Abstract
Lysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Toward this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First, we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype and reveal a role for p300s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tunable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, N.I.H., Intramural
MeSH terms
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Acetylation
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Azepines / pharmacology
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Benzimidazoles / pharmacology
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CRISPR-Associated Protein 9 / genetics
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CRISPR-Associated Proteins / genetics
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CRISPR-Cas Systems / genetics*
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Capsid Proteins / genetics
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Cytomegalovirus / genetics
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E1A-Associated p300 Protein / antagonists & inhibitors
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E1A-Associated p300 Protein / chemistry
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E1A-Associated p300 Protein / genetics
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E1A-Associated p300 Protein / metabolism*
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Enzyme Inhibitors / pharmacology
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HEK293 Cells
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Humans
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Hydantoins / pharmacology
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Interleukin 1 Receptor Antagonist Protein / genetics
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Isoxazoles / pharmacology
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Protein Domains
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RNA, Guide, CRISPR-Cas Systems / genetics
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Recombinant Fusion Proteins
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Spiro Compounds / pharmacology
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Streptococcus pyogenes / enzymology
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Transcription, Genetic / genetics
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Transcriptional Activation / genetics
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Triazoles / pharmacology
Substances
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(+)-JQ1 compound
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Azepines
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Benzimidazoles
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CBP30 compound
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CRISPR-Associated Proteins
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Capsid Proteins
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Enzyme Inhibitors
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Hydantoins
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IL1RN protein, human
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Interleukin 1 Receptor Antagonist Protein
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Isoxazoles
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RNA, Guide, CRISPR-Cas Systems
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Recombinant Fusion Proteins
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Spiro Compounds
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Triazoles
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E1A-Associated p300 Protein
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EP300 protein, human
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CRISPR-Associated Protein 9
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Cas9 endonuclease Streptococcus pyogenes