Non-specific recognition of histone modifications by H3K9bhb antibody

Abstract

Ketone bodies are short-chain fatty acids produced in the liver during periods of limited glucose availability that provide an alternative energy source for the brain, heart, and skeletal muscle. Beyond this metabolic role, β-hydroxybutyrate (BHB), is gaining recognition as a signaling molecule. Lysine β-hydroxybutyrylation (Kbhb) is a newly discovered post-translational modification in which BHB is covalently attached to lysine ε-amino groups. This protein adduct is metabolically sensitive, dependent on BHB concentration, and found on proteins in multiple intracellular compartments. Therefore, Kbhb is hypothesized to be an important component of ketone body-regulated physiology. Kbhb on histones is proposed to be an epigenetic regulator, which links metabolic alterations to gene expression. However, we found that the widely used antibody against β-hydroxybutyrylated lysine 9 on histone H3 (H3K9bhb) also recognizes other modification(s) that likely include acetylation. Therefore, caution must be used when interpreting gene regulation data acquired with the H3K9bhb antibody.

Keywords: Biochemistry; Biological sciences; Cell biology; Molecular biology.

Graphical abstract

Figure 1

Irregular band patterns of H3K9bhb antibodies (A) Structures of BHB and structurally similar butyrate. (B) Western blots of HEK293T cells and iMEFs treated with either BHB, butyrate, or TSA for 24 h. Butyrate and TSA are known deacetylation inhibitors. Ponceau S staining was used to confirm the equivalent loading of proteins. mono: monoclonal, poly: polyclonal. Data are representative of at least 3 independent experiments. (C) Upper: schematic of experimental workflow. HEK293T cells were treated with 5 mM BHB or 5 mM butyrate. At 24 h after the treatment, cellular metabolites were extracted and subjected to untargeted metabolomics by LC-MS/MS. unTx indicates untreated HEK293T cells. Lower: relative intensities of BHB in the metabolome of each treatment condition. N = 5-6 technical replicates per treatment condition. Data are represented as mean ± SD and analyzed by 1-way ANOVA (p < 0.0001).

Figure 2

Absence of H3K9bhb in butyrate-treated HEK293T cells (A) Schematic of experimental workflow. HEK293T cells were treated with 5 mM BHB or 5 mM butyrate for 24 h. Cell lysates were subjected to immunoprecipitation with α-H3K9bhb antibody or control normal rabbit IgG. The immunoprecipitants were used for Western blot analysis and SDS-PAGE, followed by Coomassie brilliant blue (CBB) staining and LC-MS/MS analysis. (B) Western blot of input and H3K9bhb IP fractions of HEK293T treated with BHB or butyrate for 24 h. Ponceau S staining was used to confirm the equivalent loading of proteins and H3 enrichment. (C) Representative tandem mass (MS/MS) spectra of the K9-BHB-ylated and K14-acetylated “PICnKSTGGKAPRR” peptides from BHB-treated samples. The detected y ions and b ions are highlighted in pink. (D) Left: a strategy of the downstream analysis. Numbers in parentheses refer to the number of detected peptides in BHB or butyrate-treated samples. Right: detected peptide numbers for each modification at the K9 position in the indicated peptides. ND: not determined.