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Abstract
The collection of known posttranslational modifications (PTMs) has expanded rapidly with the identification of various non-acetyl histone lysine acylations, such as crotonylation, succinylation and butyrylation, yet their regulation is still not fully understood. Through an unbiased chromatin immunoprecipitation (ChIP)-based approach called Epigenetics-IDentifier (Epi-ID), we aimed to identify regulators of crotonylation, succinylation and butyrylation in thousands of yeast mutants simultaneously. However, highly correlative results led us to further investigate the specificity of the pan-K-acyl antibodies used in our Epi-ID studies. This revealed cross-reactivity and lack of specificity of pan-K-acyl antibodies in various assays. Our findings suggest that the antibodies might recognize histone acetylation in vivo, in addition to histone acylation, due to the vast overabundance of acetylation compared to other acylation modifications in cells. Consequently, our Epi-ID screen mostly identified factors affecting histone acetylation, including known (e.g. GCN5, HDA1, and HDA2) and unanticipated (MET7, MTF1, CLB3, and RAD26) factors, expanding the repertoire of acetylation regulators. Antibody-independent follow-up experiments on the Gcn5-Ada2-Ada3 (ADA) complex revealed that, in addition to acetylation and crotonylation, ADA has the ability to butyrylate histones. Thus, our Epi-ID screens revealed limits of using pan-K-acyl antibodies in epigenetics research, expanded the repertoire of regulators of histone acetylation, and attributed butyrylation activity to the ADA complex.
Highlights
The collection of known posttranslational modifications (PTMs) has expanded rapidly with the identification of various non-acetyl histone lysine acylations, such as crotonylation, succinylation and butyrylation, yet their regulation is still not fully understood
In addition to a collection of ~ 4700 yeast deletion mutants[27] that we employed previously[23], we used a collection of ~ 2000 Decreased Abundance by mRNA Perturbation (DAmP) alleles for essential genes[28]. These yeast mutants were crossed with the Barcoder yeast library using Synthetic Genetic Array (SGA) technologies to create mutants containing a pair of unique barcodes, the UpTag and DownTag barcodes, surrounding a KanMX marker gene at the HO locus (Fig. 1A)[29]
We found that loss of GCN5, MET7 and CLB3, but not of SVF1, resulted in a decrease in the pan-K-acetylation and/or H3K9 acetylation levels at the UpTag barcode, whereas loss of HDA1, HDA2, MTF1 and RAD26 led to an increase in the acetylation levels at both barcodes, confirming findings from the Epi-ID screen and showing the validity of the Epi-ID approach (Fig. 6A,B)
Summary
The collection of known posttranslational modifications (PTMs) has expanded rapidly with the identification of various non-acetyl histone lysine acylations, such as crotonylation, succinylation and butyrylation, yet their regulation is still not fully understood. The collection of known PTMs has substantially increased through the use of various mass spectrometry-based approaches Among these PTMs are the non-acetyl histone lysine acylations such as crotonylation, succinylation and butyrylation[4,5,6,7,8]. We applied Epi-ID to identify regulators of histone crotonylation, succinylation and butyrylation by screening thousands of mutant yeast strains using pan-K-acyl-recognizing antibodies[5, 6, 26]. Cross-recognition of epitopes by the pan-K-acyl-recognizing antibodies was observed in various assays in yeast and human cells Given these findings, we hypothesized that the regulators identified in our Epi-ID screens are either regulators of histone acetylation or histone acylation. The Epi-ID screens revealed panK-acyl antibody aspecificity, regulators of histone acetylation, and a new role for the ADA complex in histone butyrylation
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