Epigenetic mechanisms of nicotine dependence

Smoking continues to be a leading cause of preventable disease and death worldwide. Nicotine dependence generates a lifelong propensity towards cravings and relapse, presenting an ongoing challenge for the development of treatments. Accumulating evidence supports a role for epigenetics in the development and maintenance of addiction to many drugs of abuse, however, the involvement of epigenetics in nicotine dependence is less clear. Here we review evidence that nicotine interacts with epigenetic mechanisms to enable the maintenance of nicotine-seeking across time. Research across species suggests that nicotine increases permissive histone acetylation, decreases repressive histone methylation, and modulates levels of DNA methylation and noncoding RNA expression throughout the brain. These changes are linked to the promoter regions of genes critical for learning and memory, reward processing and addiction. Pharmacological manipulation of enzymes that catalyze core epigenetic modifications regulate nicotine reward and associative learning, demonstrating a functional role of epigenetic modifications in nicotine dependence. These findings are consistent with nicotine promoting an overall permissive chromatin state at genes important for learning, memory and reward. By exploring these links through next-generation sequencing technologies, epigenetics provides a promising avenue for future interventions to treat nicotine dependence.

Smoking continues to be a leading cause of preventable disease and death worldwide.Nicotine dependence generates a lifelong propensity towards cravings and relapse, presenting an ongoing challenge for the development of treatments.Accumulating evidence supports a role for epigenetics in the development and maintenance of addiction to many drugs of abuse, however, the involvement of epigenetics in nicotine dependence is less clear.Here we review evidence that nicotine interacts with epigenetic mechanisms to enable the maintenance of nicotine-seeking across time.Research across species suggests that nicotine increases permissive histone acetylation, decreases repressive histone methylation, and modulates levels of DNA methylation and noncoding RNA expression throughout the brain.These changes are linked to the promoter regions of genes critical for learning and memory, reward processing and addiction.Pharmacological manipulation of enzymes that catalyze core epigenetic modifications regulate nicotine reward and associative learning, demonstrating a functional role of epigenetic modifications in nicotine dependence.These findings are consistent with nicotine promoting an overall permissive chromatin state at genes important for learning, memory and reward.By exploring these links through next-generation sequencing technologies, epigenetics provides a promising avenue for future interventions to treat nicotine dependence.
Tobacco smoking has decreased in many parts of the world, yet smoking-related illnesses continue to claim the lives of over 8 million people each year (Reitsma et al., 2021).Although the negative health effects of smoking are widely known, it remains challenging for many users to regulate intake with only 1 in 13 smokers successfully quitting annually (Creamer et al., 2018; U.S. Department of Health and Human Services 2020).Alternative or replacement strategies such as electronic nicotine delivery systems (ENDS) show some efficacy in reducing harm in established smokers (Pound et al., 2021), however current evidence suggests that many established smokers ultimately return to cigarettes (Dai and Leventhal, 2019).Understanding the factors that contribute to the development, maintenance and relapse of nicotine dependence remains an ongoing challenge.
A major contributing factor to nicotine dependence is the persistence of cravings across abstinence.Through associative learning processes, places, people, or paraphernalia previously associated with smoking, become predictive of nicotine intake (Janes et al., 2010;Conklin et al., 2015).In the absence of nicotine, these stimuli come to elicit strong cravings, prompt approach and procurement of nicotine/cigarettes, leading to a lapse of smoking behaviour often weeks, months or even years after cessation (Betts et al., 2020;Carter and Tiffany, 1999;Conklin et al., 2015).Increasing evidence suggests that the associations between nicotine and the stimuli that predict it are more robust and longer lasting than other memories (Puma et al., 1999;Rezvani et al., 2001) and that this is reflected in changes in brain regions involved in reward learning, including the hippocampus (Kenney and Gould 2008;Fujii et al., 1999), ventral tegmental area (VTA) (Jin et al., 2011) and prefrontal cortex (PFC) (Couey et al., 2007).Among other changes, nicotine leads to increased hippocampal activity (Due et al., 2002) and synaptic plasticity (Fujii et al., 1999), affects excitability and N-methyl-D-aspartate (NMDA) receptor expression in the VTA (Jin et al., 2011) and enhances inhibitory transmission and memory in the PFC (Couey et al., 2007), thus showing widespread regulation of connectivity and plasticity in brain regions responsible for memory storage and consolidation.
The capacity of nicotine to enhance memory consolidation arises via its direct interactions with the endogenous cholinergic system.The primary reinforcing effects of nicotine occur through its ability to increase neuronal activity in the reward circuitry of the brain (Tolu et al., 2013).By binding to nicotinic acetylcholine receptors (nAChRs), nicotine permits ion exchange, triggering depolarization and facilitating the Ca 2+ -dependent release of many other neurotransmitters (Dajas-Bailador and Wonnacott, 2004).Repeated nicotine exposure leads to changes across a more distributed brain network including cortical and subcortical regions associated with appetitive and aversive learning (Markou, 2008) and memory (Gould and Prescott, 2014).The underlying molecular changes differ with long-term use -more sustained nAChR activation and Ca 2+ influx activate calcium sensor proteins that transduce the cell nucleus (Changeux and Paas, 2009), activate transcription factors and RNA-binding proteins to ultimately influence the expression of genes relevant to memory and plasticity (Dajas-Bailador, Soliakov, and Wonnacott, 2002;Dajas-Bailador and Wonnacott, 2004;Hu et al., 2002;Kumer and Vrana, 1996;Dunckley and Lukas, 2003).Thus, activation of nAChRs leads to a range of downstream effects that when co-occurring with a learning event, can decrease the threshold required for transcriptional and translational changes necessary for plasticity and long-term memory formation (Fujii et al., 1999) an otherwise transient memory may become long-lasting.How these changes can persist across long periods of abstinence when nicotine is not present, is less clear.Across the last 15 years, a role for epigenetic regulation of gene expression has emerged as a key regulatory factor in the acquisition and consolidation of both appetitive and associative learning processes (Purva et al., 2011;Molfese, 2011).Here epigenetics refers to dynamic changes in gene expression within the lifetime of an individual that do not lead to changes in DNA sequence (Berger, 2007); although transgenerational consequences of nicotine exposure have been reported (Yohn et al., 2015).Through altering chromatin structure, epigenetic changes regulate the transcription of genes critical to the development and maintenance of long-term memories (Alaghband et al., 2016).An increasing body of work now also supports the critical role of epigenetic mechanisms in promoting the development and maintenance of dependence on various drugs of abuse (reviewed elsewhere for cocaine (Schmidt et al., 2013), opioids (Browne et al., 2020) and alcohol (Nieratschker et al., 2013).For example, cocaine self-administration has been linked to an overall permissive chromatin state through increased histone acetylation (Sadakierska-Chudy et al., 2017), decreased histone methylation (Maze et al., 2010) and altered transcription of epigenomic editor enzymes (Lionel et al., 2011;Maze et al., 2010).Furthermore, regulation of the enzymes responsible for modulating epigenetic change (e.g.histone deacetylases: HDACs) can enhance the later sensitivity to drugs of abuse (Hitchcock et al., 2019;Raybuck et al., 2013) indicating the importance of epigenetic processes in maintaining drug abstinence.Thus, drugs of abuse like cocaine appear to promote epigenetic modifications that alter the threshold required for gene expression to occur (Nestler, 2014).Whether the same holds for nicotine is less clear, however the importance of psychology rather than pharmacology in nicotine dependence (Caggiula et al., 2009;Caggiula et al., 2002;Rupprecht et al., 2015;Sorge et al., 2009), suggests that epigenetic changes to the regulation of genes important for learning and memory may be particularly relevant.
The aim of this review is to a) describe the epigenetic changes in the brain that occur in response to nicotine exposure as relevant to addiction, learning and memory and b) discuss how epigenetic modifications change the behavioural response to nicotine, with a particular emphasis on changes relevant to addiction.This review will cover rodent and human literature, as well as data from zebrafish due to the significant body of work on nicotine and epigenetics that has been published in this area (Faillace and Bernabeu 2022).Although this research area is in its infancy, we will show that there is sufficient evidence to suggest that activation of nAChRs by nicotine leads to a range of epigenetic changes that are generally consistent with a permissive chromatin state and a reduced threshold for the expression of genes important for learning and memory.We suggest that when learning (i.e.associations between stimuli and reward) occurs in the context of nicotine exposure, the epigenetic changes induced by nicotine more readily permit the instantiation of these memories into long-term memory formation than when nicotine is not present (Volkow, 2011).

Histone modifications
Epigenetic changes are many and varied.Some of the most wellstudied are modifications to the tails of histone proteins, including histone acetylation or methylation.Histone protein octamers contain pairs of 4 histone proteins (H2A, H2B, H3 and H4) to produce a spool around which DNA is coiled.The addition of functional groups to histone Nterminal tails by epigenetic writers changes chromatin structure and affects the spacing of DNA and histone proteins, impacting DNA transcription (Hong et al., 1993;Rice and Allis, 2001).Histone tail modifications can have additional functions, including the recruitment of epigenetic readers that recognize histone marks, mediate their effects and in turn influence gene expression (Grunstein, 1997).An increasing number of modifications to the histone tail have been identified (e.g.sominaliation, ubiquitization), however their role in addiction is less established (Hitchcock and Lattal, 2014) and untested for nicotine dependence.For these reasons, here we focus on histone acetylation and methylation in the context of nicotine exposure.

Histone acetylation
Histone acetylation is the most widely studied epigenetic modification in drug addiction and nicotine exposure specifically (Table 1).It involves the addition of an acetyl group to lysine residues on histone tails, reducing the electrostatic tension between histone and DNA, "opening" chromatin, recruiting transcriptional co-activators (Sanchez and Zhou, 2009) and promoting proximal gene expression (Grunstein, 1997).For this reason, histone acetylation is considered to promote a permissive chromatin state, priming gene transcription and supporting memory consolidation (Ahmad Ganai et al., 2016;Woldemichael et al., 2014).Some of the earliest indications of nicotine impacting histone acetylation come from studies of human post-mortem lung tissue (Barnes, Adcock, and Ito, 2005) and the blood of smokers (Markunas et al., 2021;Tan et al., 2016), where increased global histone acetylation is reported.Consistent with these findings, chronic nicotine administration in rodents has been linked to increased H3 and H4 acetylation in the promoter regions of genes important for learning and memory (Gozen et al., 2013;Huang et al., 2014;Levine et al., 2011).More specifically, nicotine administration via drinking water to mice is associated with increased striatal and hippocampal histone H3 (K9) and H4 (K5 to K16) acetylation globally (Huang et al., 2014) and in the promoter region of FosB, a gene important for the progression of addiction (Levine et al., 2011).Further, repeated nicotine injections in rats are associated with increased H4 acetylation and mRNA expression of the dopamine receptor gene Drd1 in the prefrontal cortex (Gozen et al., 2013), consistent with nicotine promoting chromatin accessibility in genes important for reward.A similar effect has been reported in zebrafish, where across a series of studies chronic nicotine exposure via tank water was associated with increased H3K9 acetylation in brain regions associated with reward (Faillace et al., 2018;Pisera-Fuster et al., 2020) and increased expression of mRNAs encoding nAChR subunits (Pisera-Fuster et al., 2020).These initial studies suggest first that nicotine leads to global increases in histone acetylation and therefore chromatin accessibility, and second that this is occurring in the promoter regions of genes involved in nicotine dependence.
However, the effects of nicotine on histone acetylation appear to be highly dependent on the route and duration of administration.While 7day access of nicotine to mice in drinking water increased H3 acetylation in the hippocampus, brief exposure with a single injection of nicotine does not cause lasting changes in hippocampal H3 or H4 histone acetylation 30 min later (Kenney et al., 2012).Similarly, as opposed to chronic, continuous nicotine exposure in mice (Levine et al., 2011) or zebrafish (Pisera-Fuster et al., 2020), four nicotine injections in rats do not affect H3 acetylation in the striatum (Pastor et al., 2011).These findings suggest that chronic nicotine exposure is necessary to induce permissive chromatin changes in brain regions critical for learning and memory processes.An anomaly among these findings is when nicotine is selfadministered.In a volitional model of drug intake, self-administered nicotine resulted in decreased H3K14 acetylation at the brain-derived neurotrophic factor (Bdnf) exon IV promoter (Castino et al., 2018), an immediate early gene critical for long-term potentiation and the development of long-term memories (Bramham and Messaoudi, 2005).This difference was detected in the medial prefrontal cortex 24 h after self-administration ceased, indicating persistent epigenetic changes (Castino et al., 2018).However, this was assessed the day after the last self-administration session and may reflect adaptive changes occurring following the cessation of nicotine or due to the extinction process itself (Siddiqui et al., 2017).
Volitional control over drug intake has long been established as critical for many underlying addiction-related brain changes to occur (Lepack et al., 2020;McCutcheon et al., 2011;Robinson et al., 2002).When nicotine administration is not contingent on an instrumental response, epigenetic changes observed could easily reflect acute or chronic stress occurring in response to repeated experimenter-administered drugs, or other non-drug-related learning processes (e.g.context conditioning) (Drude et al., 2011;Stankiewicz et al., 2013).These findings highlight the need for further self-administration studies in this area that capture the interplay between learning and drug exposure, better reflecting the human drug-taking experience and its consequences.

Histone acetylation-modifying enzymes
The ability of nicotine to produce changes in histone acetylation may be linked to its ability to regulate the activity and expression of histonemodifying enzymes.Histone acetylation is catalyzed by two classes of enzymes: Histone acetyltransferases (HATs) (epigenetic writers) that deposit acetylation marks on histone residues; and HDACs (epigenetic erasers) that remove these marks (Bannister and Kouzarides, 2011).Whereas the specific epigenetic activity of HATs is challenging to study due to their broad functionality, HDACs primarily deacetylase histone proteins, though research has pointed towards a more general classification as lysine deacetylases (Sengupta and Seto, 2004).Generally, inhibition of HDAC activity decreases histone deacetylation, thus increasing chromatin accessibility and gene expression (Lopez Atalaya et al., 2013).The impact of nicotine on HDAC enzymes differs based on class and tissue type (Table 1), and thus where increased histone acetylation should occur in response to the inhibition of HDACs, this is not always the case.Some of the first evidence of nicotine's interactions with HDACs came from human lung tissue, where overall HDAC activity is decreased and linked to reduced mRNA expression of HDAC2 and 3 (Cosío et al., 2004;Ito et al., 2001).Extending this to the brain, in neuronal cell culture nicotine administration leads to decreased expression of SIRT1 (Class III HDAC) and increased HDAC1 (Class I HDAC) expression (Lee et al., 2014), supporting a class-specific impact of nicotine on HDAC expression.Studies in zebrafish and rats have confirmed increased Class I HDAC (HDAC1/2) expression in the brain following chronic nicotine exposure (Faillace et al., 2015;Pastor et al., 2011;Pisera-Fuster et al., 2020), however have not directly shown that this upregulation of HDACs leads to decreased histone acetylation or the repression of gene transcription.Rather, increased HDAC1 or HDAC2 expression was found with unchanged (Pastor et al., 2011) or increased H3K9 acetylation (Pisera-Fuster, Faillace, and Bernabeu, 2020), possibly mediated by an increase in HAT CBP and decreased expression of the Class III HDAC SIRT1 (Pisera-Fuster et al., 2020).Conversely, global HDAC activity within the striatum was decreased following chronic nicotine administration in drinking water to mice, and this change was associated with increased histone acetylation (Levine et al., 2011).Such findings highlight the complexity in interpreting ChIP or gene expression data when only one histone modification is analysed or a single class of HDACs is assessed, where the net change across all modifications will ultimately dictate the accessibility of a given gene (Jenuwein and Allis, 2001).
Together these studies provide support for chronic nicotine promoting a permissive chromatin state via histone acetylation across species and tissue types, resulting in increased gene expression necessary for addiction and memory-related plasticity (Gozen et al., 2013;Levine et al., 2011).By relaxing chromatin, nicotine may be reducing the threshold necessary for the consolidation of learning and memory processes.

Histone methylation
Histone methylation involves the binding of up to three methyl groups to the N-terminal tail of histone proteins (Bannister and Kouzarides, 2011).While histone acetylation is generally permissive, the effect of histone methylation on transcription is complex.Histone methylation can repress or permit gene expression depending on the methylation state and residue affected (Kouzarides, 2002).Histone methylation changes have been widely linked to learning and memory processes (Gupta et al., 2010), and increasing evidence indicates histone methylation is also altered following drug exposure (Browne et al., 2020;Nestler, 2014;Kyzar et al., 2017;Maze et al., 2010).
As with histone acetylation, the effect of nicotine on histone methylation is consistent with promoting an overall permissive chromatin state, although here this occurs through reduced gene repression.Nicotine self-administration in rats decreased repressive H3K9 dimethylation and H3K27 trimethylation in the promoter regions of Bdnf IV and Cdk5 promoters and increased Bdnf mRNA expression (Castino et al., 2018).Similarly, acute nicotine injections in mice decreased repressive H3K9 dimethylation at the Bdnf promoter in the cortex and increased associated BDNF protein expression (Chase and Sharma 2013).Interestingly as opposed to histone acetylation patterns described above, decreased histone methylation was associated with decreased histone methyltransferase (HMT) expression (G9a, SETDB1, GLP) (Chase and Sharma 2013) suggesting that histone methylation changes following nicotine exposure were directly mediated by decreased HMT expression.Notably, Bdnf gene expression and protein levels are dysregulated following exposure to a range of drugs of abuse (Li and Wolf 2015;Logrip et al., 2015) including nicotine (Castino et al., 2018;Chase and Sharma 2013;Huang et al., 2021) and are critical for memory persistence (Bredy et al., 2007).Likewise, nicotine conditioned place preference (CPP) decreased H3K9 trimethylation and decreased expression of associated HMTs G9a and SUV39H although the change appears to be limited to dopamine-rich striatal areas (posterior tuberal nucleus (PTN), dorsal and ventral nucleus of the ventral telencephalic area (Vd/Vv)) (Pisera-Fuster, Zwiller, and Bernabeu, 2021) and was not evident in whole brain homogenates (Faillace et al., 2018).Lastly both human smokers and chronically exposed rats via osmotic minipumps showed decreased gene expression of the epigenetic writer SMYD1 in Abbreviations ChIP: chromatin immunoprecipitation; CPA: conditioned place aversion; CPP: conditioned place preference; dCTP: Deoxycytidine triphosphate; DG: dentate gyrus; dHB: dorsal habenula; dStr: dorsal striatum; ELISA: enzyme linked immunosorbent assay; F: female; FC: frontal cortex; HPC: hippocampus; IHC: immunohistochemistry; IVSA: intravenous self-administration; M: male; MeDIP: methylated DNA immunoprecipitation; mPFC: medial prefrontal cortex; MSP: methylation specific PCR; OFC: orbitofrontal cortex; PTN: posterior tuberal nucleus; PFC: prefrontal cortex; RT-PCR: reverse transcriptase polymerase chain reaction; Str: striatum; Vd: dorsal nucleus of the ventral telencephalic area; vmPFC: ventromedial prefrontal cortex; VTA: ventral tegmental area; Vv: ventral nucleus of the ventral telencephalic area; WB: Western Blot the PFC, however the impact of this change in the brain is unclear (Vargas-Medrano et al., 2023).These findings show nicotine decreased repressive histone methylation across species and explicitly indicate a potential mechanism underlying the persistence of nicotine-cue associations via epigenetic regulation of Bdnf gene expression (Chase and Sharma 2013).
Importantly, the effects of nicotine on histone methylation appear to be directly associated with nAChR activation rather than as an indirect consequence of broader neuronal activity.Nicotinic decrease in H3K9 dimethylation at the Bdnf promoter is blocked by application of the α4β2-nAChR antagonist mecamylamine (Chase and Sharma 2013).
Furthermore, nicotine-induced neuronal expression of ASH2L, a component of the permissive H3K4 methyltransferase, is abolished by co-administration with mecamylamine and the calcium channel blocker nifedipine, showing that nicotine-mediated increases in ASH2L expression are dependent on calcium influx via α4β2-nAChRs (Jung et al., 2016).Together these studies consistently support a role for nAChR-mediated reductions in repressive histone methylation marks, leading to an overall permissive chromatin state for BDNF and CDK5, providing a potential pathway enabling the persistence of nicotine-cue associations (Castino et al., 2018;Chase and Sharma 2013;Jung et al., 2016).

DNA methylation
DNA methylation involves the transfer of a methyl group to a cytosine located within a cytosine-phosphate-guanine (CpG) island in a gene promoter region of DNA.This forms 5-methylcytosine, a modification to the 5th carbon molecule of cytosine that closes chromatin into a transcriptionally inactive state, reducing gene expression (Moore, Le, and Fan, 2013).5mC can further be iteratively oxidized into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) supporting DNA demethylation and promoting gene expression (Guo et al., 2011;Wu and Zhang, 2017).
Blood from human smokers gives some indication of an overall hypomethylated DNA state following lifelong nicotine exposure, indicative of reduced gene repression and relaxed chromatin states (Lee and Pausova, 2013;Liu et al., 2018;Markunas et al., 2021).Specifically, the blood of smokers showed differential methylation of nicotine receptor subunit genes that are associated with nicotine susceptibility (CHRNA5, CHRNA3, CHRNB4) (Liu et al., 2018).While the specific differentially methylated cytosine loci often differ between tissue types, the regulated gene targets (ZIC1, PKRDC, ZCCHC24) are partially maintained between brain and blood in smokers, with stronger methylation changes observed in the brain (Markunas et al., 2021).Further, in brain samples, sites of differential methylation not identified in peripheral tissues were discovered (ABLIM3, MTMR6, CTCF, APCDD1L), indicative of unique regulation of DNA methylation in the brains of smokers (Markunas et al., 2021).
Animal studies confirm nicotine-induced regulation of DNA methylation in the brain.Chronic (12 day) nicotine exposure via miniosmotic pumps in mice led to differential DNA methylation in the promoter regions of several genes in the hippocampus, however only 2 (Smarca2, Bahcc1) out of 10 selected genes showed an associated change in gene expression (Gitik et al., 2018).This is consistent with the idea of "epigenetic priming" wherein drugs of abuse produce epigenetic changes, that initiate enhanced transcription to subsequent salient events (Nestler, 2014).Even shorter periods of nicotine exposure via injections in mice or tank water in zebrafish led to nAChR-dependent decreases in DNA methylation and increased mRNA expression of GAD67 and NMDAR1 (Pisera-Fuster et al, 2021;Satta et al., 2008).Conversely, 14-day abstinence from repeated nicotine injections in rats increased global DNA methylation and decreased gene expression, indicating potential homeostatic epigenetic repression during abstinence from nicotine (Mychasiuk et al., 2013).Together these studies are consistent with altered patterns of DNA methylation following nicotine exposure and at genes relevant to nicotine dependence.The functional or behavioural consequences of these changes in the context of nicotine dependence has yet to be determined.

DNA Methyltransferases
DNA methyltransferase (DNMTs) enzymes regulate DNA methylation.DNMT3a and DNMT3b are responsible for writing new DNA methylation marks, while DNMT1 maintains existing DNA methylation marks across cell division, all leading to transcriptional repression (Moore et al., 2013).DNA methylation marks can further recruit reader proteins such as MeCP2, binding 5mC regions and further repressing gene expression (Meehan et al., 1992).DNA demethylation is mediated by eraser enzymes including Ten-Eleven Translocation (TET) enzymes which hydroxylate 5mC marks into 5hmC, serving as an intermediate to complete demethylation (Guo et al., 2011).The activity of DNMTs is critical for learning and memory, and disruption of memory traces can occur following localized infusion of DNMT inhibitors (Day et al., 2013;Miller et al., 2010).
Studies of human smokers have shown increased DNMT3b expression in respiratory epithelial cells and the resulting DNA hypermethylation was linked to lung cancer states (Teneng et al., 2015).In postmortem brains of ever-smokers a single nucleotide polymorphism increased the expression and methylation of the DNMT3b gene and contributed to the risk of nicotine dependence (Hancock et al., 2018), suggesting a clear link between DNMT expression and the development of nicotine dependence.
DNMT3b is not expressed at high levels in the brain of most animal models (Satta et al., 2008), however preclinical studies in zebrafish establish increased DNMT3a expression following chronic nicotine exposure in striatal regions (Pisera-Fuster, Faillace, and Bernabeu, 2020).Importantly this change was dependent on the pattern of nicotine exposure, as intermittent nicotine exposure led to an opposite decreased DNMT3a expression (Pisera-Fuster et al., 2020).However, in mice, repeated injection of either nicotine or the α4β2 nAChR agonist varenicline did not change DNMT3a expression in the hippocampus or frontal cortex (Maloku et al., 2011;Satta et al., 2008), suggesting considerable variability in results depending on mode of nicotine administration.
Nicotine-induced changes in the expression of DNMT1 are more consistent, where nicotine or varenicline injections decreased DNMT1 expression in the frontal cortex and hippocampus of mice (Maloku et al., 2011;Satta et al., 2008), and a similar decrease in DNMT1 expression was observed in zebrafish striatal regions following chronic nicotine exposure (Pisera-Fuster, Faillace, and Bernabeu, 2020;Pisera-Fuster et al., 2021).Importantly, across these studies, decreased DNMT expression was associated with decreased DNA methylation (Satta et al., 2008).Conversely, when DNMT and repressive MeCP2 mRNA expression was found to be increased (Pisera-Fuster et al., 2020), DNA methylation at the learning critical Nmdar1 gene was decreased, potentially mediated by increased DNA demethylation as indicated by increased TET1 and GADD45 expression (Pisera-Fuster et al., 2020).
These findings can be challenging to interpret as histone and DNA modifying enzymes do not regulate gene expression in isolation e.g.DNMT1 forms a repressive complex with HDAC1 (Robertson et al., 2000) and 2 (Rountree et al., 2000) to mediate transcriptional repression.Thus, instead of individual changes in histone acetylation or DNA methylation, it is likely the combination of multiple repressive or permissive changes that determine the availability of a given gene for expression (Strahl and Allis, 2000).Unravelling these complexities requires more research using more comprehensive omics tools such as mass spectrometry or an Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) to assess global chromatin accessibility and provide a better overview of the regulation of total epigenomic state in response to nicotine.

Non-coding RNA
Non-coding RNA are RNA transcripts that are not translated into proteins.They have a range of regulatory functions that ultimately influence cellular function via direct or indirect interactions with the genome, or post-transcriptional interference (Cech and Steitz 2014).Noncoding RNAs, including microRNAs and long non-coding RNAs, regulate gene expression at the level of transcription and translation, interact with epigenetic modifications and are increasingly recognized as master regulators of brain function (Cech and Steitz 2014;Liau et al., 2021;Schratt et al., 2006).
MicroRNAs (miRNAs) are between 21 and 23 base pairs long and typically transcribed from intronic regions of a gene (Hammond, 2015).MicroRNAs are significantly regulated following exposure to a range of drugs of abuse (Kenny, 2014;Sim et al., 2017) and overexpression of specific miRNAs (e.g.miRNA-124, let-7d) can reduce drug-seeking behaviour (Chandrasekar and Dreyer, 2009;Schaefer et al., 2010).
Nicotine has been linked to a range of changes in miRNA expression in a multitude of organisms and cell types, including altering miRNA patterns in post-embryonic c elegans (Taki et al., 2014), T cells from human smokers (Wasén et al., 2020), perinatal miRNA in DA neurons (Kazemi et al., 2020) and other cell types (Du et al., 2019).More specifically, many of these changes are linked to the regulation of intracellular processes critical for memory formation.For example, nicotine self-administration regulates 44 miRNAs in the habenula of mice, potentially targeting the MAPK pathway critical for encoding the aversive component of nicotine withdrawal (Lee et al., 2015).Furthermore, pre-frontal cortex-specific increases in miR-221, − 483, − 199 and − 214 were observed following nicotine administration (Gomez et al., 2016;Pittenger et al., 2018).miR-221 overexpression was shown to enhance nicotine locomotor sensitivity, and this was likely mediated by decreased ERK and CREB phosphorylation (Gomez et al., 2016).In contrast, miR-199 and − 214 were only increased in nicotine-administered female rats and regulated the expression of SIRT1 (Pittenger et al., 2018), providing a potential mechanism of HDAC regulation following nicotine exposure.Together these studies show that not only does nicotine alter the expression of a range of miRNA, but that these are linked to the regulation of core learning and memory processes that can contribute to the development and maintenance of nicotine dependence.
Long non-coding RNAs are typically more than 200 nucleotides in length and can have a range of functions, activating or suppressing transcription as a mRNA sponge or influencing translation (Mercer et al., 2009).LncRNA also interact extensively with epigenetic factors, including the recruitment of HDACs or DNMTs to transcription start sites to enhance or inhibit gene expression (Huang et al., 2015;Li et al., 2016).Smoking is linked to changes in many lncRNA (Soares do Amaral et al., 2016;Ge et al., 2022) and lncRNAs expression is increasingly used to predict risk and prognosis for lung cancer in smokers (Ge et al., 2022;Zhou, Wang, and Liu, 2022).Furthermore, in rats, the long non-coding RNA antisense Bdnf (Bdnf-AS) is upregulated in the PFC during nicotine self-administration, this persists across extinction and knockdown of the transcript impairs extinction learning (Youngson et al., 2022), suggesting a possible direct functional relationship between nicotine exposure and lncRNA levels.Noncoding RNAs are emerging as important regulators of transcriptional activity in nicotine dependence, but more studies are required to understand the specific consequences of this noncoding RNA regulation for nicotine dependence.

Epigenetic manipulation and nicotine seeking
The studies discussed above provide a correlational framework of how nicotine affects epigenetic modulators.Here we will discuss studies that manipulated epigenetic mechanisms in nicotine dependence (Table 2).Such studies are important for linking broad epigenetic regulation with tangible behavioural and functional outcomes.
In paradigms of learning and memory, the administration of HDAC inhibitors has been consistently shown to facilitate memory consolidation and long-term memory formation (Ahmad Ganai et al., 2016;Stefanko et al., 2009).Similarly, HDAC inhibitors administered following extinction of nicotine self-administration in rats increased the rate of extinction and decreased reinstatement, in line with an enhanced extinction memory (Castino, Cornish, and Clemens 2015).However, this was not a general effect on learning as no change in extinction was observed following sucrose self-administration (Castino, Cornish, and Clemens 2015), showing that HDAC inhibitor-enhanced extinction was specific to nicotine extinction, potentially acting by reversing repressive epigenetic states observed during abstinence from nicotine (Mychasiuk et al., 2013).
In line with enhanced extinction, concurrent administration of HDAC inhibitor phenylbutyrate (PhB) and nicotine during nicotine CPP in rats decreased the rewarding properties of nicotine, reducing preference for the nicotine paired chamber, while not affecting conditioned place aversion (CPA) or caffeine CPP (Faillace et al., 2018;Pastor et al., 2011;Pisera-Fuster et al., 2020), although this same pattern is observed with cocaine (Romieu et al., 2008;Romieu et al., 2011).Similarly, inhibiting proteins that read histone acetylation marks via a bromodomain extra terminal inhibitor (JQ1) attenuates nicotine and amphetamine CPP but does not impact memory, reiterating the critical role of histone acetylation in the reward of stimulant drugs (Babigian et al., 2022).In

Table 2
Behavioural changes in nicotine dependence with pharmacological manipulation of epigenetic enzymes.contrast, a systemic DNMT inhibitor (AZA) enhances CPP in zebrafish while methyl donor methionine impaired CPP, showing that nicotine-induced hypomethylation is important for nicotine's reinforcing properties (Pisera-Fuster et al., 2021).However, while AZA has been shown to previously affect DNMT degradation (Ghoshal et al., 2005) and synaptic plasticity (Levenson et al., 2006), it acts partially via integration into the DNA sequence (Momparler, 2005) so should have limited efficacy in mature neurons.This highlights a general limitation of work in this area relating to cell-specificitythe majority of studies are performed on tissue homogenates and therefore it is unclear whether the effects of AZA are mediated neuronally or via integration into the DNA of replicating glial cells.Consistent with nicotine acting as an HDAC inhibitor, nicotine enhances cocaine-induced long-term potentiation (LTP) and locomotor sensitization in a similar way to administration of the HDAC inhibitor Suberoylanilide hydroxamic acid (SAHA) (Levine et al., 2011;Huang et al., 2014).These changes are specifically dependent on the ability of nicotine and SAHA to deacetylate histones (Huang et al., 2013;Huang et al., 2014;Levine et al., 2011), establishing the importance of nicotine-mediated increases in histone acetylation in nicotine memory, and consistent with nicotine acting as an epigenetic primer to later gene transcription.
Only two studies have observed HDAC inhibitors reversing nicotinic effects on behaviour, showing a reversal of depressogenic and anxiogenic effects in mice (Hayase, 2016), and reversing spatial memory enhancements in zebrafish (Faillace et al., 2017).However, the impact of nicotine on anxiety, and spatial memory is mediated by different brain regions to nicotine reward, thus the different impact of further histone deacetylation may be due to the involvement of alternate neural circuits (Molas et al., 2017).
These studies establish that both DNA methylation and histone acetylation are important for the reinforcing and rewarding properties of nicotine (Faillace et al., 2018;Pisera-Fuster et al., 2020;Pastor et al., 2011;Castino, Cornish, and Clemens 2015;Babigian et al., 2022;Pisera-Fuster et al., 2021), similar to what is observed in other stimulants, such as cocaine (Alaghband, Bredy, and Wood 2016;Kennedy et al., 2013;Malvaez et al., 2011;Romieu et al., 2011).However, such results must be interpreted with caution as the systemic methods of administration make it challenging to elucidate the exact functional mechanisms of these epigenomic editors.As opposed to research seen in other drugs of abuse (Browne et al., 2020;Anderson et al., 2019) no studies have manipulated epigenetic editors selectively to assess behavioural consequences of nicotine dependence, making the exact epigenetic enzymes, gene targets, as well as brain regions involved in the behavioural changes observed, unclear.

Discussion
While the field is still in its infancy, our understanding of nicotinic regulation of epigenetic modulators in the brain is starting to expand (as summarised in Fig. 1).The combined literature broadly supports nicotine to increase histone acetylation (Levine et al., 2011;Gozen et al., 2013;Faillace et al., 2018;Pisera-Fuster et al., 2020), decrease repressive histone methylation (Chase and Sharma 2013;Castino et al., 2018), widely regulate DNA methylation (Gitik et al., 2018;Satta et al., 2008;Pisera-Fuster et al., 2021;Mychasiuk et al., 2013) and induce Fig. 1.Summary of epigenetic changes that occur in response to nicotine administration.This includes changes to the expression of enzymes that write, read or erase epigenetic marks (e.g.histone deacetylases (HDACs), histone acetyltransferases (HATs), DNA methyltransferases (DNMTs), specific histone modifications (e.g.increased or decreased histone acetylation, histone methylation or DNA methylation) and the consequences for the expression of genes important to learning and memory.Created with BioRender.com.
C. Muenstermann and K.J. Clemens transcription of noncoding RNAs (Youngson et al., 2022;Pittenger et al., 2018;Gomez et al., 2016;Lee et al., 2015) across the brain, species and methods of administration.Importantly, these changes are likely induced via direct binding of nicotine to nAChRs in the central nervous system, require repeated or chronic nicotine exposure and result in altered expression of genes critical in addiction (Levine et al., 2011;Gozen et al., 2013) and memory (Castino et al., 2018;Pisera-Fuster et al., 2021).Systemic manipulations of epigenetic editors indicate that these changes are important for nicotine memory (Faillace et al., 2017;Huang et al., 2014;Huang et al., 2013), reward (Babigian et al., 2022;Pisera-Fuster et al., 2021) and extinction learning (Castino, Cornish, and Clemens 2015).Together these findings support a role for epigenetic changes in the development and maintenance of nicotine dependence.

Epigenetics in the persistence of nicotine memories
Epigenetics offers a stable change in chromatin that can lead to lasting changes in gene expression that persist beyond the intake of the drug itself.Although more research is required, the available evidence is consistent with chronic nAChR activation leading to the activation of intracellular processes that ultimately lead to epigenetic modifications in the promoter region of genes responsible for long-term memory formation.In this way (and much like that described in Levine et al., 2011 with cocaine), nicotine could epigenetically prime learning that occurs in the context of nicotine intake.Given that many of the changes described can be long-lasting, epigenetic change may offer a mechanism through which the associative memories forming in the presence of nicotine are enabled to persist across time.Such a synergistic relationship between nicotine and fear learning has been shown previously (Kenney et al., 2012), whereby nicotine reduces the threshold required for the expression of genes important for memory processing.This approach may well help to explain a range of phenomena in nicotine-seeking, including the incubation of craving, where motivation for the drug reward is maintained or increased with time since cessation (Bedi et al., 2011), or persistent nicotine-seeking in the absence of the drug reward (Clemens & Holmes 2018).Notably, these behavioural changes are paralleled by increases in the expression of genes such as Bdnf, that are known to be highly epigenetically regulated (Castino et al., 2018;Youngson et al., 2022;Bredy et al., 2007).In the context of drug addiction, we then propose that much like with fear learning (Kenney et al., 2012) or cross-sensitization from nicotine to cocaine (Levine et al., 2011), the persistence of nicotine-seeking is enabled due to nicotine epigenetically priming the expression of genes important for the formation and retention of nicotine-associated memories.

Challenges in understanding the role of epigenetics in nicotine dependence
In reviewing this literature there are some exceptions and conflicting results.This is due in large part to the variety of experimental approaches used for drug administration, the heterogeneity observed within brain tissue from which samples are derived, and the analytical approach employed.For example, chronic continuous nicotine versus single or occasional nicotine administration produces a range of outcomes (Pisera-Fuster et al., 2020;Kenney et al., 2012;Pastor et al., 2011), with our review indicating that chronic or repeated exposure generally produces the most robust evidence of epigenetic regulation (Table 1).Research using other drugs of abuse has further shown that contingent and volitional drug administration leads to epigenetic changes not observed utilizing other methods of administration (Lepack et al., 2020).In the case of nicotine administration, there is insufficient data yet to clearly determine the impact of different methods of administration or dosing patterns on the epigenetic changes observed.However, shifting towards more consistent and human-relevant dosing paradigms, such as volitional intake in intravenous self-administration, would offer greater insight into epigenetic regulation likely to occur in human smokers, and therefore greater translational relevance for enhanced therapeutic potential.
The variability in experimental approaches also extends to the species used.It is worth noting that the majority of reviewed studies are non-human, using predominantly rodent and zebrafish models.While these animal models are well validated and share significant genetic and behavioural homology with humans, there are significant differences that are important to highlight.In particular, zebrafish studies are limited when modelling volitional drug taking, the zebrafish brain does not share similar complexity to the mammalian brain (lacks a prefrontal cortex and telencephalon regions) and zebrafish do not have distinct sex chromosomes (Meshalkina et al., 2017).The latter is particularly relevant when considering clear sex-specific differences in nicotine metabolism and intake in both humans and rodents (Perkins et al., 1999), and sex-specific epigenetic alterations in response to other drugs of abuse (Fischer et al., 2022).This raises an important point in the literature reviewedthe majority of studies used male rodents.Extending these studies to include females will be an important and necessary next step for this field.
Concerning the analytical approach employed, many of the papers discussed in this review used a singular measure, often from a whole brain or whole region homogenate, to indicate the regulation of epigenetic marks.It is becoming increasingly apparent that changes in gene expression found via qRT-PCR do not necessarily correspond to changes in protein levels observed via Western Blot or lead to the regulation of associated epigenetic marks (Lopez-Atalaya et al., 2013;Takemon et al., 2021).In fact, multiple studies summarized in this review have shown that regulation of epigenetic writers observed via qRT-PCR following nicotine exposure did not lead to corresponding regulation of epigenetic marks observed via immunohistochemistry (IHC) (Pastor et al., 2011, Pisera-Fuster et al., 2020).Targeted analytical approaches such as qRT-PCR, IHC or Western Blots can miss less studied epigenetic regulators such as epigenetic readers that have been implicated in other drugs of abuse (e.g.BRD4) (Sartor et al., 2015).This is particularly important as is increasingly apparent that multiple epigenetic modifications occur and act to determine gene expression in concert (Strahl and Allis, 2000;Robertson et al., 2000).Recent advances have made -omics technologies such as RNA-sequencing, ChIP-sequencing or ATAC -sequencing more accessible than ever.Such approaches can provide a comprehensive assessment of epigenetic modulation in response to drug administration, by providing global overviews of changes in protein expression, non-coding RNA expression or chromatin state following drug exposure, allowing for the discovery of novel gene targets and dysregulation in larger epigenetic networks.A recent extension of these methods to single-cell resolution can further address some of the inconsistencies reported in this review by allowing for cell and region-specific analyses.This is particularly relevant for the cell type heterogeneity observed in brains.While existing literature on stimulants points towards the regulation of epigenetic mechanisms in neurons (Anier et al., 2018), research using nicotine has only used overall brain lysate, thus making it impossible to determine what cell types in the brain are being epigenetically regulated.An important future direction of this work will be the integration of increasingly sophisticated approaches for identifying the functional consequences of nicotine-induced epigenetic changes, including the use of techniques such as modifying epigenetic marks (e.g.epigenome-editing platforms using zinc-finger proteins, transcriptional activator-like effectors and CRISPR; Yim et al., 2020).

Conclusions
Together this growing body of research into the epigenetic basis of nicotine dependence is consistent with nicotine promoting an overall permissive chromatin state that reduces the threshold necessary for the expression of genes relevant to the formation and persistence of nicotine dependence.Although the interaction between nicotine and specific epigenetic changes is likely complex, dynamic and context-specific, there is consistency between the work discussed here and similar work performed using in vitro preparations (Ahmad Ganai et al., 2016;Chase and Sharma , 2013), assessing the impact of nicotine during gestation and early development (Nakamura et al., 2021) and the broad literature linking nicotine, epigenetics and various diseases including cancer (Gould, 2023).Exploring and consolidating these links with high-throughput genomics and transcriptomics offers an exciting opportunity to extend our understanding of how drugs of abuse interact with gene expression with a view towards novel therapeutic approaches for nicotine dependence.

Table 1
Epigenetic changes following nicotine administration.
(continued on next page) C. Muenstermann and K.J. Clemens Table 1 (continued )