Alcohol metabolism contributes to brain histone acetylation.

Emerging evidence suggests that epigenetic regulation is dependent on metabolic state, and implicates specific metabolic factors in neural functions that drive behaviour1. In neurons, acetylation of histones relies on the metabolite acetyl-CoA, which is produced from acetate by chromatin-bound acetyl-CoA synthetase 2 (ACSS2)2. Notably, the breakdown of alcohol in the liver leads to a rapid increase in levels of blood acetate3, and alcohol is therefore a major source of acetate in the body. Histone acetylation in neurons may thus be under the influence of acetate that is derived from alcohol4, with potential effects on alcohol-induced gene expression in the brain, and on behaviour5. Here, using in vivo stable-isotope labelling in mice, we show that the metabolism of alcohol contributes to rapid acetylation of histones in the brain, and that this occurs in part through the direct deposition of acetyl groups that are derived from alcohol onto histones in an ACSS2-dependent manner. A similar direct deposition was observed when mice were injected with heavy-labelled acetate in vivo. In a pregnant mouse, exposure to labelled alcohol resulted in the incorporation of labelled acetyl groups into gestating fetal brains. In isolated primary hippocampal neurons ex vivo, extracellular acetate induced transcriptional programs related to learning and memory, which were sensitive to ACSS2 inhibition. We show that alcohol-related associative learning requires ACSS2 in vivo. These findings suggest that there is a direct link between alcohol metabolism and gene regulation, through the ACSS2-dependent acetylation of histones in the brain.

Paternal cocaine taking elicits epigenetic remodeling and memory deficits in male progeny.

This corrects the article DOI: 10.1038/mp.2017.8.

Paternal cocaine taking elicits epigenetic remodeling and memory deficits in male progeny.

Paternal environmental perturbations including exposure to drugs of abuse can produce profound effects on the physiology and behavior of offspring via epigenetic modifications. Here we show that adult drug-naive male offspring of cocaine-exposed sires have memory formation deficits and associated reductions in NMDA receptor-mediated hippocampal synaptic plasticity. Reduced levels of the endogenous NMDA receptor co-agonist d-serine were accompanied by increased expression of the d-serine degrading enzyme d-amino acid oxidase (Dao1) in the hippocampus of cocaine-sired male progeny. Increased Dao1 transcription was associated with enrichment of permissive epigenetic marks on histone proteins in the hippocampus of male cocaine-sired progeny, some of which were enhanced near the Dao1 locus. Finally, hippocampal administration of d-serine reversed both the memory formation and synaptic plasticity deficits. Collectively, these results demonstrate that paternal cocaine exposure produces epigenetic remodeling in the hippocampus leading to NMDA receptor-dependent memory formation and synaptic plasticity impairments only in male progeny, which has significant implications for the male descendants of chronic cocaine users.

Recent Achievements in Characterizing the Histone Code and Approaches to Integrating Epigenomics and Systems Biology.

Functional epigenetic regulation occurs by dynamic modification of chromatin, including genetic material (i.e., DNA methylation), histone proteins, and other nuclear proteins. Due to the highly complex nature of the histone code, mass spectrometry (MS) has become the leading technique in identification of single and combinatorial histone modifications. MS has now overcome antibody-based strategies due to its automation, high resolution, and accurate quantitation. Moreover, multiple approaches to analysis have been developed for global quantitation of posttranslational modifications (PTMs), including large-scale characterization of modification coexistence (middle-down and top-down proteomics), which is not currently possible with any other biochemical strategy. Recently, our group and others have simplified and increased the effectiveness of analyzing histone PTMs by improving multiple MS methods and data analysis tools. This review provides an overview of the major achievements in the analysis of histone PTMs using MS with a focus on the most recent improvements. We speculate that the workflow for histone analysis at its state of the art is highly reliable in terms of identification and quantitation accuracy, and it has the potential to become a routine method for systems biology thanks to the possibility of integrating histone MS results with genomics and proteomics datasets.

Neutrophil proteomic analysis reveals the participation of antioxidant enzymes, motility and ribosomal proteins in the prevention of ischemic effects by preconditioning.

Intestinal ischemia and reperfusion injury are widely used models, which result into tissue injury and multiple organ failure also observed after trauma and surgery. Ischemic preconditioning (IPC) preceding ischemia and reperfusion (IR) was shown to attenuate this injury and has a potential therapeutic application; however the exact underlying mechanism is not clear. Neutrophils play an important role in the mechanism of injuries caused by ischemia and reperfusion while IPC led to a decrease in neutrophil stimulation and activation. The effect of preconditioning on the neutrophil proteome is unclear. Proteomic analysis has been ratified as an appropriate tool for studying complex systems. In order to evaluate the effect of IPC preceding 45min of ischemia on the proteome of neutrophils we used Wistar rats divided in four experimental groups: Control, sham laparotomy, intestinal ischemia reperfusion and ischemic preconditioning. After neutrophil separation, proteins were extracted, trypsin digested and the resulting peptides were iTRAQ labeled followed by HILIC fractionation and nLC-MS/MS analysis. After database searches, normalization and statistical analysis our proteomic analysis resulted in the identification of 2437 protein groups that were assigned to five different clusters based on the relative abundance profiles among the experimental groups. The clustering followed by statistical analysis led to the identification of significantly up and downregulated proteins in IR and IPC. Cluster based KEGG pathways analysis revealed up- regulation of actin cytoskeleton, metabolism, Fc gamma R mediated phagocytosis, chemokine signaling, focal adhesion and leukocyte transendothelial migration whereas downregulation in ribosome, spliceosome, RNA transport, protein processing in endoplasmic reticulum and proteasome, after intestinal ischemic preconditioning. Furthermore, enzyme prediction analysis revealed the regulation of some important antioxidant enzymes and having their role in reactive oxygen species production. To our knowledge, this work describes the most comprehensive and detailed quantitative proteomic study of the neutrophil showing the beneficial role of ischemic preconditioning and its effects on the neutrophil proteome. This data will be helpful to understand the effect of underlying protective mechanisms modulating the role of PMNs after IPC and provide a trustworthy basis for future studies. BIOLOGICAL SIGNIFICANCE: Preconditioning is a relevant strategy to overcome clinical implications from ischemia and reperfusion. Such implications have the neutrophil as a major player. Although many publications describe specific biochemical and physiological roles of the neutrophil in such conditions, there is no report of a proteomic study providing a broader view of this scenario. Here we describe a group of proteins significantly regulated by ischemia and reperfusion being such regulation prevented by preconditioning. Such finding may provide relevant information for a deeper understanding of the mechanisms involved, as well as serve as basis for future biomarker or drug target assays.

Identification and Quantification of Histone PTMs Using High-Resolution Mass Spectrometry.

DNA is organized into nucleosomes, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins including H2A, H2B, H3, and H4. Histones are critical regulators of many nuclear processes, including transcription, DNA damage repair, and higher order chromatin structure. Much of their function is mediated through extensive and dynamic posttranslational modification (PTM) by nuclear enzymes. Histone PTMs are thought to form a code, where combinations of PTMs are responsible for specific biological functions. Here, we present protocols to identify and quantify histone PTMs using nanoflow liquid chromatography coupled to mass spectrometry (MS). We first describe how to purify histones and prepare them for MS. We then describe three MS platforms for histone PTM analysis, including bottom-up, middle-down, and top-down approaches, and explain the relative benefits and pitfalls of each approach. We also include tips to increase the throughput of large experiments.