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 source of energy for the brain, heart, and skeletal muscle. Beyond this classical metabolic role, β-hydroxybutyrate (BHB), is gaining recognition as a pleiotropic 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 novel protein adduct is metabolically sensitive, dependent on BHB concentration, and found on proteins in multiple intracellular compartments, including the mitochondria and nucleus. 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 the β-hydroxybutyrylated lysine 9 on histone H3 (H3K9bhb) also recognizes other modification(s), which are increased by deacetylation inhibition and include likely acetylations. Therefore, caution must be used when interpreting gene regulation data acquired with the H3K9bhb antibody.

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 hours. 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 hours 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.

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 hours. 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 hours. 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.