to mass spectrometry related experiments and analysis; R.H., Z.Y. and B.R. performed the library construction and next-generation sequencing for ChIP-seq and RNA-seq; M.H. and Y.G.Z. synthesized L-lactyl-CoA. H.H. and D.Z. analyzed ChIP-seq and RNA-seq data. G.Z. provided all primary BMDM cell cultures. D.M.C. carried out the bacterial infection experiments, C.C. carried out TAM experiments. Author Information. Y.Z. is a founder, board member, advisor to, and inventor on patents licensed to PTM Bio Inc. L.B. is co-founder and CSO of rMark Bio Inc., and founder and CEO of Onchilles Pharma Inc. Readers are welcome to comment on the online version of the paper. Data availability. The ChIP-seq and RNA-seq data have been made available at the Gene Expression Omnibus (GEO) repository under the accession number GSE115354. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 31 partner repository with the dataset identifier PXD014870. All other data are available from the authors upon reasonable request.

We report the identification and characterization of a five-carbon protein post-translational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric academia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.

SUMMARY Acetylation of histones at DNA regulatory elements plays a critical role in transcriptional activation. Histones are also modified by other acyl moieties, including crotonyl, yet the mechanisms that govern acetylation versus crotonylation and the functional consequences of this “choice” remain unclear. We show that the coactivator p300 has both crotonyltransferase and acetyltransferase activities and that p300-catalyzed histone crotonylation directly stimulates transcription to a greater degree than histone acetylation. Levels of histone crotonylation are regulated by the cellular concentration of crotonyl-CoA, which can be altered through genetic and environmental perturbations. In a cell-based model of transcriptional activation, increasing or decreasing the cellular concentration of crotonyl-CoA leads to enhanced or diminished gene expression, respectively, which correlates with the levels of histone crotonylation flanking the regulatory elements of activated genes. Our findings support a general principle wherein differential histone acylation (i.e. acetylation versus crotonylation) couples cellular metabolism to the regulation of gene expression.