREBBP to chromatin. However, FRAP assays with multimerised CREBBP bromodomains resulted in a good assay to assess the efficacy of bromodomain inhibitors to this target. The bromodomain and extraterminal protein inhibitor PFI-1 was inactive against other bromodomain targets, demonstrating the specificity of the method.

CONCLUSIONS:

Viable FRAP assays were established for 11 representative bromodomain-containing proteins that broadly cover the bromodomain phylogenetic tree. Addition of SAHA can overcome weak binding to chromatin, and the use of tandem bromodomain constructs can eliminate masking effects of other chromatin binding domains. Together, these results demonstrate that FRAP assays offer a potentially pan-bromodomain method for generating cell-based assays, allowing the testing of compounds with respect to cell permeability, on-target efficacy and selectivity.

KEYWORDS:

Bromodomain; Cell-based assay; Confocal microscopy; Epigenetics; Fluorescence recovery after photobleaching; Histone acetylation; Small-molecule inhibitor

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