Optimization of Proteomics-based Substrate Trapping with Histone Deacetylase 1

Abstract

Histone writers and erasures – the proteins that add or remove histone post translational modifications – are key drivers of epigenetic transcriptional events inside the cell. Many enzymes that control epigenetic modifications are dysregulated in human diseases. For example, overexpression of the erasure histone deacetylase (HDAC) enzymes can lead to epigenetic changes in transcription and ultimately disease, such as cancer. We recently pioneered a simple method called substrate trapping to isolate HDAC1 substrates using an inactive HDAC1 mutant. Our recent publication documented that different HDAC1 mutants preferentially bound different substrates, suggesting that three mutants (H141A, F150A, C151A) should be used for efficient trapping. Based on this observation, we performed a proteomics-based trapping study of HDAC1 using all three optimal mutants simultaneously. In this study, using trapping with three mutants, 12 potential HDAC1 substrates were identified that were not observed when only a single mutant was used. Although trapping with three mutants identified novel substrates of HDAC1, the throughput of the experiment was low compared to trapping with a single mutant. Therefore, this study confirms that trapping with a single mutant is simple and effective, although three mutants may be desirable when high efficiency or a large number of substrates is necessar