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.

Summary Using an integrated approach, we here report the identification of 67 novel histone marks, a discovery that increases the current number of known histone marks by about 70%. We verified one of the newly-identified marks, lysine crotonylation (Kcr), as a novel, evolutionarily-conserved histone post-translational modification. The unique structure and genomic localization of Kcr suggest that it is mechanistically and functionally different from lysine acetylation (Kac), a previously-described post-translational modification. Specifically, in both human somatic and mouse male germ cell genomes, histone Kcr marks either active promoters or potential enhancers. In male germinal cells immediately following meiosis, Kcr is enriched on sex chromosomes and specifically marks testis-specific genes, including a significant proportion of X-linked genes that escape sex chromosome inactivation in haploid cells. These results therefore dramatically extend the repertoire of histone PTM sites and designate Kcr as a specific mark of active sex chromosome-linked genes in post-meiotic male germ cells.

SUMMARY Brown adipose tissue (BAT) can disperse stored energy as heat. Promoting BAT-like features in white adipose (WAT) is an attractive, if elusive therapeutic approach to staunch the current obesity epidemic. Here we report that gain-of-function of the NAD-dependent deacetylase SirT1 or loss-of-function of its endogenous inhibitor Deleted in breast cancer-1 (Dbc1) promote “browning” of WAT by deacetylating peroxisome proliferator-activated receptor (Ppar)-γ on Lys268 and Lys293. SirT1-dependent deacetylation of Lys268 and Lys293 is required to recruit the BAT program coactivator Prdm16 to Pparγ, leading to selective induction of BAT genes and repression of visceral WAT genes associated with insulin resistance. An acetylation-defective Pparγ mutant induces a brown phenotype in white adipocytes, while an acetylated mimetic fails to induce “brown” genes, but retains the ability to activate “white” genes. We propose that SirT1-dependent Pparγ deacetylation is a form of selective Pparγ modulation of potential therapeutic import.

SUMMARY Here we report the identification and verification of a β-hydroxybutyrate-derived protein modification, lysine β-hydroxybutyrylation (Kbhb), as a new type of histone mark. Histone Kbhb marks are dramatically induced in response to elevated β-hydroxybutyrate levels in cultured cells, and in livers from mice subjected to prolonged fasting or streptozotocin-induced diabetic ketoacidosis. In total, we identified 44 histone Kbhb sites, a figure comparable to the known number of histone acetylation sites. By ChIP-seq and RNA-seq analysis, we demonstrate that histone Kbhb is a mark enriched in active gene promoters, and that the increased H3K9bhb levels that occur during starvation are associated with genes up-regulated in starvation-responsive metabolic pathways. Histone β-hydroxybutyrylation thus represents a new epigenetic regulatory mark that couples metabolism to gene expression, offering a new avenue to study chromatin regulation and the diverse functions of β-hydroxybutyrate in the context of important human pathophysiological states, including diabetes, epilepsy, and neoplasia.

BackgroundRecent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most common inherited and some sporadic forms of Parkinson's disease (PD). The molecular mechanism underlying the pathogenic role of LRRK2 mutations in PD remains unknown.Methodology/Principal FindingsUsing affinity purification and mass spectrometric analysis, we investigated phosphorylation sites and binding proteins of LRRK2 purified from mouse brain. We identified multiple phosphorylation sites at N-terminus of LRRK2 including S910, S912, S935 and S973. Focusing on the high stoichiometry S935 phosphorylation site, we developed an anti-pS935 specific antibody and showed that LRRK2 is constitutively phosphorylated at S935 in various tissues (including brain) and at different ages in mice. We find that 14-3-3 proteins (especially isoforms γ and η) bind LRRK2 and this binding depends on phosphorylation of S935. The binding of 14-3-3, with little effect on dimer formation of LRRK2, confers protection of the phosphorylation status of S935. Furthermore, we show that protein kinase A (PKA), but not LRRK2 kinase itself, can cause the phosphorylation of LRRK2 at S935 in vitro and in cell culture, suggesting that PKA is a potential upstream kinase that regulates LRRK2 function. Finally, our study indicates that the common PD-related mutations of LRRK2, R1441G, Y1699C and G2019S, decrease homeostatic phosphorylation levels of S935 and impair 14-3-3 binding of LRRK2.Conclusions/SignificanceLRRK2 is extensively phosphorylated in vivo, and the phosphorylation of specific sites (e.g. S935) determines 14-3-3 binding of LRRK2. We propose that 14-3-3 is an important regulator of LRRK2-mediated cellular functions. Our study suggests that PKA, a cAMP-dependent kinase involved in regulating dopamine physiology, is a potential upstream kinase that phosphorylates LRRK2 at S935. Furthermore, the reduction of phosphorylation/14-3-3 binding of LRRK2 due to the common familial PD-related mutations provides novel insight into the pathogenic mechanism of LRRK2-linked PD.