This study provides a genome-wide assessment of chromatin and transcriptional alterations in the NAc in response to repeated cocaine administration. Among the many genes that show regulation by cocaine are a large number reported in previous studies to show altered mRNA or protein expression in the NAc after chronic cocaine (see examples in ). For instance, the cocaine-induced genes that encode ARC, CART, CDK5, NFκB, PDYN, σ-opioid receptor, and Period 1 and 2 (Freeman et al., 2001
; McClung and Nestler, 2003
; Yao et al., 2004
) (see also Supplemental References in Supplemental Information
) were shown in the present study to exhibit increased binding of acetylated H3 or H4, with either attenuated or unchanged methylation of H3 at K9/K27. Genes that are known to be downregulated by cocaine, such as the voltage-gated potassium channel Kv8.2 and the microtubule associated protein MTAP2 (Renthal et al., 2007
), were often associated with increased methylation of H3 at K9/K27 (). For each type of histone modification, cocaine induced increases in acetylation or methylation at many more genes than it induced decreases (). Although there are a few examples where reductions in acetylation or methylation are associated with respective changes in gene expression, our data suggest that chronic cocaine more commonly regulates transcription by either increasing histone H3 or H4 acetylation (to elevate mRNA levels), or by increasing histone H3 dimethyl-K9/27 (to reduce mRNA expression). However, these are observations of genome-wide data and exceptions likely exist. There is also a subset of genes which is highly regulated at the chromatin level but show no detectible change in steady-state mRNA expression. For example, mRNA’s for HDAC4 and myocyte-enhancer factors 2A and 2D (MEF2A and MEF2D) are not altered by cocaine (data not shown), but their promoters are dramatically altered after cocaine treatment by histone modifications and/or transcription factor binding. These are particularly interesting examples given the potent influence of HDAC4 (Kumar et al., 2005
) and of MEF2 (Pulipparacharuvil et al., 2008
) in the NAc on cocaine responses, and may illustrate a new layer of regulation not previously appreciated. Similar disconnects between gene activity and gene or protein expression have been observed during cocaine withdrawal (Self et al., 2004
is a good example where hyperacetylation of its promoter (Kumar et al., 2005
) does not correlate with an immediate increase in steady state BDNF expression, however, during cocaine withdrawal, levels of BDNF protein become significantly elevated (Grimm et al., 2003
). Similarly, gene expression arrays found increased MEF2D expression in the NAc after extinction from cocaine self-administration (personal communication, D.W. Self). Thus, histone acetylation at certain genes may represent a priming mechanism to facilitate subsequent gene induction. Taken together, our study corroborates numerous established molecular targets of cocaine action in the NAc, and demonstrates the power of ChIP-chip assays to uncover in a comprehensive manner the genomic targets through which cocaine induces neural and behavioral plasticity in this critical brain reward region. A still further advance would be to determine whether these various cocaine-induced changes in NAc occur in neurons vs. glia (Bowers and Kalivas, 2003
Likewise, results of the present study provide novel insight into the target genes through which ΔFosB and CREB contribute to the genomic effects of cocaine. Several genes previously identified as targets of ΔFosB and CREB, inferred from gene expression array studies of mice overexpressing the transcription factors or their dominant negative antagonists in the NAc (McClung and Nestler, 2003
), were identified in this study. Examples include neurogranin, Period 1, GABAA
receptor subunits, and MEF2C, to name a few. The present findings thereby indicate that many of the ΔFosB and CREB target genes determined through overexpression studies are indeed direct, physiological targets for these transcription factors in the NAc in vivo
. Although to the best of our knowledge there have been no prior genome-wide ChIP-chip studies for FosB or ΔFosB, we should note that many of the phospho-CREB-bound genes observed here after chronic cocaine exposure (e.g., Pdyn
) were also identified as CREB targets by previous CREB ChIP-chip studies (Impey et al., 2004
; Tanis et al., 2007
; Zhang et al., 2005
). While it is known that CREB targets can differ between cell types (Cha-Molstad et al., 2004
; Zhang et al., 2005
), observing many of the same genes regulated by CREB from cultured cells to brain gives us high confidence in the predictive quality of our data.
Of all the genes in the NAc that show markers of activation or repression after chronic cocaine, roughly 14% exhibit altered levels of ΔFosB binding and roughly 16% exhibit altered levels of phospho-CREB binding (; Supplemental Fig. S2
; Supplemental Tables S8
). This is interesting in that our previous DNA expression array study found that similar fractions of genes regulated in the NAc after 5 days of cocaine were also regulated upon ΔFosB overexpression or CREB overexpression, respectively (McClung and Nestler, 2003
). The number of cocaine-regulated genes influenced by ΔFosB increased to >25%, whereas that for CREB decreased to 5%, after 4 weeks of cocaine administration, which illustrates the importance of performing genome-wide ChIP-chip and gene expression studies after longer periods of cocaine exposure and different time points of cocaine withdrawal.
As stated, the dramatic cell type differences that have been reported for the genomic targets of a given transcription factor between even two types of cultured cells means that an absolutely crucial next step in the field is to define modes of chromatin regulation that occur in the brain in vivo
. Indeed, well beyond identifying lists of genes that show interesting patterns of chromatin regulation by cocaine, results of the present study reveal several novel principles by which cocaine regulates gene expression in the NAc of behaving animals. Among the lessons revealed are that most cocaine-regulated genes show altered acetylation either of histone H3 or of H4, with changes at H3 predominating, and alterations either in histone acetylation or in histone methylation, but only rarely both modifications together on the same gene. Another striking lesson is that phospho-CREB exerts complex transcriptional effects and can act as a transcriptional activator or repressor in the brain in vivo
. This is also true for ΔFosB, which has been shown previously (Kumar et al., 2005
; McClung and Nestler, 2003
; Renthal et al., 2008
) and observed in our ChIP-chip data to act as either a transcriptional activator or repressor depending on the gene promoter and conditions involved.
There have been numerous studies of cocaine regulation of gene expression in the NAc and other brain regions by gene expression arrays. This research has revealed large numbers of transcripts that are altered in response to cocaine administration. The ChIP-chip studies reported here enable the coordinated use of both approaches to identify a smaller set of genes in which the field can place greater confidence as being bona fide targets of cocaine and the key transcription factors which mediate cocaine’s effects. Moreover, this work begins to describe the specific mechanisms underlying these cocaine-induced transcriptional changes and reveals fundamentally new insight into the genome-wide patterns of chromatin regulation by cocaine in the NAc. Together, this new insight has led to the identification of a novel family of genes involved in cocaine action in the NAc, the sirtuins, which, as we have shown, play an essential role in addiction-like behavior.
We identified Sirt1 and Sirt2 from our ChIP-chip analyses of ΔFosB target genes that also were regulated by histone acetylation. We then identified significant increases in both Sirt1 and Sirt2 mRNA and protein activity in the NAc after chronic cocaine administration. We showed further that elevated sirtuin activity in the NAc increases the electrical excitability of NAc MSNs and potentiates the rewarding effects of cocaine. Finally, we demonstrated that pharmacological inhibition of sirtuins specifically in the NAc reduced both cocaine’s rewarding effects as well as the motivation to self-administer the drug. Thus, sirtuins appear to act downstream of ΔFosB and may contribute to a positive-feedback loop in which repeated drug exposure increases levels of ΔFosB and sirtuins, which in turn enhances the motivation to take additional drug. These findings raise the possibility of using SIRT1/2 inhibitors as potential treatment agents for cocaine addiction.
Taken together, our ChIP-chip analyses have revealed a novel family of proteins involved in cocaine responses and underscore the vast clinical potential of the many other new gene targets identified in this study for the development of more effective treatments of cocaine and potentially other drug addictions.