Epigenetic mechanisms in drug addiction

Abstract

Changes in gene expression in brain reward regions are thought to contribute to the pathogenesis and persistence of drug addiction. Recent studies have begun to focus on the molecular mechanisms by which drugs of abuse and related environmental stimuli, such as drug-associated cues or stress, converge on the genome to alter specific gene programs. Increasing evidence suggests that these stable gene expression changes in neurons are mediated in part by epigenetic mechanisms that alter chromatin structure on specific gene promoters. This review discusses recent findings from behavioral, molecular and bioinformatic approaches being used to understand the complex epigenetic regulation of gene expression by drugs of abuse. This novel mechanistic insight might open new avenues for improved treatments of drug addiction.

Figure 1

Regulation of chromatin remodeling by drugs of abuse. Cocaine and amphetamine increase levels of cAMP in the nucleus accumbens (NAc) and activate protein kinase A (PKA). PKA then phosphorylates cAMP-response-element-binding protein (CREB), which allows for the recruitment of the histone acetyltransferase CREB-binding protein (CBP). Examples of this are shown on the fosb and c-fos genes. Chronic cocaine or amphetamine is also known to elevate levels of ΔFosB, which can recruit histone deacetylase 1 (HDAC1) to the c-fos promoter and inhibit subsequent induction of the gene (see also Figure 3). This desensitization of c-fos also involves increased repressive histone methylation, which is thought to occur via the induction of specific histone methyltransferases. It is not yet known how cocaine regulates histone demethylases (HDMs) or DNA methyltransferases (DNMTs). Cocaine also activates the mitogen-activated protein kinase (MAPK) cascade, which through MSK1 can phosphorylate CREB and histone H3 at serine 10. In addition, stimulant drugs regulate Ca²⁺ levels in NAc neurons (perhaps via regulation of glutamatergic synapses from cortical regions). This activates CaMK (calcium/calmodulin protein kinase) signaling, which, in addition to phosphorylating CREB, also phosphorylates HDAC5. This results in nuclear export of HDAC5 and increased histone acetylation on its target genes [e.g. the NK1 receptor (also known as the neurokinin 1 or substance P receptor)]. Several other genes have been shown to display increased acetylation on their promoters after cocaine or amphetamine exposure, including cdk5, bdnf and npy. In addition, acute ethanol has been shown to reduce histone acetylation by increasing HDAC activity, whereas withdrawal from chronic ethanol increases histone acetylation by reducing HDAC activity. Figure modified with permission from [24].

Figure 2

Studying chromatin regulation in brain using chromatin immunoprecipitation. (a) Schematic of the chromatin immunoprecipitation (ChIP) protocol. ChIP is a technique used to quantify how much of a specific DNA sequence is occupied by a given histone modification or transcription factor. The technique involves lightly fixing the tissue or cells with formaldehyde to crosslink DNA with the associated histones and other associated proteins (chromatin). The crosslinked chromatin is then sonicated into ∼500 bp fragments and immunoprecipitated with an antibody raised against a specific histone modification or transcription factor. The immunoprecpitated chromatin is then reverse crosslinked from associated proteins and purified (pure DNA). Specific regions of this DNA can then be directly quantified by real time PCR (polymerase chain reaction) to determine how much of that DNA was immunoprecipitated in a drug-treated versus saline-treated animal. The final purified DNA can also be amplified for downstream use in genome-wide analysis techniques, such as microarrays (ChIP-chip) or next generation sequencing (ChIP-seq). (b) ChIP-chip data are typically displayed as an enrichment profile across each chromosome. For example, acetylated H3 (acH3)-binding on chromosome 17 from the nucleus accumbens (NAc) of a cocaine-treated mouse is displayed. One can then compare the enrichment profiles between cocaine- and saline-treated mice to determine the fold difference of acH3 on a specific chromosomal region. (c) The chromosome-wide data shown in (b) can be ‘zoomed in’ to display each gene in the genome. Displayed here is the cdk5 gene promoter in the NAc and the fold difference between cocaine- and saline-treated mice for acH3 (red), acH4 (orange) and methylated H3 (meH3) (blue). (d) The fold differences between cocaine- and saline-treated mice can be quantified for each gene and analyzed for statistical significance. The genes can then be compared and displayed, for example, using Venn diagrams to show how many genes are commonly regulated between two conditions. Shown here are the number of genes in the NAc on which cocaine commonly (overlapping area) or uniquely (non-overlapping areas) increases acH3 and acH4 binding.

Figure 3

Gene-dependent recruitment of chromatin-remodeling enzymes. (a) Cocaine- and amphetamine-induced increases in ΔFosB in the nucleus accumbens (NAc) are known to activate transcription of the cdk5 gene. This involves binding of the ΔFosB/JunD heterodimer to the cdk5 promoter and recruitment of the SWI/SNF ATP-dependent chromatin-remodeling complexes and histone acetyltransferases (HATs). This allows for the assembly of RNA polymerase II (RNA Pol II), Transcription Factor IID (TFIID) and several other protein complexes (not shown) involved in the initiation and elongation of transcription. Histone deacetylases (HDACs) are not present on the cdk5 promoter, which permits significantly higher levels of acetylated histone H3 after chronic cocaine exposure. (b) Cocaine- and amphetamine-induced increases in ΔFosB also act as a transcriptional repressor at a different gene locus, c-fos. After repeated stimulant exposure, the c-fos gene is desensitized in the NAc and much more weakly induced by subsequent drug exposures. This involves the binding of ΔFosB to the c-fos gene promoter and recruitment of HDAC1 to reduce histone acetylation and gene activity. In concert with HDAC1, chronic drug exposure increases the levels of the repressive histone methyltransferase (HMT); KMT1a/SUV39H1 and levels of histone H3K9 methylation on the c-fos promoter. Together, these enzymes and histone modifications serve to repress c-fos gene activity through a mechanism involving ΔFosB and other corepressors (Rep). Figure modified with permission from [24].