Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Histone Arginine Methylation in Cocaine Action: Essential Role of Src Signaling in the Nucleus Accumbens

ABSTRACT: Despite addiction being one of the most prevalent and debilitating disorders worldwide, effective treatments are lacking. Repeated cocaine exposure induces maladaptive transcriptional regulation within the brainâ??s reward circuitry, such as the nucleus accumbens (NAc), and epigenetic mechanisms, such as histone acetylation or methylation on Lys (K) residues, have been linked to these lasting actions of cocaine. However, in contrast to K methylation, the functional role of histone Arg (R) methylation remains unexplored in addiction models and poorly understood in brain in general. Here we show that protein-R-methyltransferase-6 (PRMT6) and its associated histone mark, asymmetric dimethylation of R2 on histone H3 (H3R2me2a), are decreased in the NAc of mice and rats after repeated cocaine exposure, as well as in the NAc of cocaine-addicted humans. PRMT6 downregulation occurs selectively in NAc medium spiny neurons expressing dopamine D2 receptors (D2-MSNs) and serves to protect against cocaine-induced addictive-like behavioral abnormalities. Using ChIP-seq, we demonstrate that reduced H3R2me2a binding at gene targets in NAc after repeated cocaine is strongly correlated with increased binding of H3K4me3, and identify Src kinase signaling inhibitor 1 (Srcin1 or p140Cap) as a key gene for these chromatin modifications. Cocaine induction of Srcin1 in NAc, which is associated with reduced Src signaling, decreases cocaine reward, the motivation to self administer cocaine, and cocaine-induced changes in NAc MSN dendritic spines. These results suggest that this suppression of Src signaling in NAc D2-MSNs, via PRMT6 and H3R2me2a downregulation, functions as a homeostatic brake to restrain cocaine action, and provide novel candidates for the development of new treatments for cocaine addiction. H3R2me2A ChIP-seq of mouse. Cocaine vs Saline, 3 biological replicates.

ORGANISM(S): Mus musculus

SUBMITTER: Eric Nestler

PROVIDER: E-GEOD-85310 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Repeated administration of the psychostimulant cocaine has been shown to produce persistent alterations in genome-wide transcriptional regulatory networks, chromatin remodeling activity and, ultimately, gene expression profiles in the brain’s reward circuitry. Virtually all previous investigations have centered on drug-mediated effects occurring throughout active euchromatic regions of the genome, such that very little is known concerning the impact of cocaine exposure on the regulation and maintenance of heterochromatin in adult brain. Here, we report that cocaine administration dramatically and dynamically alters heterochromatic histone H3 lysine 9 trimethylation (H3K9me3) in the nucleus accumbens (NAc), a key brain reward region. Furthermore, we demonstrate that repeated cocaine exposure induces persistent decreases in heterochromatization in this brain region, suggesting a potential role for heterochromatic regulation in the long-term actions of cocaine. To identify precise genomic loci affected by these alterations, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-Seq) was performed on NAc. ChIP-Seq analyses confirmed the existence of the H3K9me3 mark mainly within inter-genic regions of the genome and identified specific patterns of cocaine-induced H3K9me3 regulation at repetitive genomic sequences. Cocaine-mediated decreases in H3K9me3 enrichment at specific genomic repeats (e.g., LI repeats) were further confirmed by the increased expression of LINE-1 and IAP retrotransposons in NAc. Such increases likely reflect global patterns of genomic de-stabilization in this brain region following repeated cocaine administration and open the door for future investigations into the epigenetic and genetic basis of drug addiction. Animals received daily i.p. injections of either 'saline' (7 treatments of saline) or 'repeated' cocaine (7 treatments of 20 mg/kg cocaine). 24 hours after the last dose, chromatin immunoprecipitation was performed utilizing previously validated methods. Each experimental condition was performed in either duplicate or triplicate (2 cocaine, 3 saline). Briefly, for each ChIP, bilateral 14-gauge NAc punches (anterior and posterior) were pooled from 5 mice (20 punches). Tissue was lightly fixed to cross-link DNA with associated proteins and the material was further sheared and immunoprecipitated using sheep anti-rabbit magnetic beads conjugated to an antibody that specifically recognizes H3K9me3. Resulting immunoprecipitated DNA and total (input) genomic DNA were prepared for ChIP-sequencing using an Illumina kit according to manufacturer’s instructions. 20 ng of starting material, as determined by PicoGreen concentrations, was used in each case. Briefly, each sample underwent end repair followed by addition of an A base to the 3’ end. Proprietary adapters were then ligated to the ends, followed by size selection on a 3% agarose gel. The range of excision was 175-225 bp. Following DNA clean up, samples were amplified with 21 cycles of PCR. Amplification and size selection were confirmed with a BioAnalyzer. 5 pM concentrations were used to generate clusters for sequencing analysis.

Project description:Many of the long-term effects of cocaine on the brain's reward circuitry have been shown to be mediated by alterations in gene expression. Several chromatin modifications, including histone acetylation and methylation, have been implicated in this regulation, but the effect of other histone modifications remains poorly understood. Poly(ADP-ribose) polymerase-1 (PARP-1), a ubiquitous and abundant nuclear protein, catalyzes the synthesis of a negatively charged polymer called poly(ADP-ribose) or PAR on histones and other substrate proteins and forms transcriptional regulatory complexes with several other chromatin proteins. Here, we identify an essential role for PARP-1 in cocaine-induced molecular, neural, and behavioral plasticity. Repeated cocaine administration, including self-administration, increased global levels of PARP-1 and its mark PAR in mouse nucleus accumbens (NAc), a key brain reward region. Using PARP-1 inhibitors and viral-mediated gene transfer, we established that PARP-1induction in NAc mediates enhanced behavioral responses to cocaine, including increased self-administration of the drug. Using chromatin immunoprecipitation sequencing, we demonstrated a global, genome-wide enrichment of PARP-1 in NAc of cocaine-exposed mice and identified several PARP-1 target genes that could contribute to the lasting effects of cocaine. Specifically, we identified sidekick-1-important for synaptic connections during development-as a critical PARP-1 target gene involved in cocaine's behavioral effects as well as in its ability to induce dendritic spines on NAc neurons. These findings establish the involvement of PARP-1 and PARylation in the long-term actions of cocaine. c57bl/6 mice were given IP injections of chronic cocaine 20mg/kg once per day for 7 days and sacrificed 30 minutes after the final dose of cocaine. Control animals were given saline for 7 days and sacrificed 30 minutes after the final dose of saline. Nucleus accumbens (NAc) tissue was collected and then PARP-1 ChIP-seq was performed. Three sequencing replicates were performed on each group.

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Histone Arginine Methylation in Cocaine Action: Essential Role of Src Signaling in the Nucleus Accumbens

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