Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Apr 22;15(4):R65.
doi: 10.1186/gb-2014-15-4-r65.

Chronic cocaine-regulated epigenomic changes in mouse nucleus accumbens

Jian Feng, Matthew Wilkinson, Xiaochuan Liu, Immanuel Purushothaman, Deveroux Ferguson, Vincent Vialou, Ian Maze, Ningyi Shao, Pamela Kennedy, JaWook Koo, Caroline Dias, Benjamin Laitman, Victoria Stockman, Quincey LaPlant, Michael E Cahill, Eric J Nestler, Li Shen

Chronic cocaine-regulated epigenomic changes in mouse nucleus accumbens

Jian Feng et al. Genome Biol. .

Erratum in

Abstract

Background: Increasing evidence supports a role for altered gene expression in mediating the lasting effects of cocaine on the brain, and recent work has demonstrated the involvement of chromatin modifications in these alterations. However, all such studies to date have been restricted by their reliance on microarray technologies that have intrinsic limitations.

Results: We use next generation sequencing methods, RNA-seq and ChIP-seq for RNA polymerase II and several histone methylation marks, to obtain a more complete view of cocaine-induced changes in gene expression and associated adaptations in numerous modes of chromatin regulation in the mouse nucleus accumbens, a key brain reward region. We demonstrate an unexpectedly large number of pre-mRNA splicing alterations in response to repeated cocaine treatment. In addition, we identify combinations of chromatin changes, or signatures, that correlate with cocaine-dependent regulation of gene expression, including those involving pre-mRNA alternative splicing. Through bioinformatic prediction and biological validation, we identify one particular splicing factor, A2BP1(Rbfox1/Fox-1), which is enriched at genes that display certain chromatin signatures and contributes to drug-induced behavioral abnormalities. Together, this delineation of the cocaine-induced epigenome in the nucleus accumbens reveals several novel modes of regulation by which cocaine alters the brain.

Conclusions: We establish combinatorial chromatin and transcriptional profiles in mouse nucleus accumbens after repeated cocaine treatment. These results serve as an important resource for the field and provide a template for the analysis of other systems to reveal new transcriptional and epigenetic mechanisms of neuronal regulation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Chronic cocaine-induced transcriptome in mouse NAc. (A) Cocaine-regulated transcriptomic events versus total events illustrated as percentages (for example, 92 out of 22,000 genes show significant expression changes), which are based on Cuffdiff prediction (see Materials and methods). (B) Gene Ontology (GO) enrichment for differentially spliced genes is illustrated as an enrichment map [26]: each node represents an enriched GO term and its size represents the number of genes; the color intensity of the node represents the statistical significance; the strength of the green lines represents the number of common genes shared between two nodes; the GO terms that are clustered together are circled and summarized with a name. Singletons are not shown; a full list of enriched GO terms is in Additional file 6.
Figure 2
Figure 2
Role of A2BP1 in cocaine responses. (A) Co-immunoprecipitation shows A2BP1 western blotting of H3K4me3 or IgG pulldown of whole NAc lysate. Two repeated cocaine-treated samples and two saline control samples are shown. (B) The two Venn diagrams show overlap between: A2BP1-motif containing genes versus H3K4me3 differential site-containing genes (upper); A2BP1 motif- and H3K4me3 differential site-containing genes versus cocaine-regulated differential and spliced genes (lower). The table shows the top five enriched functional terms of the 478 overlapped genes from the lower Venn diagram. (C) Western blot analysis of A2BP1 in nuclear lysates of NAc after repeated cocaine. Error bars are mean ± standard error of the mean (SEM) derived from eight cocaine treated and eight saline treated control mice, respectively. (D) Conditioned place preference (CPP) scores of AAV-Cre-GFP-injected A2bp1loxP/loxP mice (cre) with AAV-GFP-injected A2bp1loxP/loxP mice as control (con). Error bars are mean ± SEM derived from seven cre and eight control samples. (E) Four predicted A2BP1 target candidate genes were chosen for Nanostring validation. All show the same direction of transcription change after chronic cocaine treatment as observed in RNA-seq. Error bars are mean ± SEM derived from 14 cocaine and 14 saline treated samples. (F) Cocaine-induced transcription change as observed in (E) are lost (Rps6kb2, Zfp26, Dvl1) or in one case reversed (Ece2) when A2bp1 was conditionally knocked down by using AAV-Cre viral injection in NAc. Error bars are mean ± SEM derived from five cocaine and six saline treated samples. *P < 0.05, **P < 0.01.
Figure 3
Figure 3
Repeated cocaine-regulated epigenome measured by ChIP-seq. (A-C) Averaged coverage plots of different biological replicates of H3K9me2 and DNA input for different genomic regions. The x-axis represents the genomic region from 5’ to 3’. The y-axis represents coverage that has been normalized to the number of aligned reads per million mapped reads (RPM). (A) Genebody of the entire mouse genome. (B) Differential sites that show decreased H3K9me2 binding in genic regions. (C) Differential sites that show increased H3K9me2 binding in genic regions. (D) Pie charts show the genome-wide distribution patterns of the differential sites of the seven chromatin marks.
Figure 4
Figure 4
Heatmap of cocaine-regulated chromatin modifications of the 29 signature clusters that associate with transcriptional regulation. In the left panel, each row represents a cluster and each column represents an epigenomic mark at a specific genomic region; each square represents the averaged chromatin modification change in log2 scale; purple and orange colors indicate increased or decreased binding and the darkness indicates the magnitude of change. The heatmap in the right panel illustrates the statistical significance of each cluster’s association with transcriptional change. N.S., not significant.
Figure 5
Figure 5
The functional enrichment of the 29 signature clusters and identification of significant chromatin modification regions for an example cluster. (A,B) Each row represents a cluster and each column represents a functional term or canonical pathway. The color of each square represents the statistical significance of enrichment. The enriched terms are ranked by co-occurrence and only the top 10 are shown. (A) Biological functions. (B) Canonical pathways. N.S., not significant. (C) The chromatin modification heatmap for cluster 9. Each column represents a transcript and each row represents a chromatin mark at a specific genomic region. Only 10 example transcripts are shown here. Each square represents the chromatin change in log2 scale. The transcriptional change is simply indicated as a binary value. The significantly modified regions are marked by asterisks to the right of their names. An example transcript, ENSMUST00000141539 of gene Dvl1 (a protein in the WNT/β-catenin pathway), is further illustrated in (D-F). (D) Genome browser screenshot for the demo transcript with H3K4me3 and H3K27me3 in the top and bottom two tracks, respectively. The exon structures of the demo transcript and another transcript of the same gene are shown below the chromatin tracks. The genomic regions are marked by letters ('u', upstream intergenic; 'p', promoter exon; 'c', canonical exon; 'v', variant exon; 'i', intron), and are further followed by numbers to distinguish different regions. (E,F) Mean and standard error of the mean of the significant regions with some neighbors are shown as line plots with error bars. The x-axis represents different regions from 5’ to 3’ and the y-axis represents normalized coverage. (E) H3K4me3 at u, p, i1, i2 and c. (F) H3K27me3 at v1, i3, i4, v2 and i5.
Figure 6
Figure 6
Splicing and transcription factors inferred from the 29 signature clusters induced by repeated cocaine and their interacting epigenomic marks. Splicing and transcription factors are indicated on the right panel by different colors. Green means the enrichment of a splicing factor on the left; red means the enrichment of a transcription factor on the right; blue means the enrichment of both. All 32 splicing factors and the top 32 transcription factors are shown here. Each column represents a signature cluster. The gene expression level for the regulators is labeled as 'Very High' (RPKM >20), 'High' (RPKM >5 and ≤20), 'Medium' (RPKM >1 and ≤5), and 'Low' (RPKM ≤1) based on our RNA-seq data. The chromatin marks that interact with the splicing factors are shown on the left panel by pink color.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Nestler EJ. Molecular basis of long-term plasticity underlying addiction. Nat Rev Neurosci. 2001;2:119–128. doi: 10.1038/35053570. - DOI - PubMed
    1. Robison AJ, Nestler EJ. Transcriptional and epigenetic mechanisms of addiction. Nat Rev Neurosci. 2011;12:623–637. doi: 10.1038/nrn3111. - DOI - PMC - PubMed
    1. Day JJ, Sweatt JD. DNA methylation and memory formation. Nat Neurosci. 2010;13:1319–1323. doi: 10.1038/nn.2666. - DOI - PMC - PubMed
    1. Peixoto L, Abel T. The role of histone acetylation in memory formation and cognitive impairments. Neuropsychopharmacology. 2013;38:62–76. doi: 10.1038/npp.2012.86. - DOI - PMC - PubMed
    1. Jonkman S, Kenny PJ. Molecular, Cellular, and Structural Mechanisms of Cocaine Addiction: A Key Role for MicroRNAs. Neuropsychopharmacology. 2013;38:198–211. doi: 10.1038/npp.2012.120. - DOI - PMC - PubMed

Publication types