The regulatory enzymes and protein substrates for the lysine β-hydroxybutyrylation pathway

Abstract

Metabolism-mediated epigenetic changes represent an adapted mechanism for cellular signaling, in which lysine acetylation and methylation have been the historical focus of interest. We recently discovered a β-hydroxybutyrate-mediated epigenetic pathway that couples metabolism to gene expression. However, its regulatory enzymes and substrate proteins remain unknown, hindering its functional study. Here, we report that the acyltransferase p300 can catalyze the enzymatic addition of β-hydroxybutyrate to lysine (Kbhb), while histone deacetylase 1 (HDAC1) and HDAC2 enzymatically remove Kbhb. We demonstrate that p300-dependent histone Kbhb can directly mediate in vitro transcription. Moreover, a comprehensive analysis of Kbhb substrates in mammalian cells has identified 3248 Kbhb sites on 1397 substrate proteins. The dependence of histone Kbhb on p300 argues that enzyme-catalyzed acylation is the major mechanism for nuclear Kbhb. Our study thus reveals key regulatory elements for the Kbhb pathway, laying a foundation for studying its roles in diverse cellular processes.

Fig. 1. p53- and p300-dependent histone Kbhb assay on recombinant chromatin templates.

(A) Illustration of enzymatic reaction for acetyl-lysine and the hypothesized mechanism for β-hydroxybutyryllysine. (B) Schematic of the p300-dependent in vitro transcription assay. NTPs, nucleoside triphosphates. (C) The histone acyltransferase assay with indicated factors, ac-CoA and bhb-CoA. The Kbhb and Kac levels of histones were analyzed by immunoblotting with indicated antibodies. (D) p300-dependent histone Kbhb facilitates active transcription by p53 on recombinant chromatin templates, which can be abolished by histone H3 and H4 K-R mutations. RNA products were visualized by autoradiography. “Oct” represents octamer.

Fig. 2. p300 has histone lysine β-hydroxybutyryltransferase activity in vivo.

(A) p300 knockdown by siRNA transfection impairs histone Kbhb in HCT116 cells. Histone Kbhb and Kac levels were analyzed by immunoblotting with indicated antibodies. NS, nonsilencing. (B) p300 knockout (KO) decreases histone Kbhb levels in HCT116 cells. The indicated histone PTMs were analyzed by immunoblotting with indicated antibodies. WT, wild type. (C) p300 inhibitor A485 reduces histone Kbhb and Kac levels dose-dependently in HCT116 cells. HCT116 cells were treated with A485 for 24 hours, and the Kbhb and Kac levels of histones were analyzed by immunoblotting with indicated antibodies.

Fig. 3. HDAC1 to HDAC3 and SIRT1 and SIRT2 can remove Kbhb in vitro.

(A) In vitro screening of HDAC and sirtuins Kbhb deacylase activities. (B) Kbhb deacylase activity assay of HDAC1 to HDAC3 and SIRT1 and SIRT2 using Kbhb-containing peptides as substrates.

Fig. 4. HDAC1 and HDAC2 remove Kbhb in cells.

(A) Class I/II HDAC inhibitors (NaBu, TSA, and FK228), but not a class III HDAC inhibitor (NAM), elevate H3K9bhb and H3K9ac levels in HEK293 cells. DMSO, dimethyl sulfoxide. (B) TSA treatment increases histone Kbhb levels in HEK293 cells. Cells were treated with TSA at the indicated concentrations for 18 hours, and Kbhb and Kac levels were analyzed by immunoblotting with indicated antibodies. (C) Joint knockdown of HDAC1 and HDAC2 increases histone Kbhb levels in HEK293 and HeLa cells. Kbhb and Kac levels were detected by immunoblotting using indicated antibodies. Immunoblot of histone H3 was used as loading control. (D) An HDAC1/2/3 selective inhibitor, MS275, dose-dependently increases Kbhb and Kac levels in 293T cells. Cells were treated with MS275 for 24 hours, and the Kbhb and Kac levels were analyzed by immunoblotting with the indicated antibodies.

Fig. 5. Systematic profiling of the Kbhb proteome.

(A) Schematic representation of experimental workflow for the identification of Kbhb-containing protein substrates in HEK293 cells. (B) MS/MS spectra of two representative Kbhb peptides derived from HEK293 core histones. (C) Distribution of the Kbhb protein (left) and Kbhb sites (right) based on the site number per protein.

Fig. 6. Characterization of the Kbhb proteome in HEK293 cells.

(A) Sequence motif logo shows a representative sequence for all Kbhb sites. (B) Venn diagram shows the cellular compartment distribution of Kbhb proteins. (C) Protein class enrichment of Kbhb proteins. (D) Protein complexes enriched in the Kbhb proteome. The color bar depicts the protein abundance percentile in HEK293 cells, and the size stands for the number of Kbhb sites identified in each protein. Circles and squares represent bait and prey nodes, respectively.