Epigenetics Identifier screens reveal regulators of chromatin acylation and limited specificity of acylation antibodies

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.

Figure 1

Epi-ID identifies regulators of chromatin. (A) Construction of the Epi-ID library. Knock-out and DAmP libraries of yeast mutants were crossed to a Barcoder library of yeast strains using SGA technology. Each strain in the Barcoder library contains a KanMX selection gene flanked by unique 20 bp UpTag and 20 bp DownTag barcodes integrated at the HO locus. (B) Outline of Epi-ID. Strains from the Epi-ID library were pooled, grown in liquid medium and subjected to ChIP using pan-K-acyl antibodies. The barcodes were amplified from ChIP and input DNA. (C) Next-generation sequencing of barcodes. Barcodes counted in immpunoprecipitates were normalized to counts in input. (D) Outcome of Epi-ID for acetylation on approximately 6500 mutant strains. Scatter plots present log2-transformed acetyl ChIP/input ratios normalized to H3 ChIP/input ratios for UpTag versus DownTag. Averages of two independent Epi-ID screens are shown. Each dot represents a single mutant strain. Mutants of the ADA (red), RPD3 (green), HDA1 (green) complexes and newly identified mutants selected for follow-up (blue) are highlighted. (E) Spearman correlations plotted for all Epi-ID screens, unnormalized for H3.

Figure 2

Dot-blots of modified BSA to test pan-K-acyl antibody specificity. (A–D) Immunoblot analysis of serially diluted and differently modified BSA with the indicated antibodies. Ponceau staining in (C) is for both succinylation and butyrylation blots.

Figure 3

Western blot analysis of pan-K-acyl antibodies with BSA competitors on yeast and HeLa cell extracts. Whole-cell extracts of wild-type yeast cells (A–C) and HeLa cells (D–F) were immunoblotted with the indicated antibodies in the presence of (modified) BSA. All lanes contain the same yeast or HeLa whole-cell extract. 0.5 µg BSA per microliter of antibody was added. Asterisk highlights background band. Whole-cell extracts of wild-type yeast cells (G–I) were immunoblotted with the indicated antibodies in the presence of (acetylated) H3 peptide. All lanes contain the same whole-cell extract. Below the blots is a quantification of the acylation signal over H4 of at least two independent experiments. (J) Western blot analysis of in vitro histone acetyltransferase assay with recombinant wild-type and catalytic-dead (Gcn5-E173H) ADA complex and wild-type Piccolo NuA4 complex immunoblotted with the indicated antibodies.

Figure 4

Immunofluorescence assays using pan-K-acyl antibodies with BSA competitors on HeLa cells. (A) Representative immunofluorescence images of HeLa cells showing nuclei stained with DAPI and pan-K-acetyl-recognizing antibodies against (A) acetylation, (B) crotonylation, (C) succinylation or (D) butyrylation in the presence of the indicated BSA competitors. (E–H) Quantification of data, representing the mean intensity of individual nuclei from (A–D), respectively. For each microliter of antibody, one microgram of BSA was added as competitor.

Figure 5

ChIP-qPCR analysis of pan-K-acyl antibodies with BSA competitors in yeast. (A) Overview of the KanMX cassette integrated at the HO locus on chromosome IV. Binding sites of primers at the TEF promoter and TEF terminator used for ChIP-qPCR are indicated. These sites are located 43 and 119 bases away from the barcoded region, respectively. (B–I) ChIP-qPCR analysis of TEF promoter and TEF terminator regions of the KANMX selection gene at the HO locus in wild-type yeast using the indicated pan-K-acyl antibodies in combination with high and low amounts of the indicated BSA competitors. In high BSA conditions, 6 µg of BSA was added per microliter of antibody. In low BSA conditions, 0.5 µg of BSA was added per microliter of antibody. Data represent the mean fold enrichment over background (IgG) from two independent experiments + s.e.m.

Figure 6

Epi-ID reveals potential regulators of acetylation and ADA complex displays histone butyryltransferase activity. (A–C) ChIP-qPCR analysis of pan-K-acetylation (A), H3K9ac (B) and pan-K-crotonylation (C) at UpTag and DownTag in a selection of hits from the Epi-ID screens. Averages of at least three independent experiments + s.e.m. are presented. Pan-K-acetylation and H3K9ac levels were normalized to H3 occupancy and signals at a telomeric region, and compared to wild-type (set to 1) using a one-sample T test for UpTag or DownTag. (D) Western blot analysis of global pan-K-acetylation in whole-cell extracts from the indicated yeast strains, dotted line indicates a cropped blot. All uncropped blots are shown in Supplementary Figure 5. (E) RT-qPCR analysis of KANMX expression in wild-type, clb3Δ and met7Δ strains. Expression is normalized to ACT1.

Figure 7

ADA complex displays histone butyryltransferase activity. (A,B) Western blot analysis of in vitro histone butyryltransferase assay with recombinant wild-type with (A) histone octamers and (B) mononucleosomes as a substrate. (C) Mass spectrometry spectrum of a peptide corresponding to a region spanning K9-R17 in histone H3 showing butyrylated K14 (red) and deuterated acetic acid (d) acetylations (green). (D) Schematic depicting residues 1–37 of H3 with acetylated (ac, red) and butyrylated (bu, yellow) lysine residues as identified by mass spectrometry. (E, F) Wild-type and gcn5Δ strains were treated with increasing concentrations of sodium butyrate (1, 2, 5, and 10 mM; pH 7.5) or sodium acetate (10 mM; pH 7.5) (E) or sodium butyrate (10 mM; pH 7.5) (F) for 3.5 h in the presence of 0.8 m sorbitol. Whole-cell extracts were immunoblotted with the indicated antibodies. (G) Western blot analysis using the indicated antibodies of nuclei isolated from wild-type and gcn5Δ strains. Nuclei were incubated with butyryl-CoA for 1 h.