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Persistent variations in neuronal DNA methylation following cocaine
self-administration and protracted abstinence in mice
Danay Baker-Andresen a, Qiongyi Zhao a, Xiang Li a,b, Bianca Jupp c,d, Rose Chesworth c,Andrew J. Lawrence c,⁎, Timothy W. Bredy a,b,⁎⁎a Queensland Brain Institute, The University of Queensland, Brisbane 4072, Australiab Center for the Neurobiology of Learning and Memory and Department of Neurobiology and Behavior, University of California, Irvine, CA 92617, United Statesc Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville 3052, Australiad Behavioural and Clinical Neuroscience Institute, University of Cambridge, Cambridge CB2 2QQ, UK
⁎ Correspondence to: A. J. Lawrence, Florey Institute of NUniversity of Melbourne, Parkville 3052, Australia.⁎⁎ Correspondence to:T. Bredy,QueenslandBrain InstitutBrisbane 4072, Australia.
A central factor in the development of cocaine addiction is thepathological overlearning of associations between cues in the druguse environment and the rewarding effects of the drug. Memories ofthese associations are extraordinarily persistent and readily reacti-vated following re-exposure to cocaine-paired cues, which canprompt powerful cravings for cocaine and relapse despite prolongedabstinence (Childress et al., 1999; O'brien et al., 1998). However, themolecular features that sustain cocaine-associated memories andenhanced vulnerability to relapse during abstinence remain enigmat-ic. Accumulating evidence suggests that persistent learning-inducedepigenetic modifications support the maintenance of long-termmemories (Miller et al., 2010; Mizuno et al., 2012) and long-termbehavioral adaptation (Champagne and Curley, 2009; Weaver et al.,
euroscience andMental Health,
e, TheUniversity ofQueensland,
.J. Lawrence),
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2005). In particular, relatively stable inducible epigenetic modifica-tions, such as DNA methylation, may mediate the maintenance ofmemory and enduring behavioral propensities (Griffith and Mahler,1969; Miller et al., 2010). Therefore, persistent learning-inducedchanges in DNA methylation could function as a conserved mecha-nism of memory maintenance and underlie both the persistence ofcocaine-related memories and enduring cocaine-seeking behaviorduring abstinence.
Long-lasting changes in DNA methylation induced by cocaineself-administration may arise in a brain region- and cell type-specificmanner. Enduring changes in DNA methylation likely occur through-out reward-related neural circuitry during drug abuse, yet thosearising within the medial prefrontal cortex (mPFC) may be particu-larly relevant to the maintenance of enduring cocaine-relatedmemories. In self-administering animals and human users, exposureto previously cocaine-paired cues is associated with hyperactivitywithin the mPFC (Childress et al., 1999; Ciccocioppo et al., 2001) andthe mPFC is further required for the reinstatement of cocaine seekingduring abstinence (Capriles et al., 2003; Di Pietro et al., 2006).Together, this suggests that lasting neuroadaptive changes in themPFC underpin continued cocaine seeking during abstinence. More-over, the enduring contribution of the mPFC to drug-seeking behavior
nder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Fig. 1. Mice continue to seek cocaine during protracted abstinence. A two-way ANOVArevealed that mice press the previously cocaine-paired lever significantly more thanthe inactive lever after 1 and 21 days of forced abstinence (F1,128 = 198.6, p b 0.0001,IVSA (Day 10–12): n = 34, IVSA 1: n = 18 and IVSA 21: n = 15). There was nodifference in the number of cocaine-paired lever presses performed during the last3 days of cocaine self-administration or after 1 or 21 days of forced abstinence(F2,128 = 0.40, not significant). Data are displayed as mean lever presses ± SEM;IVSA 1: mice tested after 1 day of abstinence, IVSA 21: mice tested after 21 daysof abstinence.
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is echoed in steadfast transcriptional and proteomic changes that persistfor up to 100 days of forced abstinence following cocaine self-adminis-tration (Freemanet al., 2008, 2010; Lull et al., 2009). Finally, at the cellularlevel, theneuronal genomecould represent amore suitable repository forpersistent acquired epigenetic modifications, as mature neurons are notsubject to cell division (Griffith and Mahler, 1969).
Recent evidence indicates that cocaine exposure can inducechanges in DNA methylation (Anier et al., 2010; Barros et al., 2011;Carouge et al., 2010; Laplant et al., 2010; Massart et al., 2015; PolBodetto et al., 2013; Tian et al., 2012; Wright et al., 2015) and otherbase modifications (Feng et al., 2015); however, the majority ofinvestigations fail to distinguish between modifications arising fromsimple cocaine exposure and those that are associated with learnedcocaine-seeking. This distinction is important, as passive involuntarycocaine exposure is aversive (Twining et al., 2009) and fails to producethe persistent changes in long-term potentiation (Chen et al., 2008;Martin et al., 2006) and sharp rise in extracellular dopamine (Hembyet al., 1997) and acetylcholine (Mark et al., 1999) that may underlieoverlearning and the extreme persistence of cocaine-associatedmemories as well as the development of addiction. We thereforeemployed a mouse model of intravenous cocaine self-administration(IVSA) that contrasts learned cocaine self-administration with passivecocaine exposure to identify modifications in DNAmethylation that areuniquely associated with learned cocaine-seeking and enduringcocaine-seeking during abstinence.
2. Materials and methods
2.1. Animals
Adult male C57BL/6J mice (8–9 weeks of age at the start ofexperiments, 20–24 g) were singly housed under a 12 h reverselight–dark cycle (lights off at 7 am) in standard housing conditionswith ad libitum access to standard rodent chow and water unlessotherwise specified. All experiments were performed with approval
from the Animal Ethics Committee of the University of Melbourne inaccordance with the Prevention of Cruelty to Animals Act (1986) andthe Australian Code of Practice for the Care and Use of Animals forScientific Purposes.
Operant self-administration of cocaine (5 mg/kg/infusion) wasperformed with minor modifications to previously described proto-cols (Brown et al., 2009; Mcpherson et al., 2010). Briefly, the animalswere divided into three experimental subgroups (naïve: n = 13;self-administering (IVSA), n = 33; yoked controls: n = 7). Naïveanimals remained in their home cages for the duration of operantconditioning. The instrumental response (lever press) was estab-lished in IVSA animals using 10% w/v oral sucrose under a FR1 (fixedratio 1) schedule, as previously described (Brown et al., 2009). Yokedcontrol animals received equivalent amounts of sucrose, contingentupon the responses of their paired self-administering counterparts.Following the acquisition of the instrumental response by IVSAanimals, both yoked and self-administering animals were surgicallyimplanted with an indwelling jugular catheter (Brown et al., 2009).
After a two-day recovery period, IVSA mice self-administeredcocaine intravenously under a FR1 schedule, over 12 daily, 2 hsessions. Responses on the reward-paired lever resulted in theinfusion of 0.5 mg/kg of cocaine hydrochloride (Sigma Aldrich, inphysiological saline) over 1.7 s (infusion volume of 19 μl) and theconcurrent presentation of a 5 s light cue. To minimize the risk ofoverdose, cocainewas unavailable during the ongoing presentation of thelight cue (“time out” period, responses recorded) and a within-sessionmaximum of 80 infusions was applied. Yoked animals receivedcocaine intravenously, concurrent with earned infusions deliveredto paired self-administering mice. Following self-administrationtraining, mice were assigned to groups (IVSA 1: 1 day of abstinence +relapse test, n = 18; IVSA 21 R: 21 days of abstinence + relapsetest, n = 15; IVSA 21 NR: 21 days of abstinence + no relapse test,n = 7) counterbalanced for cocaine-seeking behavior (determinedby the average number of infusions received over the final 3 daysof cocaine self-administration). Yoked animals were culled aftereither 1 (Yoked 1, n = 3) or 21 days (Yoked 21, n = 4) of forcedabstinence. During the abstinence period all animals remained intheir home cages.
2.3. Cue-induced cocaine seeking during abstinence
With the exception of IVSA 21 NR mice, self-administeringmicewere subject to a 1 h cocaine-seeking (relapse) test in the absenceof cocaine after either 1 (IVSA 1) or 21 days (IVSA 21) of forcedabstinence (Brown et al., 2009; Madsen et al., 2012). All cocaine-pairedcues were present and cocaine-seeking behavior was measured as thesum of all responses on the previously cocaine-paired lever. Yokedanimals underwent simple contextual re-exposure for 1 h. The animalswere killed by cervical dislocation either immediately after testing(next-generation sequencing) or 2 hpost-test (validationof sequencingand gene expression). Their brains were then removed, snap-frozenover liquid nitrogen and stored at−80 °C.
2.4. Identification of genome-wide changes in DNA methylation by MBDUltra-Seq
Individual mPFCs were isolated by Palkovits punch (Palkovits,1973). Neuronal genomic DNA was isolated from the mPFC ofindividual animals, as previously described (Li et al., 2014). Next-generation sequencing was performed by Methyl CpG BindingDomain (MBD) Ultra-Seq (Li et al., 2014) with two minor modifica-tions: 75 ng of genomic DNA from each animal was used for librarypreparation and the final pooled, amplified methyl-enriched library
Fig. 2. Representative heatmap of 5mC enrichment. All RMEs that were supported by enrichment (relative to background genomic coverage as identified by MACS) in at least 50% ofbiological replicates in naïve (n = 5), IVSA 1 (n = 7) or IVSA 21 (n = 6) animals are plotted. White indicates that no biological replicates displayed enrichment for 5mC, while darkred indicates that all biological replicates had enrichment for 5mC at the given genomic locus. The ratio is indicative of the number of animals within a group that displayed anenrichment for 5mC at a given RME. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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for each treatment group was loaded in duplicate, in different lanes ofthe flow cell, to prevent the production of artificial differences byinter-lane discrepancies. Regions of methylation enrichment (RMEs,or peaks) were identified using Model-based Analysis of ChIP-Seq(MACS) (Zhang et al., 2008) and previously established thresholds(Li et al., 2014).
2.5. Candidate DMR selection
To identify differentially methylated regions (DMRs), RMEscovered by a minimum of 5 normalized reads in at least one of thetreatment groups were selected. From this selection, RMEs within600 bp of each other were grouped and defined as a single RME.Student's t-tests were performed to compare the relative level of5-methylcytosine (5mC) enrichment at each RME (as measured bythe normalized read counts) between naïve and cocaine-treatedanimals (IVSA 1, IVSA 21, Yoked 1, Yoked 21). The Benjamini–Hochberg false discovery rate (FDR) correction was used to accountfor the effects of multiple testing and to calculate adjusted p-values,allowing for a false discovery rate of 10%. The level of 5mC enrichmentwas considered significantly different between groups if the FDR-adjusted p-value from the Student's t-test was less than 0.1 (Ellis et al.,
2012; Non et al., 2014). Persistent DMRs arising from cocaineself-administration were those that were significantly different inthe IVSA 1 and IVSA 21 groups, but not in the yoked cocaine controls,relative to naïve animals. Levels of 5mC enrichment at abstinence-associated DMRs were significantly different from those in naïveanimals after 21 days of abstinence from cocaine self-administration,but not following 21 days of abstinence from yoked cocaine exposure.Furthermore, we verified that the genomic region (RME summit+/−150 bp) surrounding each DMR of interest contained at least5 CpG dinucleotides, as the MBD2b/3L1 complex captures fragmentsof DNA containing a minimum of 5 methylated CpGs (Active Motif).Finally, we confirmed that the 300 bp region overlapping eachDMR corresponded to a unique location in the mouse referencegenome (mm9) to avoid artifacts produced by poor mappingof repetitive regions. Associations between significant DMRs(persistent and abstinence-associated) and regulatory features ofthe genomewere examined using EpiExplorer (Halachev et al., 2012),a web-based tool that identifies genes and regulatory featuresoverlapped by user-defined genomic regions. DMRs wereconsidered to co-localize with regulatory features if at least 10% ofthe DMR (peak summit +/−150 bp) was shared by the regulatoryfeature annotation.
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2.6. Validation of select DMRs by MBD quantitative PCR (MBD qPCR)
MBD pull-downs were performed using 150 ng of neuronal DNAderived from the mPFCs of individual animals from a second cohort ofanimals. Genomic DNA was extracted as previously described (Li et al.,2014), although two 250 μl aliquots were retained for the analysis ofgene expression following the homogenization of the tissue. Briefly,genomic DNA was sheared by sonication in 130 μl of ultrapure H2O(Covaris S2, bath temperature: 4 °C, duty: 10%, intensity: 6, cycle/burst:100, time: 180 s) to fragments of ~300 bp in length, as verified byBioanalyzer HS (Agilent). 13 μl of fragmented DNAwas diluted in a totalvolume of 80 μl of DNAse/RNase-free TE buffer (pH 8) and retained asinput to control for slight variations in the amount of DNA used in eachpull-down. MBD pull-downs were performed according to themanufacturer's protocol (Methylcollector Ultra, ActiveMotif). Capturedmethylated fragments were eluted in 60 μl of TE buffer (pH 8).
A 300 bp region surrounding each candidate DMR was retrieved(UCSC genome browser, mm9), and primers (Supplementary Table 1)were designed using Primer3 (Untergasser et al., 2012) to amplify120–300 bp regions overlapping the peak summit of select candidateDMRs. qPCR was performed in duplicate using a Rotor gene Q(Qiagen) in 10 μl reactions (5 μl 2X SYBR-green master mix (Qiagen),1 μl 10 μM primer (forward + reverse), 3 μl H2O, 1 μl MBD DNA/input DNA). Ct values for methyl-enriched DNA were normalized toinput and the relative enrichment between groups was calculatedusing the ΔΔCT method, relative to naïve animals.
2.7. Analysis of gene expression
250 μl of whole mPFC homogenate was retained from each animal inthe secondbiological cohort following homogenization of the brain tissue.RNA was extracted by Trizol LS (Invitrogen) according to the manufac-turer's protocol and quantified by spectrophotometry (Nanodrop).250 ngofRNAwas reverse transcribed(QuantiTectReverse Transcription,Qiagen). cDNA was diluted to approximately 100 ng/μl prior to qPCR(Nanodrop).WholemPFChomogenatewasusedbecause it is not possibleto obtain mRNA (which resides in the cytoplasm) from neuronal nuclei.
Primers (IDT) for gene expression analysis were designed usingPrimer3 (Untergasser et al., 2012) or AutoPrime (Wrobel et al., 2004)or obtained from PrimerBank (Wang et al., 2012) (SupplementaryTable 2). In select cases, particularly when intragenic methylation waspresent, the expression of multiple isoforms of a gene was examined.qPCR was performed in triplicate using a Rotor gene Q (Qiagen) in10 μl reactions (5 μl of 2X SYBR-greenmaster mix, 2 μl of 5 μMprimer(forward + reverse), 2 μl H2O, 1 μl 100 ng/μl cDNA). Relativeexpression was quantified using the ΔΔCT method relative to naïveanimals. Within each animal, expression levels of genes of interestwere normalized to the level of dynein expression.
3. Results
3.1. Mice continue to seek cocaine during protracted abstinence
We first verified that mice continue to seek cocaine duringabstinence, as continued cocaine-seeking behavior is indicative of
Fig. 3. Validationof candidateDMRsandexpressionof co-localizing genes. Inan independent cohselect DMRs and qPCR was used to examine expression of co-localizing genes. (a) In accordanself-administration; F3,17 = 3.57, p b 0.05. (b) The decreased DNA methylation was correlatedregardless of relapse testing; F3,26 = 5.42, p b 0.01. (c) TheDMR locatedwithinKctd16 displayecocaine self-administration; F3,16 = 2.73, p = 0.07. (d) Interestingly, a trend towards a significrelapse testing; F3,26 = 6.55, p b 0.01, Holm–Sidak post hoc test IVSA 21 R vs. naïve, p = 0.07.relapse testing (IVSA 21 R) compared to those that were simply sacrificed (IVSA 21 NR), p b
relationship between altered DNA methylation and gene expression. (e) Demethylation was(f) Decreased DNA methylation was not associated with a significant change in the expressreproduced following 21 days of abstinence regardless of relapse testing; F3,17 = 5.59, p b 0.0significant reduction in the expression ofGlra1 (all isoforms) after 21 days of abstinence;WelchSEM, and p-values are derived from Holm–Sidak post hoc tests relative to naïve animals, exce
the presence of long-lasting neuroadaptations. Continued cocaine-seeking behavior manifests as a sustained and significant preferencefor the previously cocaine-paired lever relative to the inactive lever. Inour paradigm, mice trained to self-administer cocaine continued toseek cocaine during abstinence, with high discrimination for thecocaine-paired lever over the inactive lever during days 10–12 ofintravenous self-administration and following 1 or 21 days ofabstinence (F1,128 = 198.6, p b 0.0001, Fig. 1). Cocaine-seekingbehavior did not differ across the end of self-administration trainingor throughout period of enforced abstinence (Holm–Sidak post hoctests, IVSA (Day 10–12) vs. IVSA1, IVSA (Day 10–12) vs. IVSA 21 andIVSA 1 vs. IVSA 21, not significant). Therefore, this behavioralparadigm produces enduring cocaine-seeking behavior and is suitablefor the examination of persistent neuroadaptations following cocaine-self administration.
To identify changes inDNAmethylation specific to self-administeringanimals, we performed genome-wide sequencing byMBD Ultra-Seq (Liet al., 2014) on neuronal DNA derived from the mPFCs of naïve animals(n = 5), mice that had self-administered cocaine and been sacrificedafter either 1 or 21 days of abstinence (n = 7 and 6 respectively) andyoked cocaine controls sacrificed at the same time points (n = 3 and 4respectively). Comparisons between self-administering animals andyoked cocaine controls served to isolate modifications of DNAmethylation produced by voluntary learned cocaine-seeking fromthose produced by passive cocaine exposure. MACS (Zhang et al.,2008) identified 46,464 RMEs across the genome (Supplementary File1). A representative heatmap of 5mC enrichment revealed that passivecocaine exposure and cocaine self-administration produced distinctpatterns of 5mC enrichment (Fig. 2).
3.3. Persistent changes in DNA methylation
Patterns of DNA methylation that arise during cocaine self-administration and persist over time are consistent with the enduringnature of cocaine-seeking behavior and may mediate the mainte-nance of long-lasting cocaine-related memories. Twenty-nine geno-mic regions became differentially methylated in a persistent manner(relative to naïve animals) in response to cocaine self-administrationbut not passive cocaine exposure (p b 0.1 in IVSA 1 and IVSA 21animals, p N 0.1 in Yoked 1 and Yoked 21 animals, FDR-adjustedp-values derived from Student's t-tests relative to naïve animals,Supplementary File 2). Five persistent DMRs became demethylatedfollowing cocaine self-administration, whereas the remaindershowed an increase in DNA methylation. The genomic coordinatesof the persistent DMRs and associated or proximal genes are listed inSupplementary Table 3.
EpiExplorer (Halachev et al., 2012) was used to identify associa-tions between persistent IVSA-associated DMRs and genomic regula-tory features. Fifteen of 29 DMRs were embedded within genes(coding and non-coding) and a further 5 were located distal (b10 kb)to a gene (NCBI Ensembl Build 37). A chief function of persistentIVSA-associated modifications of DNA methylation may therefore be
ortof animals,MBDqPCRwasperformed to assess the relative levelsof 5mCenrichmentatce with sequencing results, the locus within Golgb1 was demethylated following cocainewith a significant reduction in the expression of Golgb1 (all isoforms) at all time points
d a near-significant trend towardspersistentmethylation in all treatment groups followingant decrease in Kctd16 gene expressionwas observed only after 21 days of abstinence andAfter 21 days of abstinence, Kctd16 expression was decreased in animals that underwent0.05 (Holm–Sidak post hoc test), which suggests that the relapse test may influence thealso replicated at the intergenic locus located proximal to Snw1; F3,17 = 4.35, p b 0.05.ion of Snw1; F3,26 = 0.07. (g) Finally, demethylation of the Glra1-associated DMR was1. (h) Demethylation of the Glra1-associated DMR was associated with a trend towards a's F3,13.36 = 7.52, p b 0.01, Games–Howell post hoc tests. All data are displayed asmean ±pt where specifically indicated. *p b 0.05, and **p b 0.01.
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the regulation of gene transcription. Moreover, several (13 of 29)persistent DMRs overlapped DNase I-hypersensitive sites (DNAse I HSsites). This further suggests that persistent changes in DNA methyl-ation could regulate gene transcription, as DNAse I HS sites areclassically associatedwith open chromatin and transcriptional activity(Weintraub and Groudine, 1976). Interestingly, gene-associatedDMRs may regulate the expression of specific splice variants, as 12of 15 persistent gene-associated DMRs were located within intronicregions or non-coding loci, whereas principal promoter regions(−5 kb to 1 kb from transcription start sites) and exons wererelatively devoid of changes. Persistent IVSA-associated DMRs wereoften located within repetitive elements (19 of 29 DMRs overlappedrepetitive elements identified by RepeatMasker); however, asrepetitive elements tend to be heavily methylated, it is often easierto discern changes in DNA methylation at these loci when usingenrichment-based methods to detect DNA methylation (Hardcastle,2013). Nevertheless, cocaine exposure leads to repetitive elementunsilencing (Maze et al., 2011), and it is therefore possible thatpersistent IVSA-associatedmodifications of DNAmethylation regulaterepetitive element activity.
3.4. Abstinence-associated changes in DNA methylation
Abstinence-associated changes in DNAmethylation denote chang-es in DNA methylation that were observed in animals subject to21 days of forced abstinence following cocaine self-administrationtraining, but not in those subject to 1 day of abstinence or in yokedcocaine controls (relative to naïve animals). Abstinence-associatedDMRs may arise as a consequence of withdrawal, but could equally bepertinent to the maintenance of cocaine-related memories. Duringabstinence, interoceptive cues (such as withdrawal states) mayprompt the retrieval and reconsolidation of cocaine-related memo-ries. The retrieval and reconsolidation of memories could drive furtherchanges in DNA methylation (Maddox and Schafe, 2011), which maybe associated with an increased persistence of memory, as reconso-lidation can strengthen memory (Tronson and Taylor, 2013).Abstinence-associated DNA modifications that are unique to self-administering animals could arise from the retrieval and reconsolida-tion of cocaine-related memories and therefore be relevant to themaintenance of these memories as well as enduring cocaine-seekingbehavior. Twenty-eight genomic regions (Supplementary Table 4)became differentially methylated during abstinence from cocaineself-administration but not passive cocaine exposure (p b 0.1 in IVSA21 animals, p N 0.1 in IVSA 1, Yoked 1 and Yoked 21 animals,FDR-adjusted p-values relative to naïve animals, p b 0.05 IVSA 21 vs.IVSA 1 and Yoked 21, follow-up Student's t-tests, Supplementary File3). Eight abstinence-associated DMRs became demethylated duringprolonged abstinence whereas the remainder was methylated.Thirteen of 28 DMRs were located within genes and a further 3were located within 10 kb of a gene. Again, gene-associated DMRspredominantly arose within introns or non-coding loci; only 5 of 19gene-associated DMRs were located within exons or promoterregions. A greater percentage of abstinence-associated DMRs wasfound within 10–100 kb of annotated genes (39% of abstinence-associated DMRs as opposed to 14% of persistent DMRs) which mayindicate a more pronounced role for abstinence-associated DMRs inthe regulation of enhancer or insulator regions across the genome.Once more, abstinence-associated DMRs frequently occurred within
Fig. 4. Expression of genes co-localizing with DMRs that cannot be validated by MBD qPCR. (awithin Mctp1 (left panel). Increased DNAmethylation correlated with a concomitant decrease(right panel). (b) Cocaine self-administration resulted in a persistent increase in DNAmethylatassociated with a long-lasting upregulation of Cpeb4 (001/201 isoform) expression; F3,26 = 5.9Nkain3was observed after 21 days of abstinence (left panel) and the increase inDNAmethylatioabstinence and relapse testing (right panel), though this decreasewas not observedwhen the re21 NR, P b .01. Data are displayed as mean ± SEM, all P values are derived from Holm-Sidak p
repetitive elements (16 of 28 DMRs overlapped repetitive elements).Surprisingly, 24 of 28 abstinence-associated DMRs overlapped orwere located proximal to nuclear lamina domains (13 overlapped and11 were located within 1 kb of nuclear lamina domains). The unusualassociation of abstinence-associated DMRs with lamina domainssuggests extensive repositioning of the genome during abstinence,potentially with profound transcriptional consequences.
3.5. Validation of changes in DNA methylation
A number of DMRs were validated by MBD qPCR in an independentcohort of animals. An additional group that did not undergo relapse testingafter 21 days of abstinence (IVSA 21 NR) was included to ensure thatabstinence-associated changes in DNA methylation were not simply theproduct of the cocaine-seeking (relapse) test. Select DMRs included threeregions that were persistently differentially methylated following cocaineself-administration and one that became differentially methylated duringabstinence. Themeannormalized read distribution at each candidateDMRis provided in Supplementary Fig. 1. All candidate DMRs selected forvalidation by MBD qPCR validated reliably (Fig. 3), as was an additionalcandidateDMRwithinCdh13 (Supplementary Fig. 2, this candidatedidnotmeet the final statistical criteria for selection), indicating that MBDUltra-Seq and the statistical limitations employed reliably identify changesin DNAmethylation induced by cocaine self-administration.
3.6. Altered DNAmethylation is associatedwith changes in gene expression
In addition to propagating enduring changes in gene transcription,persistent experience-induced modifications of DNA methylationcould act as silent signatures of cocaine-related learning and primethe transcription of the affected locus upon reactivation of cocaine-related memories, representing a form of genomic metaplasticity(Baker-Andresen et al., 2012). To examine this possibility, weexamined gene expression in animals that were subject to a relapsetest after 21 days of abstinence (IVSA21 R) and others that weresimply sacrificed at the same time point (IVSA21 NR). The relapse testserved to explicitly reactivate cocaine-associated memories, whereasthese memories would have remained relatively dormant in animalsthat were not subject to a relapse test. Golgb1 and Glra1 expressionswere decreased regardless of whether or not relapse occurred(Golgb1: F3,26 = 5.42, p b 0.01, Holm–Sidak post hoc test, naïve vs.IVSA 1, IVSA 21 R and IVSA 21 NR all p b 0.01, GLRa1: Welch'sF3,13.36 = 7.52, p b 0.01, Games–Howell post hoc test, naïve vs. IVSA21 R and IVSA 21 NR, p b 0.1) (Fig. 3b and h respectively). Conversely,Kctd16 expression was altered only in animals that underwent arelapse test (F3,26 = 6.55, p b 0.01, Holm–Sidak post hoc test, naïvevs. IVSA 21 R, p b 0.1, IVSA 21 R vs. IVSA 21 NR, p b 0.05) (Fig. 3d),despite increased DNA methylation across all groups followingcocaine self-administration (Fig. 3c). The intergenic change in DNAmethylation located distal to Snw1 (Fig. 3e) had no effect on itsexpression (F3,26 = 0.07, not significant; Fig. 3f). The change in DNAmethylation within Cdh13 was associated with a trend towardsreduction in the expression of this gene (F3,26 = 2.53, p = 0.07;Supplementary Fig. 2) regardless of relapse testing. Therefore,IVSA-related changes in DNA methylation are associated with alteredexpression of co-localizing genes, although in some instances thisassociation is modulated by the reactivation state of the cocaine-related memories.
) Cocaine self-administration yielded a persistent increase in DNA methylation at a locusin the expression of Mctp1 (−001 isoform) across treatment groups; F3,26 = 4.07, P b .05ion within an intragenic region of Cpeb4 (left panel) and increased DNAmethylation was6, P b .01 (right panel). (c) A significant increase in DNA methylation within an intron ofnwas associatedwith a decrease in the expression ofNkain3 (all isoforms) after 21 days oflapse test did not occur; F3,26=4.56, P b .05, naïve vs. IVSA21R, P b .05, IVSA21R vs. IVSAost hoc tests relative to naïve animals, *P b .05 and **P b .01.
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A number of significant DMRs were located within lengthyrepetitive elements and unsuited to validation by MBD qPCR.However, these DMRs merit mention, as they are located withingenes that have been implicated in addiction, including Mctp1(multiple C2 domains, transmembrane 1), Cpeb4 (cytoplasmicpolyadenylation element binding protein 4) and Nkain3 (Na+/K+transporting ATPase interacting 3).Mctp1- and Cpeb4-associated DMRswere persistently methylated following cocaine self-administration(Fig. 4a,b), whereas the DMR within Nkain3 becamemethylated duringprolonged abstinence from cocaine self-administration (Fig. 4c). Thepersistent increase in DNA methylation within Mctp1 was associatedwith an enduring and significant decrease in the expression ofMctp1(−001 isoform; F3,26 = 4.07, p b 0.05, Holm–Sidak post hoc test,naïve vs. IVSA 1, IVSA 21 R and IVSA 21NR all p b 0.05)while a similarincrease in DNA methylation within Cpeb4 was associated with asignificant increase in the expression of this gene (−001 isoform;F3,26 = 5.96, p b 0.01, Holm–Sidak post hoc test, naïve vs. IVSA 1 and21 NR, p b 0.01, naïve vs. IVSA 21 R, p b 0.05; Fig. 4a,b). Interestingly,the expression of Nkain3 was exclusively decreased in mice that hadundergone relapse testing after 21 days of abstinence (F3,26 = 4.56,p b 0.05, Holm–Sidak post hoc test, naïve vs. IVSA 21 R, p b 0.05, IVSA21 R vs. IVSA 21 NR, p b 0.01; Fig. 4c), which provides furtherpreliminary evidence of a complex relationship between learning-induced changes in DNA methylation, gene expression and thereactivation state of the memory.
3.7. Differential DNA methylation regulates the expression of specificsplice variants
A principal function of intragenic methylation is the regulationof alternative splicing (Oberdoerffer, 2012). Accordingly, DMRslocated within Cdh13, Cpeb4 andMctp1were associated with changesin the expression of specific isoforms of each gene (Fig. 5). Therewas atrend towards the overall decreased expression of Cdh13 (F3,26 =2.53, p = 0.07; Fig. 5a), however, this is likely due to the regulationof non-coding transcripts of Cdh13, as the expression of theprotein-coding transcript (Cdh13-001) was not significantly alteredfollowing cocaine self-administration (F3,26 = 1.35, not significant;Fig. 5b). The overall expression of Cpeb4 (all isoforms) increasedfollowing cocaine self-administration (F3,26 = 5.96, p b 0.01; Fig. 5c),yet the principal isoform (Cpeb4-001, Ensembl 37) exhibited adifferent pattern of expression (F3,26 = 4.32, p b 0.01; Fig. 5d). Thepersistent increase in methylation within Mctp1 was also associatedwith an enduring decrease in the expression of Mctp1-001 (F3,26 =4.07, p b 0.05; Fig. 5f), but the overall expression of all protein-codingtranscripts of this gene was only significantly different between theanimals that underwent relapse testing after 21 days of abstinence(IVSA 21 R) and those that were simply sacrificed (IVSA 21 NR)(F3,26 = 3.62, p b 0.05, Holm–Sidak post hoc test, IVSA 21R vs. IVSA21 NR; p b 0.05; Fig. 5e). Together, these data suggest that theintragenic changes in DNA methylation produced as a result ofcocaine self-administration may regulate the expression of associ-ated genes in an isoform-specific manner.
Fig. 5. Regulation of alternative splicing and isoform-specific expression by intragenic DNself-administration may contribute to the differential regulation of select splice variants. Tisoforms transcribed from the genomic region proximal to the DMR of interest. (a) There w(b) However, this is likely due to the regulation of non-coding transcripts of Cdh13, as the exself-administration; F3,26 = 1.35, not significant. (c). Subsequent to self-administration,(d) Nevertheless, the expression of the common isoform, Cpeb4-001, displayed a different patexpression being between animals subject to relapse testing at 21 days of abstinence compHolm–Sidak post hoc test). (e) When the collective expression of all protein-coding isoforsubject to relapse testing at 21 days and those that were not (F3,26 = 3.62, p b 0.05, Holindividually, the expression of one protein-coding isoform (Mctp1-001, Ensembl 37) was peData are displayed as mean ± SEM; all p-values are derived from Holm–Sidak post hoc tes
4. Discussion
Taken together, the findings of this study reveal that cocaineself-administration produces distinct and enduring DNA methylationstates within neurons of the mPFC. Functionally, IVSA-associatedchanges in DNA methylation are associated with concomitant isoform-specific modifications of gene expression. In select cases the associationbetween IVSA-associated DMRs and transcription is only evidentfollowing reactivation of cocaine-associated memories, which providesa putative demonstration of experience-dependent genomic metaplas-ticity in the brain, and suggests that persistent changes in DNAmethylation could contribute to memory maintenance in ways thatextend beyond the perpetuation of altered patterns of gene expression.
A growing body of evidence indicates that cocaine interacts withcomponents of the DNA methylation machinery to give rise to brainregion-specific changes in DNA methylation (Anier et al., 2010;Laplant et al., 2010; Pol Bodetto et al., 2013; Tian et al., 2012).However, few of the aforementioned reports distinguished betweenepigenetic modifications associated with voluntary cocaine-seekingand self-administration and those that are simply induced by passivecocaine exposure. Although examining the epigenetic consequencesof passive cocaine exposure can yield important insight into thepharmacological effects of cocaine, this approach cannot be used todetermine the epigenetic changes underlying the cognitive adapta-tions that give rise to addiction-like behavior.
We found that the majority of IVSA-related modifications of DNAmethylation were located within, or proximal to, genes (both codingand non-coding) and predominantly within introns, which isconsistent with the observation that the majority of neuronalactivity-induced changes in DNA methylation arise within introns(Guo et al., 2011). Equally predictable was the relative enrichment ofDMRs within repetitive elements; approximately 42% of the mousegenome corresponds to repetitive elements (Church et al., 2009) andchanges in DNA methylation within these elements are often morereadily detected due to high levels of basal DNA methylation.However, the myriad of abstinence-associated DMRs within orproximal to nuclear lamina-associated domains was surprising. Thenuclear lamina is a protein framework located beneath the nuclearenvelope, to which heterochromatin is often tethered. Approximately35–40% of the genome comprises lamina-associated domains, whichare key determinants of higher-order chromatin architecture and areclassically transcriptionally silent (Peric-Hupkes and Van Steensel,2010). Importantly, the association of the genome with the nuclearlamina is altered following neuronal activity (Walczak et al., 2013)and dysregulation of genome–lamina associations has been implicat-ed in the etiology of neuropsychiatric disorders (Ito et al., 2014;Wilczynski, 2014). Moreover, DNAmethylation directly influences theassociation between genomic regions and the nuclear lamina, as themethyl-CpG binding protein MeCP2 interacts with inner nuclearlamina-associated proteins (Guarda et al., 2009). Therefore, absti-nence-associated changes in DNA methylation may alter genome–lamina associations and heterochromatin stability, although anexplanation for the specific enrichment of abstinence-associatedDMRs within lamina-associated domains remains elusive.
A methylation. Intragenic modifications of DNA methylation arising during cocainehe isoforms selected include the major protein-coding isoform of the select gene andas a trend towards the overall decreased expression of Cdh13, F3,26 = 2.53, p = 0.07.pression of the protein-coding transcript was not significantly altered following cocaineoverall Cpeb4 expression increased in all treatment groups; F3,26 = 5.96, p b 0.01.tern of expression relative to that in naïve animals, with the sole significant difference inared to those that were not (F3,26 = 4.32, p b 0.01, IVSA 21 R vs. IVSA 21 NR, p b 0.01,ms of Mctp1 was explored, the sole significant difference was again between animalsm–Sidak post hoc, IVSA 21 R vs. IVSA 21 NR, p b 0.05). (f) However, when exploredrsistently decreased at all time points relative to naïve animals; F3,26 = 4.07, p b 0.05.ts relative to naïve animals except where indicated, *p b 0.05, and **p b 0.01.
10 D. Baker-Andresen et al. / Neuroepigenetics 4 (2015) 1–11
A number of gene-associated DMRswere locatedwithin genes thathave been implicated in learning, memory and addiction, and theirpotential biological functions merit discussion. KCTD16 can act as anauxiliary subunit of the GABAB receptor, enhancing its sensitivity andaccelerating responses to agonists (Schwenk et al., 2010). Activationof the GABAB receptor, which is equally subject to persistent changesin methylation following cocaine self-administration (data notshown), is associated with a reduction in cocaine self-administration(Roberts and Brebner, 2000). By altering the kinetics and sensitivity ofthis receptor, the differential methylation within Kctd16 and Gababmay therefore contribute to the modulation of cocaine seekingbehavior. CDH13 is a particularly striking candidate, as polymor-phisms within this gene have been implicated in substance depen-dence (Uhl et al., 2008), vulnerability to addiction (Johnson et al.,2011), and disorders of impulse control (Arias-Vasquez et alc, 2011),although no studies have explored the epigenetic regulation of thisgene in cocaine seeking and addiction. Polymorphisms within Mctp1are associated with bipolar disorder (Scott et al., 2009), and theexpression ofMctp1 is altered during abstinence from several drugs ofabuse (Le Merrer et al., 2012), though the cellular changes that giverise to this alteration remain to be determined. Together with theother DMRs identified, these changes in DNAmethylation have a clearpotential to contribute to the development of addiction and, given themalleable nature of epigenetic modifications, may represent promis-ing targets for pharmacological interventions in the treatment of thiscondition. Nevertheless, it will be necessary to establish that theseDMRs regulate addiction and not simply cocaine self-administration.Moreover, as many DMRs are located within repetitive regions,further verification will require the application of novel techniquesthat currently in development, such as SMRT sequencing (PacBio).Future experiments will focus on epigenetic changes in neuronsselectively engaged by cocaine-related memories, using inducibleneuron-specific activity-driven reporter proteins (i.e. E-SARE-drivenGFP (Kawashima et al., 2013) and fluorescence activated cell sorting).Excitingly, with emergent technologies, it may be possible to directlyedit or reverse locus-specific changes in DNAmethylation incurred byself-administration in recently activated neurons and directly dem-onstrate their functional role in behavior.
Repeated exposure to cocaine can generate ‘silent’ glutamatergicsynapses that do not influence the basal efficacy of synaptictransmission but are pronounced sites of plasticity in response tosubsequent stimulation (Lee and Dong, 2011). This investigationprovides preliminary evidence of an analogous phenomenon withinthe genome, whereby persistent IVSA-induced modifications of DNAmethylation do not necessarily produce long-lasting changes in thetranscription of co-localizing genes, but instead prime transcription inresponse to subsequent neuronal and memory reactivation. In asubset of genes (Kctd16, Cpeb4 and Nkain3), an association betweendifferential methylation and altered gene transcription was onlyevident following the explicit re-activation of cocaine-related mem-ories through relapse testing, which suggests that a number ofIVSA-associated changes in DNA methylation represent a form ofgenomic metaplasticity (Baker-Andresen et al., 2012). The Kctd16-associated data are particularly persuasive as there was no differencein methylation in animals that underwent relapse testing after21 days of abstinence compared to those that were simply sacrificed.However, caution must be used when interpreting the Nkain3 andCpeb4-related data, as it is possible that changes in DNA methylationwere produced by the relapse test, resulting in the differences inexpression between the animals that underwent testing and thosethat did not. Additional experiments will extend these findings andconclusively demonstrate that the observed metaplastic priming ofgene transcription is a direct consequence of memory reactivation andDNA methylation at a specific locus.
Finally, IVSA-associated changes in intragenic DNA methylationare associated with the regulation of alternative splicing and the
expression of specific isoforms of co-localizing genes. Importantly, wedemonstrated that the absence of an overall change in the expressionof a gene does not preclude the possibility that the expression ofindividual splice variants is altered. Although the overall expression ofMctp1 appeared unchanged, the expression of a single isoform(Mctp1-001) was differentially regulated in this paradigm. Inprinciple, opposing changes in the expression of specific isoforms ofgenes could obscure global changes in the expression of a given gene.Importantly, each isoform of a gene may produce proteins with vastlydifferent functional capabilities and it is therefore critical that theexpression of specific splice variants of candidate genes is determinedand acknowledged. At the level of individual DMRs, exploring theexpression of each splice variant of co-localizing genes may be tootime-consuming and expensive; it would therefore be preferable tosimultaneously perform whole-transcriptome sequencing to broadlyexplore the regulation of the expression of alternative splice variants.
In summary, this investigation has revealed persistent andabstinence-associated changes in DNA methylation in neurons ofthe mPFC that are specific to voluntary cocaine self-administration.These long-lasting changes in DNAmethylation could in turn underliethe maintenance of cocaine-related memories and continued cocaineseeking during abstinence. Moreover, this study provides preliminaryevidence of memory-related genomic metaplasticity: the priming oftranscription in response to memory reactivation by enduringepigenetic modifications. Future experiments will functionally estab-lish the role of these time-dependent changes in DNA methylation incocaine seeking and memory maintenance and explore theircontribution to the long-term regulation of transcriptional changes.
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nepig.2015.10.001.
Acknowledgments
The authors gratefully acknowledge grant support from theNational Health and Medical Research Council (APP1023127), andthe Australian Research Council (DP1096148). A postgraduate awardfrom the National Sciences and Engineering Research Council ofCanada supports DBA, and XL is supported by a University ofQueensland postgraduate scholarship. The authors would also liketo thank Ms. Virginia Nink for the assistance with FACS, Dr. RobynBrown and Ms. Nicola Chen for the assistance with the behavioralparadigm and Ms. Rowan Tweedale for the helpful editing ofthe manuscript.
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