Biomedicine & Pharmacotherapy 170 (2024) 115951

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Review

Drug addiction and treatment: An epigenetic perspective

Arunkumar Singh Koijam a, Kabrambam Dasanta Singh a, Bunindro Singh Nameirakpam a,
Reena Haobam b, Yallappa Rajashekar a, *
a
Insect Bioresources Laboratory, Animal Bioresources Programme, Institute of Bioresources & Sustainable Development, Department of Biotechnology, Govt. of India,
Takyelpat, Imphal 795001, Manipur, India
b
Department of Biotechnology, Manipur University, Canchipur, Imphal 795003, Manipur, India

A R T I C L E I N F O A B S T R A C T

Keywords:
Drug addiction is a complex disease affected by numerous genetic and environmental factors. Brain regions in
Addiction reward pathway, neuronal adaptations, genetic and epigenetic interactions causing transcriptional enhancement

Abbreviations: ADPr, ADP-ribose; Airn, antisense Igf2r RNA; ANP, Atrial natriuretic peptide; ATP, Adenosine triphosphate; AVP,, Vasopressin; BDNF, Brain-
derived neurotrophic factor; CamKIIa, Calcium/calmodulin-depencent kinase II a; cAMP, Cyclic AMP; CBP, CREB binding protein; CCAT1-L, Colon cancer associated
transcript 1 large; CCAT1-S, Colon cancer associated transcript 1 small; ccr2, C-C chemokine receptor 2; Cdk5, Cyclin-dependent kinase 5; cFos, cFos protooncogene;
CREB, cAMP response element binding; CRISPR-Cas9, Clustered Regularly Interspaced Short Palindromic Repeats/ Cas9; crRNA, CRISPR RNA; CYP1A1, Cytochrome
P450 Family 1 Subfamily A Member 1; D3R, Dopamine receptor D3; dCas9, Dead Cas9; DLK1, Delta-like 1 homolog; DNMTs, DNA Methyltransferases; DNMT1, DNA
Methyltransferase 1; DNMT3A, DNA Methyltransferase 3 A; DNMT3B, DNA Methyltransferase 3B; DPF, Double plant homeodomain finger; DRD2, Dopamine receptor
D2; DSB, Double strand break; DUBs, Deubiquitinating enzymes; EGR1, Early growth response; Ehmt1, Euchromatic histone-lysine-N-methyltransferases 1; Ehmt2,
Euchromatic histone-lysine-N-methyltransferases 2; FDA, Food and drug administration; FosB, FosB protooncogene; G9a, G9a gene or Ehmt2 gene; GAD1, Glutamate
Decarboxylase 1; GID, Gibberellin insensitive dwarf; GLP, G9a like protein; gRNA, guide RNA; GTP, Guanosine triphosphate; GWAS, Genome wide association study;
H2AK119, H2A lysine 119; H2AK119ub1, H2A lysine 119 monoubiquitylation; H2BK34ub, H2B lysine 34 ubiquitylation; H2AX, histone H2A variant; H2AXS139,
H2AX Serine 139; H2AXY142, H2AX tyrosine 142; H2BS10, H2B serine 10; H2BS32, H2B serine 32; H2BS36, H2B serine 36; H3R2me2a, H3 arginine 2 dimethylation
asymmetric; H3K122cr, H3 lysine 122 crotonylation; H3K4me3, Histone H3 trimethylation; H3K27ac, Histone H3 Lysine 27 acetylation; H3K27cr, Histone H3 Lysine
27 crotonylation; H3R17me1, H3 Arginine 17 monomethylation; H3K4, histone H3 lysine 4; H3K36, histone H3 lysine 36; H3K79, histone H3 lysine 79; H3K9,
histone H3 lysine 9; H3K27, histone H3 lysine 27; H3S10, H3 serine 10; H3S28, H3 serine 28; H3Y41, H3 tyrosine 41; H3T3, Histone H3 threonine 3; H3T6, H3
threonine 6; H3T11, H3 threonine 11; H3T45, H3 threonine 45; H3Q5ser, H3 glutamine 5 serotonylation; H3K9K14ac2, histone H3 lysine 9 lysine 14 diacetylation;
H3K4me3, H3 Lysine 4 trimethylation; H3K4me3Q5ser, H3 Lysine trimethylation; H3Q5dop, H3 glutamine 5 dopaminylation; H4H18, H2 histidine 18; H4H75, H4
histidine 75; H4K20, histone H3 lysine 20; H4K20me1, histone H4 lysine 20 monomethylation; H4S1, H4 serine 1; HATs, Histone acetyltransferases; HCT, Histone
crotonyltransferases; HDACs, Histone deacetylases; HDCRs, Histone decrotonylases; HDMs, Histone demethylases; HDR, Homology directed repair; HERP,, Homo­
cysteine-induced endoplasmic reticulum protein; HMTs, Histone methyltransferases; HOTAIR, HOX Transcript Antisense RNA; HP1, Heterochromatin protein 1; ICR,
Imprint control region; IEGs, Immediate early genes; IG-DMR, Intergenic differentially methylated region; KCr, Lysine crotonylation; LINE, Long interspersed ret­
rotransposable elements; Lnc RNAs, long non-coding RNAs; MEG3, Maternally expressed 3; miRNA, MicroRNA; MIAT, Myocardial Infarction Associated Transcript;
EMX2O, Empty spiracles homeobox 2 O; MEF2, Myocyte enhancer factor-2; MOF, K(lysine) acetyltransferase 8 (KAT8) gene; MAOA,, monoamine oxidase A;
MARylation, Mono-ADP-ribosylation; MBD, Methyl-CpG-binding domain; MeCP2, Methyl CpG binding protein 2; MEG3, Maternally expressed gene 3; MEF2,
Multiple myocyte-specific enhancer factor 2; MIAT, Myocardial infarction associated transcript; MSN, Medium spiny neurons; MT1, Metallothionein 1; MT2, Met­
allothionein 2; NAc, Nucleus Accumbens; NAD, Nicotinamide adenine dinucleotide; NEAT1, Nuclear paraspeckle assembly transcript 1; NEAT2, Nuclear paraspeckle
assembly transcript 2; NF-κB, Nuclear factor-kappa B; NGF, Nerve growth factor; NHEJ, Non-homologous end joining; NMDA, N-methyl D Aspartate; NR2B, NMDA
receptor 2B; OFC, Orbitofrontal cortex; OPRM1,, Mu opioid receptor 1; PAM, Protospacer Adjacent Motif; PARylation, Poly-ADP-ribosylation; PCAF, P300/CBP-
associated factor; PDYN, Prodynorphin; PFC, Prefrontal cortex; Pitx3, Pituitary homeobox 3; piRNAs, P-element-induced wimpy testis-interacting RNAs; PIWI, P-
element-induced wimpy testis; PIWIL1, PIWI like protein 1; PKA, Protein kinase A; Pomc, Proopiomelanocortin; PTMs, Post translational modifications; Raf-1, Raf1,
protooncogene; RRBS, Reduced representation bisulfite sequencing; ROS, Reactive oxygen species; RVDs, Repeat variable diresidues; SAHA, Suberoylanilide
hydroxamic acid; SAM, S-adenosyl methionine; sg RNA, single guide RNA; siRNAs, Short interfering RNAs; SNCA,, α-synuclein; Srcin1, Src kinase signaling inhibitor
1; SRF, Serum-response factor; SUMO, Small ubiquitin-related modifier; TAB-Seq, Tet-assisted bisulfite sequencing; TALENs, Transcription activator-like effector
nucleases; TET1, Ten Eleven Translocase 1; Tet3, Ten Eleven Translocase 3; TORC, Transducer of Regulated CREB; tracrRNA, Trans Activating crRNA; UHRF,
Ubiquitin-like, with PHD and RING finger domains; UTR, Untranslated region; VS, Ventral Striatum; VTA, Ventral Tegmental Area; WDR5, WD repeat containing
protein 5; WGBS, Whole genome bisulfite sequencing; ZFNs, Zinc-finger nucleases; ZFP, Zinc finger proteins.
* Corresponding author.
E-mail address: rajacftri77@gmail.com (Y. Rajashekar).

https://doi.org/10.1016/j.biopha.2023.115951
Received 16 September 2023; Received in revised form 23 November 2023; Accepted 27 November 2023
Available online 2 December 2023
0753-3322/© 2023 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
A.S. Koijam et al. Biomedicine & Pharmacotherapy 170 (2024) 115951

Epigenetic or repression of multiple genes induce different addiction phenotypes for varying duration. Addictive drug use
Epigenome editing complexes causes epigenetic alterations and similarly epigenetic changes induced by environment can promote addiction.
CRISPR-Cas9 Epigenetic mechanisms include DNA methylation and post-translational modifications like methylation, acety­
TALEN
lation, phosphorylation, ubiquitylation, sumoylation, dopaminylation and crotonylation of histones, and ADP-
ZFN
ribosylation. Non-coding RNAs also induce epigenetic changes. This review discusses these above areas and
stresses the need for exploring epidrugs as a treatment alternative and adjunct, considering the limited success of
current addiction treatment strategies. Epigenome editing complexes have lately been effective in eukaryotic
systems. Targeted DNA cleavage techniques such as CRISPR-Cas9 system, CRISPR-dCas9 complexes, transcrip­
tion activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs) have been exploited as targeted
DNA recognition or anchoring platforms, fused with epigenetic writer or eraser proteins and delivered by
transfection or transduction methods. Efficacy of epidrugs is seen in various neuropsychiatric conditions and
initial results in addiction treatment involving model organisms are remarkable. Epidrugs present a promising
alternative treatment for addiction.

1. Introduction
Drug addiction is a complex disease of recurring psychological and
physiological impairment on persistent use of psychoactive drugs. It is
characterized by behavioural transition from positive reinforcement of
drug rewarding effects to negative reinforcement of countering with­
drawal symptoms, as presented in Fig. 1 [1]. Addiction is influenced by
multiple mechanisms of genetic, epigenetic, molecular and
neuro-endocrine entities, environmental influences, behavioural,
cognitive, emotional and motivational conditions, and brain responses
to reward and stress cues [2]. Genetics involve alterations in DNA,
genes, nucleotide sequences, expression changes arising thereof and its
heritability while epigenetics involve expression changes and herita­
bility due to regulatory effects arising out of changes in DNA and
DNA-associated proteins but not DNA sequence. Onset of drug use is
fundamentally dependent upon the genes and environment to a lesser
degree, while progression towards addiction is dependent significantly
upon genetic [3] and environmental factors [4,5]. While addiction and
its phenotypes have been associated with various genes, environmental
factors affecting addiction include family conditioning, physical,
emotional and sexual abuse, peer pressure, disease conditions etc [6].
Sex, age and ethnicity have reports of significant association while
socio-economic status, lower educational achievement, religion, marital
status and region showed varying degrees of association with different
addictive drugs [7]. Drug exposure induces expression changes in
numerous genes through epigenetic mechanisms within various brain
regions, accompanied by changes in structure, function and neuro­
circuitry. They are retained for long durations spanning months or years
[8]. Familial interaction, genetic vulnerability, neuroendocrine changes
and behaviours preceding addiction onset interlink through epigenetic
modifications in protective and risk polymorphisms on endogenous
opioid, monoaminergic, oxytoninergic and hypothalamic pituitary ad­
renal stress response systems [9]. Considering the limited success of
current addiction treatment strategies, reversible and inducible nature
of epigenetic mechanisms and initial remarkable results with model
organisms offer tremendous possibilities of epigenetic treatment for
addiction.

Fig. 1. The cycle of Addiction and drug-induced neuronal effects. Intoxication/ Binge stage- characterized by rewarding sensation and positive reinforcement with
subsequent learning and memory formation. Reward mediating neurotransmitters and associated processes in Nucleus accumbens, Ventral and Dorsal striatum,
Global pallidus and Thalamus are engaged. Withdrawal/ Negative effect stage- characterized by negative emotions and stress with involvement of Amygdala, Bed
nucleus of stria terminalis, and ventral striatum. Preoccupation/ craving- characterized by processing conditioned reinforcement, contextual inputs and execution
involving Prefrontal cortex, Anterior cingulate, Orbitofrontal cortex, Hippocampus and Basolateral nucleus of amygdala [1]. Drug-induced short term and long term
effects are executed by processes involving neurotransmitters, neuronal proteins and gene expression changes specifically in neurons of the reward circuit.

2
A.S. Koijam et al.
2. Brain functions and neuronal changes in addiction
The Reward system or Mesolimbic system in the brain is the centre
for cognitive and physiological processing of reward [10]. Brain struc­
tures involved in Reward Pathway are crucial in determining the degree
of addiction, which include Prefrontal cortex (PFC), orbitofrontal cortex
(OFC), Ventral Tegmental Area (VTA), Nucleus Accumbens (NAc),
Dorsal Striatum, Ventral striatum (VS), Amygdala and Hippocampus.
Much of these structures are a mixture of distinct cell types with varied
signaling and electrophysiological properties offering differential tran­
scription leading to a variety of drug responses [11]. Imaging studies
have demonstrated that intoxication/ binge stage of addiction cycle are
primarily influenced by VTA and VS, while negative/ withdrawal stage
is influenced by extended amygdala, and Anticipation/ preoccupation
stage is influenced by OFC, DS, PFC, basolateral amygdala and hippo­
campus [1]. Drugs cause rewarding stimuli by activating the dopami­
nergic mesolimbic system leading to dopamine release in target nuclei
[10]. Dopamine levels are increased in the NAc by almost all addictive
drugs via indirect or direct action on dopaminergic neurons. Dopamine
signaling in NAc is a crucial mechanism to drug reinforcement. Drugs
increase dopamine release in brain and continued use decrease natural
production levels of dopamine, inducing drug dependency for main­
taining normal dopamine levels. Of the various molecular mechanisms
of drug action, few prominent ones include the downregulation or
upregulation of their receptors or transporters, changes in membrane
permeability or ion conductance, inhibition of endogenous opioids and
peptides by feedback mechanism, effects on neurotransmitter systems
and neuronal plasticity of reward circuit [12]. Glutamatergic-mediated
neuroadaptations in dopamine striato-thalamo-cortical and limbic
pathways are triggered by chronic exposure to drugs [13]. There are
certain general effects of various drugs on NAc and VTA of brain.
Alcohol and opiates stimulate DA production indirectly through inhi­
bition of GABAergic neurons of VTA. Stimulants, on the other hand,
enhance DA transmission directly. Nicotine induces DA neuron firing
through action on acetylcholine receptors. Cannabinoids dysregulate
normal endocannabinoid signaling of dopaminergic neurons to
GABAergic and glutamatergic neurons through regressive suppression of
inhibition and regressive suppression of excitation respectively [13].
Neurotransmitters, their distribution, excitatory or inhibitory nature
and timespan in synapse also contribute to addiction phenotypes.
Epigenetic processes, drug-induced neuronal and synaptic plasticity has
a crucial role towards learning, memory, addiction development and
relapse [14,15]. Drugs, neurotransmitters and neuronal proteins interact
to cause alterations in gene expression particularly the immediate early
genes (IEGs). Epigenetic changes may be induced in these genes based
on the degree and phase of drug exposure or during withdrawal [16].
Neurons acquire high plasticity via such epigenetic changes to store and
integrate new inputs or information. Neuronal changes in terms of
morphology and dendritic spine density of medium spiny neurons
(MSN) within the nucleus accumbens (NAc) and prefrontal cortex of rats
are induced by various drugs of abuse [17,18]. Methamphetamine
exposure induces the expression of synapsins, syntaxin 1 A and syn­
aptophysins which causes change in synaptic plasticity [19]. Various
transcription factors have crucial roles in drug related synaptic plas­
ticity. They contain DNA-binding domains specific to sequences in
promoter or enhancer regions of genes that promote or block RNA po­
lymerase II transcriptional complex recruitment. Apart from other
functions, cAMP response element binding (CREB) mediates dendritic
spine formation and generates silent synapses vital to synaptic devel­
opment linked with intensified drug seeking [20,21]. Nuclear
factor-kappa B (NF-κB) regulates synaptic plasticity, crucial for dendritic
spine induction in Nucleus Accumbens Medium Spiny Neurons [22].
Multiple myocyte-specific enhancer factor 2 (MEF2) modulates tran­
scription activation or repression of genes engaged in excitatory synapse
regulation and cell survival. It may negatively regulate neuronal plas­
ticity. Serum-response factor (SRF) mediates cocaine rewarding
3
Biomedicine & Pharmacotherapy 170 (2024) 115951
properties in NAc via regulation of ΔFosB expression and also regulates
actin cytoskeletal remodelling for spinogenesis. ΔFosB overexpression
in NAc amplifies drug reward and self administration while transcrip­
tional silencing has the reverse effects [23].
3. Epigenetics and mechanisms
Epigenetics involves the study of changes in gene expression and
their heritability due to chemical modifications in DNA and DNA-
associated proteins but not DNA sequence changes. Epigenetic pro­
cesses contribute to the unique differential gene expressions in various
cells and correlate to nucleosomal changes regulating transcription and
biological processes. Changes in chromatin structure, particularly
condensation and decondensation of nucleosomes, leads to concealment
or exposure of specific gene regions. These changes inhibit or allow
access of transcriptional machinery to the chromatin scaffold and thus
gene expression is repressed or activated. A nucleosome is a basic DNA
compaction unit made of approximately 147 bp DNA coiled around a
core histone octamer of two copies each of H2A, H2B, H3 and H4.
Adjacent nucleosomes are linked together by 20–60 bp linker DNA and
H1 histone. Histones of nucleosome have a carboxyl-terminal, a globular
domain and a relatively disorganised amino-terminal bearing 20–35
amino acid residues where majority of the post translational modifica­
tions (PTMs) occur. Nucleosomal restructuring after replication involves
daughter chromosomes receiving distributed parental histones at
around the same dislodged locus and newly synthesized histones
replenishing to replicate covalent modification states of parental nu­
cleosomes [24]. Epigenetic mechanisms include methylation of DNA;
acetylation, methylation, sumoylation, phosphorylation, ubiquitylation,
serotonylation, dopaminylation and crotonylation of histones; ADP
ribosylation and gene expression regulation by non-coding RNAs. Pro­
tein machineries affecting epigenetic modifications are broadly classi­
fied as Writers, Readers and Erasers, based on their respective catalytic
activity of addition, recognition (through specific binding motifs and
triggering various signaling pathways) and removal of various func­
tional groups. Fig. 2 provides a brief illustration of various environ­
mental factors and protein machineries affecting epigenome. Readers
contain various protein domains which bind to epigenetically modified
sites to direct or guide relevant protein complexes for downstream
effects.
Epigenetic changes can be induced by numerous chemical and
environmental factors [25]. Epigenetic inheritance in somatic cells is
linked to long-term behavioural changes. Germ cells display parental
imprinting and with progression from zygote to later embryonic stages,
there is de-novo methylation. Imprinted genes exhibit specific DNA
methylation patterns and retain molecular memory of their germline but
are also contingent on epigenetic reprogramming post environmental
exposures. The epigenome of primordial germ cells reset to regain
developmental totipotency where a limited number of genes can escape
the epimark erasure process [26]. Various drugs of abuse are known to
induce intergenerational effects through epigenetic processes acting at
the level of gametogenesis or early embryogenesis [27–29] as detailed in
Section 4. Various factors account for epigenetic marks to be either
tissue specific or tissue independent and studies that can provide a
consensus remains elusive.
3.1. DNA methylation
DNA Methylation has distinct role in tissue-specific gene expression,
X-chromosome inactivation, parental imprinting, chromatin organisa­
tion and overall organism development. There is extreme complexity in
patterns of DNA methylation and are correlated with genomic locations.
The nucleobases Cytosine, Guanine and Adenine display natural enzy­
matic methylation tendency which modifies into 5-methylcytosine, 5-
hydroxymethylcytosine, N7Methylguanine, N4-methylcytosine and N6-
methyladenine. 5-methylcytosine is the most commonly found DNA
A.S. Koijam et al. Biomedicine & Pharmacotherapy 170 (2024) 115951

Fig. 2. Environmental factors and Epigenetic regulatory mechanisms. Various environmental factors modulate gene expression and corresponding phenotypes
through epigenetic mechanisms. Proteins/ enzymes grouped under Writers add chemical moieties or epigenetic tags to DNA or histones which can be interpreted by
Readers to help various factors to mediate cellular processes and Erasers remove the epigenetic tags. For instance, histone acetylation relaxes chromatin, activate
transcription and increase gene expression.

methylation in humans, followed by a tenfold lower level of 5-hydroxy­
methylcytosine while other methylations are rare or limited to pro­
karyotes [30]. Methylation mainly occurs at cytosine residues
particularly at the CpG dinucleotides. CpG dinucleotides are concen­
trated in genomic regions called CpG islands (regions of about 1 kb with
more than 55% GC) and CpG shores (0–2 kb from promoter) and to a
lesser degree in CpG shelves (2–4 kb from promoter) and open sea (other
regions in gene promoter). The human genome is estimated to contain
around 29000 CpG islands with locations near transcription start site or
first exon [31]. Methylation of CpG islands and CpG shores is associated
with underexpression of genes [32]. In dividing cells, methylation of the
gene region downstream of first exon known as gene body, have been
correlated with overexpression while the same is not exhibited by
slow-dividing and non-dividing cells [33]. Generally, hypomethylation
occurs in the active gene regulatory elements and methylation di­
minishes the binding affinity of transcription factors (TFs) thereby often
resulting in underexpression of the relevant gene. Interestingly, analysis
with methylation-sensitive SELEX (systematic evolution of ligands by
exponential enrichment) involving 542 human TFs have shown that CpG
methylated sequences are preferential for many TFs [34]. Methylation of
cytosine is also reported in CpH (where H= A, C or T) dinucleotides
restricting mostly to neurons, muscles and embryonic stem cells. After
birth, synaptic pruning and synaptogenesis coincides with CpH
methylation accumulation in brain [35]. Human primary astrocytes
exposed to cocaine have been reported to change CpG and non CpG
methylation levels of mtDNA [36]. Methylation changes may occur at a
specific locus or globally or both and may be an epigenetic driver of
various conditions [37]. Demethylation and de-novo methylation
executed by Demethylases and DNA Methyltransferases (DNMTs) during
embryonic development allow establishment of unique and stable
methylation patterns in genes of differentiated tissues [33]. The brain
exhibits one of the maximum methylation levels in comparison to other
human parts [38,39] mCH frequency were found to be highest in adult
neurons while glial mCH level was observed to be same as cortical tis­
sues of foetus. Out of the total methylation in the genome of adult
human neurons, about 53% is accounted to mCH while about 47% is
accounted to mCG [39]. Rizzardi et al. [40] reported that differentially
methylated regions (DMRs) within neuronal population are greatly
enhanced for heritability of addictive behaviour. While DNA methyl­
ation patterns vary between tissues and within cells, methylation studies
involving peripheral tissues other than cells in various brain regions,
have indicated towards possible usage as biomarkers of addiction
[41–43]. Pilot scale study involving 27 participants with substance use
disorder have demonstrated differential methylation of 5532 CGs with

4
A.S. Koijam et al.
1074 CG hypermethylation and 4458 CG hypomethylation in T-cells
[41]. A similar observation of 186 differentially methylated positions
was observed in a study involving 23 cocaine use disorder participants
and 24 healthy controls [43]. Hypermethylation of specific genes like
OPRM1 has also been observed in opioid use disorder individuals [42].
DNA methylation is the catalytic transfer of a methyl group from S-
adenosyl methionine (SAM) to a cytosine or adenine residue. Three
prominent DNMTs viz. DNMT1, DNMT3A and DNMT3B methylate DNA.
DNMT1 maintains de-novo DNA methylation, with its deletion inducing
global demethylation and death in human embryonic stem cells [44], or
overexpression causing embryonic lethality [33]. DNMT3A regulates
genome-wide de-novo methylation and methylation patterns during
parental imprinting, embryonic and haematopoietic stem cell differen­
tiation. DNMT3B is the main de-novo DNA methyltransferase actively
expressed during implantation and embryonic stages. DNMT3A and
DNMT3B have been largely linked to structural changes in DNA ac­
counting to long-term memory formation [45]. Three different protein
families viz. methyl-CpG-binding domain (MBD) proteins, ubiquitin-like
containing plant homeodomain and RING finger domain (UHRF) pro­
teins and Zinc finger proteins recognize DNA methylations and aid in
reading by acting along with various protein complexes. Chromatin
remodelers such as methylases and histone deacetylases are recruited to
methylated DNA by MBD proteins, but they also tend to bind to regu­
latory regions and unmethylated DNA [46]. On repeated cocaine
exposure, DNMT3A and DNMT3B overexpression was observed. Further,
in the caudate putamen of rats, increased methylation of PP1Cb gene,
and subsequent repression of PP1Cb gene was observed along with its
MeCP2 binding [47]. An isoform of DNMT3A is the DNMT3A2 and its
overexpression in NAc shell of rats but not NAc core, has been reported
following acute cocaine administration. While cocaine-reinforcing ef­
fects are not mediated by DNMT3A2, it regulates cocaine-cue memories
driving reinstatement to seek cocaine, following early abstinence and
incubation of cocaine craving [48]. The same study [48] used primary
striatal cultures to demonstrate the activation of DNMT3A2 by dopa­
mine D1 receptor signaling and the induction of IEGs viz. Arc, FosB and
Egr2 is impaired by DNMT3A2 knockdown. Demethylation during
developmental processes and cellular differentiation occurs through two
mechanisms viz. Passive DNA demethylation and Active DNA deme­
thylation. Replication-dependent 5mC dilution or Passive DNA deme­
thylation results from lack of functional maintenance methylation.
Replication-independent Active DNA demethylation is an indirect pro­
cess which brings about 5mC replacement with unmodified cytosine
preceded by enzyme-mediated 5mC modification [49]. Another set of
enzymes called Ten eleven translocation (TET) proteins are responsible
for oxidation of 5-methylcytosine and potentially remove DNA
methylation [50,51]. TET1, TET2 and TET3 oxidise 5-methylcytosine
into 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcyto­
sine. TET1 but not TET2 and TET3, has been demonstrated to be
downregulated in the nucleus accumbens following repated cocaine
administration [52]. Further, differential levels of 5-hydroxymethylcy­
tosine were reported at 11,511 regions across autosomes following
repeated cocaine exposure [52]. TET1 and TET3 have also been asso­
ciated with morphine seeking behaviour as epigenetic modulators and
present options in addiction treatment [51].
Paternal epigenome is globally demethylated before the first cell
division while maternal epigenome gets passively demethylated with
subsequent cleavage [53]. Epigenetic marks in Imprint control region
(ICR) of genes regulate genomic imprinting and are heritable resulting in
monoallelic, parent of origin dependent gene expression. Maternal
methylation is predominant in ICRs with consequence of inheritance
and reprogramming through maternal line [54]. Transcription factors
which can be repressors or activators, interact with methylated cyto­
sines. CRE sequence methylation promotes C/EBPα binding to DNA
consequently activating a promoter set specialised for differentiation of
adipocytes. Methylated CpG dinucleotide bind to methyl-binding
domain (MBD) proteins and induces deacetylation of histones leading
5
Biomedicine & Pharmacotherapy 170 (2024) 115951
to condensation of chromatin and repression of genes [55].
3.2. Histone methylation
Histone methylation involves incorporating methyl groups donated
by SAM in the lysine and arginine residues present at histone tails.
Histone tails do not exhibit change in overall charge but hydrophobicity,
basicity and anionic affinity are modified on histone methylation. His­
tone methylation and demethylation processes are catalyzed by Histone
methyltransferases (HMTs) and Histone demethylases (HDMs) respec­
tively (represented in Fig. 3). Histone methylation regulate gene
expression at multiple transcriptional levels including chromatin ar­
chitecture and loci-specific regulation by loosening or binding their tails
around the DNA thus granting or blocking access to cell-specific tran­
scription factors, initiation factors, elongation factors and associated
proteins [56]. Lysine 4 of histone H3 can be mono, di or tri methylated
[57]. In many cell types, H3K4me1 is linked to gene repression.
H3Kme2/3 recruits chromatin readers or interactors such as PHD fingers
of ING1 and ING2 [58]. Gene activation is promoted by H3Kme3
through transcription activation effector molecules [57]. Complex
combinatorial possibilities of histone methylation regulate gene
expression and phenotypic outcomes including disease development.
For instance, monomethylation on lysine 20 of histone H4 (H4K20me1)
leads to chromatin contraction [59,60]. H4K20me1 may also facilitate
chromatin openness and subsequently promote transcription [61] while
H3R17me1 induces transcriptional activation [59]. Lysine methylation
at H3K4, H3K36 and H3K79 induces transcriptional activation while
H3K9, H3K27 and H4K20 cause transcriptional silencing [62].
H3K9me3 and H3K9me2 have been associated with transcriptionally
silent, while H3K4me3 and H3K4me2 are associated with actively
transcribing genes [63]. Intermittent subcutaneous methamphetamine
injection in mice leading to methamphetamine-induced behaviour,
caused elevated mRNA expression of chemokine receptor CCR2, which
in turn has been attributed to an increase in trimethylation of H3 histone
at lysine 4 (H3K4me3) in CCR2 promoter [64]. Increased H3K4me3 has
been correlated with methamphetamine-associated memory. Knock­
down of the methyltransferase MLL1 and subsequent reduction of
H3K4me3 disrupt methamphetamine-associated memory while knock­
down of the histone demethylase, KDM5C leading to H3K4 hyper­
methylation prevents expression of methamphetamine-associated
memory [65]. Numerous biological processes such as transcription, cell
cycle, DNA repair, differentiation, stress response, long-term memory
formation and aging are influenced by histone methylation [56,66].
3.3. Histone acetylation
Histone acetylation exhibits transfer of acetyl group from donor
Acetyl CoA to ε-amino group (–NH3 +) of lysine of histones. Acetylation
especially lysine in N-terminal histone tails reduce DNA-histone contacts
by neutralizing the electrostatic interaction between positively charged
amino groups on lysine and negatively charged DNA. This loosens the
nucleosomal structure for easy access to transcriptional machinery and
thus gene expression is activated. Lysine deacetylation on the other
hand, has the reverse effect of chromatin condensation and transcription
inhibition [62]. Two groups of counteracting enzymes viz. histone
acetyltransferases (HATs) and histone deacetylases (HDACs) regulate
acetylation by adding or removing acetyl groups respectively (Fig. 3).
HATs are classified on the basis of sub-cellular location as Type A HATs
(nuclear localization) that acetylate histones and chromatin associated
proteins and Type B HATs (predominantly cytoplasmic localization)
which acetylate newly synthesized histones. The 18 membered HDAC
superfamily has four classes, broadly grouped into two protein families:
Zinc-dependent metalloproteins (Class I, Class II and Class IV HDACs)
and NAD+ dependent proteins (Class III HDACs) [67]. Histone acety­
lation has been observed on all the core histones in human viz. lysine
residues at positions 5, 9, 13, 15, 36, 74, 75, 95, 118, 127 and 129 in
A.S. Koijam et al. Biomedicine & Pharmacotherapy 170 (2024) 115951

Fig. 3. Histone post translational modifications, PTM enzymes and substrates. Histone ADP-ribosylation- Reversible single or multiple ADP-ribose addition on lysine
(K)/ arginine (R)/ aspartic acid (D)/ glutamic acid (E)/ cysteine (C)/ serine (S) causing Mono-ADP-ribosylation (MARylation) and Poly-ADP-ribosylation (PAR­
ylation). Sumoylation- Adding SUMO to Lysine residue in histones in a 3-step process of activation, conjugation and ligation. Ubiquitylation- Adding ubiquitin to
lysine residues in histone. Phosphorylation- adds phosphate from ATP/GTP to serine, tyrosine and threonine in histones. Methyltransferase adds methyl group from
SAM to lysine and arginine in histones. Acetylation- Counteracting HATs and HDACs add and remove acetyl groups respectively in lysine of histones.

histone H2A; lysine at 5, 11, 12, 15, 16, 20, 23, 24, 43, 46, 57, 85, 108,
116, 120 and 125 in histone H2B; lysine at 4, 9, 14, 18, 23, 27, 36, 37,
56, 64, 79, 115 and 122 in histone H3; and lysine at 5, 8, 12, 16, 20, 31,
77, 79 and 91 in histone H4 [68,69]. Chronic exposure to psychosti­
mulants has been associated with increased histone acetylation at pro­
moters of various genes involved in synaptic transmission including
Cdk5, CamKIIa, NFkB subunits [70,71]. Although a few inconsistencies
have been reported [72,73], acetylation of lysine at 5, 8, 12 and 16 of H4
at Fos and Fosb is increased with acute cocaine administration but not at
lysine 9 and 14 at H3. Cdk5, Bdnf and Fosb gene promoters displayed
increased acetylation at H3K9, H3K14 but not H4K5, H4K8, H4K12,
H4K16, following chronic cocaine administration [70]. CREB-binding
protein (CBP) which is a histone acetyltransferase is responsible for
mediating transcriptional activation by histone acetylation and
recruitment of basal transcription machinery. It regulates expression of
genes required for memory and long-term plasticity [73]. CBP deletion
in mice NAc has been correlated with impaired cocaine sensitivity and
context-cocaine associated memory [73]. In a study [74], the NAc of

6
A.S. Koijam et al.
chronic cocaine treated mice or saline were taken to map the
genome-wide promoter interaction of modified histones using ChIP and
antibodies directed against polyacetylation of H3 and H4. Chronic
cocaine administration induced hyperacetylation of histones H3 and H4
at 959 and 692 gene promoters respectively while hypoacetylating 83
and 123 gene promoters respectively [74]. Autoacetylation of P300 and
acetyltransferase action has been reported to be upregulated by NEAT1
along with changes in H3K27ac and H3K27cr [75]. Maurice et al., [76]
demonstrated that P300/CBP-associated factor (PCAF) homozygous
knockout mice have memory and learning impairments, indicating the
inseparable role of HATs. At 2 months maturity, these knockout mice
indicated short term memory deficits that were correlated to hippo­
campal changes, Fos levels and activation of MAP kinase. Memory and
learning worsened with age but differences based on gender were not
observed [76]. Considering the extensive overlapping of learning and
memory with neural processes underlying addiction [77], HATs may
play a role through PCAF. In striatal neurons, D2-like receptors blockade
cause phospho-acetylation of H3 (H3pS10-acK14) through
cAMP-dependent PKA and NMDA receptor signaling [78]. HDACs can be
recruited by MBD proteins to methylated DNA, but also tend to bind to
regulatory regions and unmethylated DNA through nucleosome
remodeling deacetylase (NuRD/Mi-2) complex members [46]. In­
hibitors of HDAC (HDACi) such as Valproic acid and Magnesium val­
proate have been approved by FDA and used for treatment of neurologic
disorders and seizures. Valproic acid acts on gamma amino butyric acid
(GABA) levels and most HDACs [79]. The expression of Class III HDACs
like SIRT1 and SIRT2 within mouse NAc is increased on chronic cocaine
exposure. Chronic morphine exposure causes SIRT1 overexpression
[80]. Rewarding effects of both cocaine and morphine were enhanced by
viral-mediated SIRT1 and SIRT2 overexpression in NAc of mice. On the
other hand, drug reward decreased in mice with SIRT1 knockdown in
NAc. SIRT1-induced behavioural effects are regulated through various
synaptic proteins in NAc and SIRT1-induced dendritic spines on medium
spiny neurons of NAc [80]. In a study [81] involving rats withdrawn
from methamphetamine self administration and footshocks for several
weeks, HDAC1 mRNA significantly decreased in the dorsal striatum of
sensitive rats as against control rats. Sensitive rats displayed a significant
decline in the mRNA levels of HDAC2 and HDAC8 as compared to
control and resistant rats. In comparison to control rats, the dorsal
striatum of sensitive rats showed a significant decline in HDAC class IIa,
IIb and IV. HDAC9 mRNA was significantly downregulated in sensitive
rats compared to control and resistant rats. The dorsal striatum of
resistant rats showed significant increase in the mRNA of the lysine
acetyltransferases KAT4 and KAT5 in comparison to sensitive and con­
trol rats [81]. Downregulation was observed for SIRT2, SIRT3 and SIRT4
in the dorsal striatum of shock resistant and shock sensitive rats as
compared to controls. The effects seen on few epigenetic enzymes in the
NAc is suggestive of the fact that dorsal striatum participates in
behavioural manifestations of forced abstinence following compulsive
meth administration [81]. Cadet et al., [82] have reported that mRNAs
of HDACs were differentially expressed in the NAc of rats with
compulsive methamphetamine and abstinent rats. These were analysed
on the basis of responses to footshocks during the period of metham­
phetamine self administration. In the study [82], HDAC1, HDAC3,
HDAC6, and HDAC8 expressions in NAc were observed to be signifi­
cantly different between shock resistant (SR) and shock sensitive (SS)
rats. SS rats displayed significantly higher SIRT1 and SIRT7 expression
compared to SR rats. SS rats also exhibited significantly higher mRNA
levels compared to both SR and control rats [82]. HDAC inhibitors are
potential epidrugs for addiction treatment and are discussed in later
section.
3.4. Histone phosphorylation
Histone phosphorylation involves catalytically adding phosphate
group from ATP or GTP to serine, tyrosine and threonine amino acids of
7
Biomedicine & Pharmacotherapy 170 (2024) 115951
histones by protein kinases while phosphatases cause counteractive
dephosphorylation (Fig. 3). Histone phosphorylation in general, has a
transcriptional activation effect due to repulsion between negatively
charged phosphates in histones and DNA backbone as well as HAT
recruitment, specific transcription factor association and removal of
proteins or PTMs engaged in gene silencing [83]. In human H2AX his­
tone variant, phosphorylation has been reported on serine at 139 and
tyrosine at 142 (H2AXS139 and H2AXY142). Similarly, serine at 32 and
36 position in H2B (H2BS32, H2BS36); threonine at 3, 6, 11, 45 (H3T3,
H3T6, H3T11, H3T45), serine at 10 and 28 (H3S10, H3S28) and tyrosine
at 41 (H3Y41) in H3, and serine at 1, histidine at 18 and 75 (H4S1,
H4H18 and H4H75) in H4 histones are phosphorylated [84]. It has been
linked to various cellular processes such as transcription regulation
involving H2BS36, H2BY37, H3S10, H3S28, H2BS32, H3T6, H3T11,
H3Y41, H4S1 and H4S47; meiosis involving H2BS10 and H4S1; mitosis
involving H3S10 and H3S28; DNA damage involving H2AX S139 and
H4S1; apoptosis involving H2AXS139, H2BS10 and H3T45 [85]. Pro­
moter regions of rapidly inducible ‘immediate early’ genes like c-fos
exhibit histone phosphorylation [85]. H2AX phosphorylation is a tran­
sient modification resulting from DNA damage. Although there are no
direct experimental evidences of H2AX phosphorylation with drugs,
certain drugs like opioids are known to induce DNA damage in the NAc
and PFC in heroin exposed mice [86]. MeCP2 phosphorylation at Ser421
is known to be induced by psychostimulants, and this phosphorylation
has role in limiting circuit plasticities in NAc which relate to addictive
behaviours [87].
3.5. Histone ubiquitylation
Histone ubiquitylation or ubiquitination is a PTM involving enzy­
matic covalent ligation of an ubiquitin protein to histones wherein an
isopeptide bond gets established between ε-amino group (–NH+ 3 ) of
lysine in histones and carboxylic acid group of the last glycine (glycine
76) in ubiquitin. Three groups of enzymes process ubiquitylation viz.
Ubiquitin-activating enzymes (E1s), Ubiquitin-conjugating enzymes
(E2s) and Ubiquitin ligases (E3s). The structure of chromatin is affected
by ubiquitinylation through mechanisms such as regulating DNA access
to transcriptional machinery, providing binding site for transcriptional
activators or inhibitors, trans-histone cross talk and associating other
PTMs. Although all histones including H1 can be ubiquitinylated, H2A,
H2B and H3 are most commonly ubiquitinylated in the nucleus. Ubiq­
uitinylation can be reversed by ubiquitin-specific peptidases called
Deubiquitinating enzymes (DUBs) (Fig. 3). H2A ubiquitination has a
repressive effect on transcription while H2B ubiquitination has an
activating or permissive effect on transcription [88]. Ubiquitination has
been reported for H2AK119ub1, H2AK13/15ub1, H2AK127/129ub1,
H2BK120ub1, H3K18/23ub1, H3K14ub1 and H3K23/36/37ub1 [89].
H2B is ubiquitinated by PAF1 complex in the course of transcription
elongation. PAF1C controls ubiquitination of H2BK120ub and
H2BK34ub. H2BK120ub is required for H3K79me2/3 and H3K4me2/3.
H2BK34ub enhances H4K16ac via MOF action [90].
3.6. Histone sumoylation
Histone Sumoylation is another reversible PTM where isopeptide
bond is formed between the ε-amino group (–NH+ 3 ) of lysine in histones
and C-terminal Glycine of small ubiquitin-related modifier (SUMO)
thereby covalently attaching SUMO group. Humans express four distinct
SUMO isoforms viz. SUMO1, SUMO2, SUMO3 and SUMO4. Sumoylation
process is achieved through an activating heterodimer enzyme (E1:
Aos1/Uba2), a conjugating enzyme (E2: Ubc9) and SUMO ligase (E3)
(Fig. 3). Sumoylation occurs on all histone types including variants H2A.
Z and H2A.X. affecting transcriptional repression by recruiting HDAC
and HP1. A study [91] demonstrated that sumoylation has been linked
to various processes such as chromatin organization, protein-protein or
protein-DNA interactions, transcriptional regulation, nuclear-cytoplasm
A.S. Koijam et al.
transport, genome stability, protein stability, stress response, cell cycle
progression, apoptosis and cellular localization [92,93].
3.7. Histone ADP ribosylation
ADP-ribosylation is a reversible PTM characterized by enzymatic
transfer of ADP-ribose (ADPr) from donor nicotinamide adenine dinu­
cleotide (NAD+) to amino acid side chains with nucleophilic nitrogen,
oxygen or sulphur (such as lysine, arginine, aspartic acid, glutamic acid,
cysteine and serine) on histone protein forming N-, O- or S- glycosidic
linkage to ribose and releasing nicotinamide. It is affected by ADP-
ribosyltransferases that covalently attaches ADPr and ADP-
ribosylglycohydrolases which remove ADPr (as represented in Fig. 3).
There are two ways in which ADP-ribosylation can occur viz. Mono-
ADP-ribosylation (MARylation) and Poly-ADP-ribosylation (PAR­
ylation). ADP-ribosylation provides numerous options to change the
functions and properties of substrates (histones) due to the fact that
ADPr can cause hydrogen bonding and hydrophobic interactions as it
contains an adenine ring, two ribose moieties and bears two negative
charges at physiological pH. ADP-ribosylation regulates numerous key
processes such as transcriptional regulation, DNA repair, cellular
transport, immune response, cell survival etc. [94]. PARylation by
PARP1 are vital for viability in neurons, learning and memory. Transient
decondensation of chromatin is induced by PARylation leading to
transcription of genetic components required for long term memory
[95]. Cocaine-induced PARylation has been reported in several studies
[96–100]. A study [96] demonstrated the global increase in PARP1 and
Poly(ADP-ribose) or PAR in mice NAc following repeated cocaine
exposure. Further, H1 and H3 were selectively PARylated with chronic
cocaine use, indicating a permissive chromatin structure. PARP1 has
also been associated with cocaine-paired cues and acts by binding at
promoter of a putative transposase inhibitor [97] PARP1 is thus a po­
tential target for cocaine relapse prevention. PARP1 is known to be
post-transcriptionally regulated by miR-125b and miR-124. Acute and
chronic cocaine exposure, both in cultured neurons and in mice,
persistently downregulates miR-125b and miR-124, which in turn in­
creases PARP1 expression [98,99]. PARP1 acts through positive or
negative regulation of promoters and enhancers along with transcrip­
tional modulation via structural changes in chromatin. PARP1 enzyme
action is crucial for activation of NF-κB [101]. The regulation of gene
expression is attributed to the competition between histone H1 and
PARP1 to bind to specific gene promoters [102,103]. PARP1 also con­
tributes to open chromatin formation through prevention of H3K4me3
demethylation. Binding of KDM5B to chromatin is inhibited by its
PARylation and this enhances RNA polymerase II binding at promoter
regions of specific genes with positive regulation [104].
3.8. Histone Serotonylation
Histone serotonylation is the PTM involving the addition of serotonyl
group to histones. The 5th position of Histone 3 occupied by glutamine is
of specific importance in terms of serotonylation (H3Q5ser) as it has a
permissive role in gene activation in conjunction with H3K4me3 during
differentiation of neurons. Cellular differentiation is associated with
increased levels of H3K4me3Q5ser. Mutant RN46A-B14 cells incapable
of H3Q5ser demonstrated inability for cellular differentiation [105].
WDR5 is an H3Q5ser reader whose interaction mediates epigenetic
regulation and tumour development [106].
3.9. Histone dopaminylation
Histone dopaminylation is another PTM wherein dopamine mole­
cules are directly attached to glutamine in histones by Transglutaminase
2 and was first reported by Lepack et al., [107]. In association with
cocaine administration, dopaminylation of histones in the VTA play a
major role in dysregulated gene expression within neurons and
8
Biomedicine & Pharmacotherapy 170 (2024) 115951
contributes to vulnerability and relapse. Administration of cocaine
acutely enhances dopamine production in NAc synapses and cause
dopaminylation of glutamine 5 at histone H3 (H3Q5dop). H3Q5dop in
VTA is strongly associated with cocaine-induced transcriptional plas­
ticity. H3Q5dop attenuation in the VTA following prolonged cocaine
access decreases the release of dopamine in ventral striatum. H3Q5dop
reduction in VTA impairs cocaine seeking [107]. H3Q5dop and dopa­
mine increases following acute cocaine exposure, resulting in under­
expression of DRD2 and finally leads to cocaine withdrawal. Further,
during cocaine withdrawal, Src kinase signaling inhibitor 1 (Srcin1 or
p140CAP) reduces H3R2me2a binding. This is followed by inhibition of
dopaminylation and diminished NAc Src signaling, cocaine reward and
motivation [108].
3.10. Histone crotonylation
Histone crotonylation was first identified in the core histones (H2A,
H2B, H3 and H4) by Tan et al., [68] and has been associated with gene
activation. Crotonyl-CoA is the substrate for crotonylation. Much like
acetylation, crotonylation at lysine residue of histones neutralize its
positive charge weakening DNA interactions and loosening chromatin
for access to various DNA-interacting proteins. It is thus suggested to
have a role in transcription activation of genes [109]. Lysine crotony­
lation (KCr) has been reported to involve in replication stress response.
Replication stress reversibly regulates crotonylation and ubiquitination
of H2A at lysine 119 (H2AK119). Nucleosome destabilisation capability
of Kcr has been suggested to involve in chromatin to nucleoprotamine
transition in the course of spermatogenesis [110]. Crotonylation occurs
on all the five histones in humans viz. lysine residues at positions 33, 63,
84, 89, 96, 158 and 167 in histone H1; lysine at 36, 118, 119 and 125 in
histone H2A; lysine at 5, 11, 12, 15, 16, 20, 23 and 34 in histone H2B;
lysine at 4, 9, 18, 23, 27 and 56 in histone H3; and lysine at 5, 8 and 12 in
histone H4 [68,69]. Crotonylation of 122 lysine in H3 (H3K122cr)
causes hindrance in water-mediated hydrogen bond formation with
DNA backbone thereby reducing DNA-histone interaction and eventu­
ally favouring transcriptional activation. Although crotonylation mainly
promotes transcription, crotonylation at H3K9 and H3K27 on
Pro-growth genes and endocytosis-related genes respectively, have been
reported to inhibit transcription [111]. MOF, CBP and p300 are the main
histone crotonyltransferases (HCT) in the mammalian system while
Class I HDACs are the main histone decrotonylases (HDCRs). Decroto­
nylation is also processed by Sirt1 via multiple targets [109]. While
Bromodomain proteins are also identified Kcr readers, Double plant
homeodomain finger (DPF) domain has been identified as histone
Kcr-preferential reader [69,112]. Lysine crotonylation level is suggested
to be impacted by intracellular crotonyl-CoA concentration, dynamics of
crotonyltransferase and decrotonylase, and ratio of
Acetyl-CoA/crotonyl-CoA [69]. Kcr has been reported to occur in
non-neuronal and neuronal cells in spinal cord, medulla oblongata, tri­
geminal ganglia and dorsal root ganglia. Kcr has been suggested to be
involved in regulation of neuropathic pain and neuroinflammation
[113] which are also associated with chronic opioid use [114].
3.11. Non-coding RNA induced epigenetic changes
Non-coding RNAs represent the group of RNAs which do not code for
any functional protein but are engaged in post transcriptional gene
regulation. They include long non-coding RNAs (lncRNAs), P-element-
induced wimpy testis (PIWI)-interacting RNAs (piRNAs), microRNAs
(miRNAs) and short interfering RNAs (siRNAs). They play a vital role in
epigenetic regulation. Various drugs of abuse regulate the expression of
non-coding RNAs. Non-coding RNAs are crucial regulators of chromatin
regulating proteins and transcription factors which in turn affect
neuronal adaptations and subsequent memory formation [115].
lncRNAs are more than 200 nucleotide long RNA which are spliced,
capped at 5′ and polyadenylated. With over 30,000 lncRNAs known in
A.S. Koijam et al.
humans and mice, lncRNAs such as MIAT, MEG3, Airn, CCAT1-L,
CCAT1-S, H19, HOTAIR etc. have been associated with epigenetic
regulation of various genes and cellular processes [116]. Heroin abusers
display upregulated MIAT, MEG3, NEAT1 and NEAT2 in NAc. NEAT1,
NEAT2 and MIAT are known to link with nuclear domains and thus
likely affect gene expression changes [117]. MEG3 is a cAMP-sensitive
lncRNA which is maternally imprinted and expressed in specific neu­
rons. Few splice variants of MEG3 act as transcriptional co-activators
[118]. GWAS study have also associated MEG3 with risk for heroin
addiction [119]. mRNA levels of many synaptic genes and synaptic
density is likely regulated by NEAT2 [118].
piRNAs, miRNAs and siRNAs are associated with chromatin-
mediated gene silencing, rearrangement of DNA and mRNA transcript
regulation. piRNAs are 26–31 nucleotide long, small non-coding RNAs
that interact with PIWI proteins. piRNAs are involved in regulation of
chromatin, altering specific sites on a gene and silencing transposon.
They interact in clusters at euchromatin-heterochromatin junctions and
contain copies of ancient fragmented transposon. The piRNA in­
teractions and its pathway have also been associated with transgene
silencing, transcriptional silencing, formation of heterochromatin,
modification of histones and HP1α [120]. piRNAs inhibit transposon in
mice through DNA methylation of CpG islands in transposable elements.
The repressive H3K9me3 is maintained at high levels in long inter­
spersed retrotransposable elements (LINE) region of germ cells through
piRNA pathway [121]. PIWIL1 activation in piRNA-PIWIL1 pathway
can cause specific localized change in hypermethylation while losing
hypomethylation globally [122]. Endo-siRNAs such as RDR2-dependent
siRNAs can silence retroelements, transposons and DNA methylation
[123]. piRNA is known to regulate target miRNA genes positively and
target mRNA genes negatively [124].
miRNA is a group of 21–23 nucleotide long small noncoding RNA
molecules capable of repressing translation and gene silencing through
specific complementary binding of the 3′ untranslated region (UTR) of
mRNAs. MicroRNA mediated translational regulation induces synaptic
remodeling that is likely involved in transition from recreational drug
use to compulsive use. Various miRNAs target functional protein com­
plexes and epigenetic enzymes. For instance, miR-200a targets 3′ UTR of
HDAC4 mRNA repressing HDAC4 expression and upregulating acety­
lation levels of H3 histones. MiR-27a-5p directly targets Tet3 gene
involved in paternal methylation reprogramming [125]. MiR-29b is
known to target DNMT3A and DNMT3B directly and DNMT1 indirectly
to eventually induce global DNA hypomethylation. miRNAs are also
under epigenetic control as they are transcribed by specifically assigned
mRNA genes and undergo post-transcriptional processing [126].
Cocaine upregulates MIAT, MEG3, NEAT2 and EMX2O in NAc along
with miR-324, miR-369, miR-212, miR-181a, Ago2, miR-7, miR-8, miR-
142 and let-7 families, while downregulating miR-124, let-7d, miR-
200c, miR125a-5p, miR-200b, miR770–5p and miR200a. miR-324 and
miR-369 associated with cocaine administration, modulates MEF2 and
FosB transcription factors involved in memory development and reward
[115]. Long term cocaine administration induces upregulation of
miR-212 in the dorsal striatum resulting in reduced motivation. miR-212
intensifies Raf-1 action sensitizing adenylyl cyclase and overexpression
of Transducer of Regulated CREB (TORC), eventually increasing CREB
signaling [127]. MiR-212 interacts with MeCP2 in regulating cocaine
administration through striatal brain-derived neurotrophic factor
(BDNF) [128]. Ago2 is involved in generating miRNA and miRNA
mediated gene silencing. Drd2-expressing neurons devoid of Ago2 im­
pairs motivation for cocaine self administration in mice [129]. Chronic
cocaine inhibits let-7d and miR-124 while inducing miR-181a. let-7d
and miR-124 markedly downregulates D3R and BDNF respectively,
indicating a complex network with cocaine-sensitive plasticity genes
[130]. miR181a negatively regulates GluA2 post transcription, along
with involvement in cocaine-induced synaptic plasticity [131]. Cocaine
exposure induces differential expression of miRNA clusters that encode
miR8 family members (miR-429/200a/b and miR-141/200c), let-7
9
Biomedicine & Pharmacotherapy 170 (2024) 115951
family members cluster (let-7 f/ miR-98) and clusters miR-1/133a,
miR-182/96/183, and miR-216a/217, indicating a coordinate regula­
tion controlling similar mRNA targets [132]. miR-125b and miR-124
post-transcriptionally regulate PARP1 during acute and chronic
cocaine exposure [88,89]. Nicotine is known to promote the expression
of miR-140 and miR-504, while alcohol upregulates miR-9, miR-497 and
miR-302b. miR-23b and let-7 is upregulated while miR-190 and
miR-133b is downregulated by opioids. Neuronal differentiation is
regulated by morphine and one of the mechanisms is through down­
regulation of miR133b. Being a target of miR133B, the transcription
factor Pitx3, which activate dopamine transporter and tyrosine hy­
droxylase, is overexpressed [133].
4. Addiction-induced epigenetic alteration and epigenetic
alteration-induced addiction
Addiction or repeated exposure to various psychoactive drugs is
known to induce epigenetic effects. Numerous genes are influenced by
addictive drugs directly or indirectly at an epigenetic level, which in
turn induce expression changes that contribute to addiction phenotypes.
Parental health, stress, exposure to various chemicals, diet and envi­
ronments influence the epigenetic pattern of offspring development,
health and disease outcomes [134]. Cigarette and alcohol consumption
during pregnancy induce alteration in DNA methylation and microRNA
expression [135,136]. Significant increase in CYP1A1 expression of
placenta along with differential methylation of critical xenobiotic
response element has been observed following in-utero tobacco expo­
sure [137]. Germline and sperm epigenomes have been reported to
change due to paternal alcohol use and exposure to chemicals like
chromium chloride and vinclozolin [134]. Long term epigenetic modi­
fications contributing to addictive phenotype in later life can be induced
by prenatal exposure to cannabinoids [138]. Various studies involving
paternal cocaine administration have demonstrated enhancement in
drug susceptibility and related reward-based behaviours in their off­
springs through epigenetics processes [27–29]. Paternal voluntary
administration of cocaine has demonstrated to cause epigenetic germ­
line reprogramming with increased Brain-derived neurotrophic factor
(Bdnf) mRNA and BDNF protein in medial prefrontal cortex of only male
offsprings. This was also associated with H3 acetylation at BDNF pro­
moters [27]. Further studies with similar cocaine exposure also indi­
cated hippocampal epigenetic remodeling resulting in NMDA receptor
–dependent memory formation and impairments in synaptic plasticity of
male offsprings only [29]. Analysis involving targeted next-generation
bisulphite sequencing (TNGBS) of human astrocytes exposed to
cocaine demonstrated changes in levels of DNMT1, DNMT3a, DNMT3b,
TET1, TET2 and TET3 along with lower mtDNA methylation levels [36].
A study demonstrated the overexpression of HDAC3 in Drd1 but not
Drd2 cells of mice NAc, after chronic cocaine exposure. Mice with dis­
rupted HDAC3 enzyme activity within the NAc displayed changes in
gene expression and synaptic plasticity following cocaine administra­
tion. However, there was no effect on cocaine-induced behaviour [139].
Cocaine exposure induces overexpression of MeCP2, HDAC2 and
HDAC11 accounting to increased acetylation of histones in NAc [140]
while significantly downregulating GLP (Ehmt1) and G9a (Ehmt2) in
NAc [11]. Chronic cocaine exposure also caused hyperacetylation at
CamKIIa gene. Chronic alcohol and cocaine administration induces
regulatory changes of various genes like FosB, Cdk5 and Bdnf at chro­
matin level [119]. A study [70] observed hyperacetylation of H4 histone
at cFos promoter on acute cocaine administration but not chronic
administration while histone H3 at bdnf and cdk5 promoters displayed
hyperacetylation on chronic cocaine administration but not acute
administration. DNMTs significantly regulate synaptic plasticity induc­
tion in hippocampus while their inhibition changed methylation states
in promoter region of BDNF and reelin genes which have been associated
with synaptic plasticity induction [141]. Cocaine administration in­
duces DNMT3A overexpression causing enhanced dendritic spine
A.S. Koijam et al.
density in NAc [11]. Offsprings of mothers exposed to cocaine exhibited
higher DNA methylation and overexpression of DNMTs [119]. Acute
exposure of ethanol in mice caused lower expression of GAD1, HDAC2
and HDAC11 leading to hypoacetylation of histone at GAD1 and HDAC2
promoters. Further MT1, MT2, EGR1 expression increased and associa­
tion was observed with increased H3K4me3 levels at MT2 promoter and
decreased H3K27me3 levels MT1 promoter [142]. Long-term ethanol
consumption induces significant DNA hypomethylation due to
decreased methionine and SAM production caused by excess ROS for­
mation following ethanol metabolism. Ethanol also causes site-specific
methylation, phosphorylation and acetylation of histones [143]. His­
tone H3 and H4 acetylation increased in amygdala following acute
alcohol exposure whereas withdrawal decreased acetylation [144].
Chronic alcohol use induces DNA demethylation of NMDA NR2B gene
with elevated NR2B expression in the cortex of mice. DNA hyper­
methylation of PDYN have been observed in cultured neurons and cortex
of alcoholics. Their lymphocytes exhibited increased DNA methylation
in α-synuclein (SNCA), homocysteine-induced endoplasmic reticulum
protein (HERP), monoamine oxidase A (MAOA) and vasopressin (AVP)
genes while ANP gene was hypomethylated. Withdrawal of alcohol
displayed DNA hypermethylation of NGF gene promoter in blood.
Hypomethylation of imprinted IG-DMR region of DLK1 gene has been
observed in sperms obtained from alcoholics [119]. Alcohol probably
induces epigenetic effects in human, as significant global hyper­
methylation was observed among alcoholics than in normal controls
[145]. PPMG1 gene hypermethylation was found to be associated with
alcohol use disorder [146]. Significant association was also reported for
MAOA gene DNA methylation and nicotine and alcohol dependence in
women [147]. Cigarette smoking downregulates DNMT3B in lung can­
cer cells and causes demethylation of metastatic genes [148]. Meth­
amphetamine use induces histone H3 methylation (H3K4me3) on C-C
chemokine receptor 2 (ccr2) gene promoter and knocking out this gene in
mice results in impaired sensitization to methamphetamine [149].
Chronic methamphetamine has been reported to induce expression of
SIRT1, SIRT2, HDAC1, HDAC2, repressor element 1 silencing tran­
scription factor (REST) and REST corepressor proteins in dorsal striatum
of rats [150]. Hypoacetylation of H4K5, H4K12 and H4K16 at promoter
or enhancer regions of GluA1 and GluA2 genes has been observed on
chronic methamphetamine exposure [150]. Prenatal drug exposure
changes synaptic plasticity, neural development and phenotypes along
with transgenerational inheritance pointing towards the role of epige­
netic marks in addiction [151]. Rat sperms of F1 offsprings exposed to
alcohol at fetal stages exhibited hypermethylation at Pomc gene and
male germline transmitted the same upto F3 generation [152]. Epige­
netic changes in the germline of male rats voluntarily administering
cocaine induce histone H3 acetylation (H3K9K14ac2) at Bdnf promoter
of medial PFC brain region and their male offsprings exhibited reduced
cocaine intake [153]. Chronic opioid exposure induces hyper­
methylation in OPRM1 gene promoter with variations among different
ethnic groups [119]. Many non-coding RNAs have been associated with
addiction and related phenotypes as discussed above. Enhanced resis­
tance to conventional treatment modalities and vulnerability to relapse
have been observed in people with drug use disorder, with epigenetic
modifications mediated by addictive drugs [138]. High relapse rate
among addicts also necessitates establishing its connection with long
lasting and stable epigenetic changes. The undeniable evidences pre­
sented by the above studies linking addiction and epigenetic changes
simply direct the exploration of epidrugs for possible addiction
treatment.
5. Prospects of epidrugs in addiction treatment
Current addiction treatment varies from medications, treating co-
morbid conditions, counselling, behavioural therapies, exercise, yoga,
food supplements, vocational training to socialization depending on the
drug/ addictive substance to be recovered from. Current medications to
10
Biomedicine & Pharmacotherapy 170 (2024) 115951
treat addiction and withdrawal of various psychoactive drugs vary
depending on the type of drug. Common treatment medications include
buprenorphine, methadone, extended release naltrexone and lofexidine
for opioid addiction treatment. Bupropion, varenicline and nicotine
replacement therapies are used for nicotine addiction treatment. For
alcohol addiction treatment disulfiram, naltrexone and acamprosate are
used [154]. Medications generally mimic compounds that provide
intermediary relief or prolong reward sensation or treat withdrawal
symptoms, whose dosage can be gradually reduced to finally abstaining
it. Limited success of current treatment strategies with 40–60% relapse
rate [155] calls for newer effective alternatives and adjuncts like epi­
drugs. Epigenetic drugs or epidrugs are molecular complexes which
harness the potential of targeting epigenetic effector domains and the
possibility to change epigenetic marks within the human genome.
Presently, epidrugs specifically aimed for addiction treatment are very
limited. RG108 is a DNA methylation inhibitor acting through direct
binding and inhibition of DNMT1 enzyme [67]. Rat models injected
with RG108 within NAc demonstrated significant attenuation of
cue-induced cocaine seeking after upto 1 month treatment. Similar trials
with S-adenosyl methionine (SAM) displayed opposite effects with long
term cue-induced cocaine seeking [156]. Addictive behaviours of
cocaine-dependent rodents have been reported to be inhibited by
administering L-methionine. Multiple piRNAs regulating Vav2 genes, in
PFC following cocaine treatment have been reversed by L-methionine
treatment [124]. Methionine administration has also been reported to
reverse demethylation of c-MYB binding site, inhibit cocaine condi­
tioned place preference (CPP) and induce Bdnf IV expression by cocaine
CPP [157]. Methionine treatment reverses the reduced CpG methylation
in the c-Fos gene promoter and c-Fos expression induced by cocaine
[158]. However, FDA-approved epidrugs are in use for various cancer
and neuropsychiatric disease treatment as presented in Table 1. Many
epidrugs with different targets and action mechanisms are under study
at various experimental stages and presented in Table 2. Epidrugs have
evolved upto three generations with better understanding [159]. Epi­
drugs may range from targeting or interacting individual gene to set of
genes involved in one or more pathways. Most of the FDA-approved
epidrugs have shown remarkable efficiency as adjuncts to existing
medications. FDA-approved atypical antipsychotics such as clozapine,
olanzapine and quetiapine have reports of inducing epigenetic effects.
Sulpiride and Clozapine activate DNA demethylation while the latter
also induces acetylation of H3K9 in PFC [160]. 5-azacitidine has been
reported to prevent excess intake of alcohol in mice through its action of
reducing DNA methylation by inhibiting DNMT. FDA-approved SAHA,
HDAC inhibitor, has also been observed to inhibit alcohol-seeking
motivation [161]. Preclinical studies with HDAC inhibitors viz Tri­
chostatin A and Phenylbutyrate, have reported reduction in motivation
and cocaine intake in self-administering rats [162]. Pretreatment with
Valproic acid, an HDACi, in mice failed to reduce ethanol-induced
conditioned place preference (CPP) but increased the level of BDNF in
ventral striatum, indicating its modulatory effect on neutrophic mech­
anisms and addiction-like phenotype [163]. Another study [164] on
twenty individuals with cocaine-use disorder found that valproic acid
treatment was unable to reduce spontaneous and cue-induced cocaine
craving. In mice, valproic acid has been reported to inhibit
methamphetamine-induced behavioural sensitization when it is acutely
or repeatedly co-administered with methamphetamine [165]. Valproic
acid co-treatment in methamphetamine administered rats prevented
methamphetamine-mediated hypoacetylation of H4K5, H4K12 and
H4K16 at GluA1, GluA2 and GluN1 genes through HDAC1 and HDAC2
inhibition [150]. Valproic acid has shown to block
methamphetamine-induced underexpression of the glutamate receptors
viz alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid re­
ceptors (AMPARs) and N-methyl-D-aspartate receptor (NMDAR) [150].
Another HDAC inhibitor, RGFP966 caused cocaine CPP and
drug-seeking behaviour to become extinct, particularly through HDAC3
inhibition. RGFP966 significantly enhanced acetylation at lysine 14 on
A.S. Koijam et al. Biomedicine & Pharmacotherapy 170 (2024) 115951

Table 1
FDA approved epidrugs and their characteristics [182–184].
Epidrug Type FDA Approved Disease Approved Characteristics
Epidrug

DNA Methyltransferases inhibitors Decitabine Myelodysplastic syndrome Anti-cancer, anti-metabolite drug, cytotoxic to cancer cells.
(DNMTi) 5-Azacytidine Myelodysplastic syndrome Cytidine analog, incorporation in nucleic acids causing faulty
methylation
Hydralazine Hypertension Antihypertensive, inhibits DNMT1 activity, adverse immune
reactions observed
Procainamide Ventricular arrhythmias Inhibits DNMT1 activity
Tazemetostat Epitheloid sarcoma, refractory or Inhibits zeste homolog 2
relapsed follicular lymphoma
Histone Demethylase inhibitors (HDMi) Tranylcypromine Major depression disorder Irreversibly inhibits monoamine oxidase, antidepressant.
Deferiprone Treatment of iron Iron chelator
overload in thalassemia
Deferasirox Treatment of iron Iron chelator
overload
Histone methyltransferase inhibitors Phenelzine Depression Irreversibly inhibits monoamine oxidase.
(HMTi)
Histone Deacetylase inhibitor (HDACi) Vorinostat/SAHA Cutaneous T-cell lymphoma Inhibits all class I and II HDAC proteins
Panobinostat Multiple myeloma Inhibits all HDACs
Nicotinamide Pellagra, As food additives. Inhibits Sir2 protein non-competitively, inhibits deacetylation
action of sirtuins through reaction with an intermediate
Belinostat Peripheral T-cell lymphoma Inhibits HDACs and cell proliferation
Romidepsin Cutaneous T-cell lymphoma Strong inhibitor of HDAC1 and HDAC2, less potent inhibitor of
HDAC4 and HDAC6
Valproic acid Neurologic disorders, Antidepressive Anti-epileptic, anticancer, chemosensitizer, inhibit sodium
channels, increases GABA availability in synapse of brain
Magnesium Seizures Selective inhibition of HDAC2, under tests for treating
Valproate chemoresistance
Carbamazepine Epilepsy, trigeminal neuralgia, episodes Anti-epileptic, inhibits HDAC3 and HDAC7
of bipolar disorder
Bromodomain & Extra terminal motif Dinaciclib Different types of cancer Cyclin dependent kinase inhibitor
proteins inhibitors (BETi)
Poly(ADP-ribose) Olaparib Breast cancer, Ovarian cancer, Prostrate Inhibits PARP-1, PARP-2, PARP-3
polymerase cancer
(PARP1) inhibitor (PARPi) Niraparib Prostrate cancer Inhibits PARP-1, PARP-2
Rucaparib Metastatic castrate-resistant prostate Inhibits PARP-1, PARP-2, PARP-3
cancer
Talazoparib Metastatic castrate-resistant prostate Inhibits PARP-1, PARP-2
cancer
Non-coding RNAs Patisiran Familial amyloid polyneuropathy Direct sequence-specific degradation of Transthyretin (TTR) mRNA
in the liver
Givosiran Hepatic porphyria siRNA directed against 5-aminolevulinic acid synthase 1
Pegatanib Macular degeneration Binds and inhibits extracellular Vascular Endothelial Growth Factor
(VEGF165)
Protein arginine streptomycin and Rheumatoid arthritis inhibition of the binding of interleukin 1-beta to its cell surface
deiminase inhibitor methotrexate receptor
(PADi)

H3 histone (H3K14Ac) within NAc, hippocampus and infra-limbic cor­
tex, and H3K14Ac has been correlated with learning. H4K8Ac is known
to be regulated by HDAC3, and on RGFP966 treatment, acetylation of
H4 histone at lysine 8 (H4K8Ac) increased in infra-limbic cortex but not
in hippocampus and NAc [166]. BIX-01294 is an investigational epidrug
inhibiting histone H3K9-specific methyltransferases G9a and to a lesser
extent, Glp84. It induces autophagy, can attenuate neuronal gene
expression and direct infusion into ventral striatum causes increased
cocaine reward phenotype [167]. Epigenetic changes associated with
use of various investigational epidrugs and psychotropic drugs have
already been reported [168].
The realization of epidrugs as alternative or adjunct is not without
challenges. The numerous mechanisms of actions in epigenetics
involving an even more numerous epigenetic effectors add to the
complexity of addiction, but at the same time, also presents multiple
approaches through which therapeutic models can be addressed. In spite
of the knowledge of various compounds with epigenetic activity or
epidrugs, the transition from preclinical studies to therapeutics of
addiction is limited by issues of blood-brain barrier, tissue specific
epigenetic landscape and global or non-targeted epigenetic effects
among others. The limited understanding of epigenome and variations
in epigenetic landscapes affected by addiction across cells, tissues, or­
gans and individuals is also a big hurdle. While striking the key
epigenetic sites for a desired effect is imperative, it requires extensive
and advanced studies ranging from in-silico, in-vitro to in-vivo systems.
High throughput technologies for studying the complex milieu of epi­
marks at genome-wide level and advanced artificial intelligence tools for
decoding epigenome data are expected to provide deeper insights.
Various next generation sequencing platforms such as Whole genome
bisulfite sequencing (WGBS), reduced representation bisulfite
sequencing (RRBS), targeted bisulfite sequencing, Tet-assisted bisulfite
sequencing (TAB-Seq) and RNA sequencing (RNA-Seq), need to be
extensively exploited to produce more effective data relating to epige­
netics and gene expression in addiction. Data from such studies will
eventually pave the epigenetic approaches to addiction treatment. There
is also a need for cost effective tools and processes that can reliably and
effectively screen epidrugs. Site-specific and targeted delivery of epi­
drugs to exert their effects without any unwanted side-effects, non-tar­
geted or otherwise global epigenetic effects is another big challenge
towards epidrug realization. However, this has been largely overcome
with recent developments in epigenome engineering tools such as Zinc
finger nucleases (ZFNs), Transcription activator-like effector nucleases
(TALENs) and CRISPR/Cas 9, with characteristics (Table 3) that enable
targeting specific epigenetic mark to single gene [169]. Targeted DNA
cleavage techniques such as CRISPR-Cas9 system, transcription
activator-like effector nucleases (TALENs) and zinc-finger nucleases

11
A.S. Koijam et al. Biomedicine & Pharmacotherapy 170 (2024) 115951

Table 2 Table 3
Epidrugs in experimental stages for various disease conditions [182,183]. A list of characteristics of the epigenetic engineering tools CRISPR-Cas9, TALENs
Epidrug Type Experimental Epidrugs
and ZFNs.
Characteristics CRISPR-Cas9 TALENs ZFNs
DNA Methyltransferases inhibitors Epigallocatechin-3-gallate, Zebularine,
(DNMTi) Equol, Genistein, SGI-110, Procaine, Target site 22 bp+ PAM 30–40 bp 9–18 bp
Nanaomycin A, Disulfiram, Lomeguatrib, RG- length sequence
108, SGI-1027, MG98, CP-4200, Hinokitiol, Target range Any genomic site Upto 18742 Limited by ZFP
DC_517, DC-05 preceding PAM human genes combinations
Histone Methyltransferases (HMTi) UNC0321, UNC0224, EPZ-6438, DZNep, sequence based on TALEN
GSK343, Chaetocin, BIX-01338, BIX-01294, plasmid library
UNCO638, EPZ005687, GSK126, EPZ-5676, (Ho et al.,2018)
EPZ004777, SGC0946, E72, A-366, Off target Low High Moderate
UNC1999, CPI360, UNC0965, BIX-01337, Dimerization Not required Required Required
EI1, GSK503, BCI-121, LLY-507, EPZ015666, Recognition/ DNA-RNA DNA-Protein DNA-Protein
AZ505 ditrifluoroacetate, GSK3326595, Interaction/
MS023, UNC0642, JNJ-64619178, CM-579, type
EED226, MI-503, EPZ015866, MI-463, MI- Endonuclease Cas9 FokI FokI
538, MS049, CPI-169, BRD9539, LLY-283, Design and Relatively Easy Labour intensive Labour intensive
EML741, OTS186935, SGC3027, CPI-1205, assembly
Domatinostat (4SC-202), Tazemetostat. Specificity Moderate, High, few Moderate, more
Histone Demethylase inhibitors Pargyline, Clorgyline, Bizine, GSK2879552, comparatively more mismatches mismatches
(HDMi) KDMC-C70, JIB-04, ORY-1001, SID mismatches tolerated tolerated
85736331, Namoline, CBB1007, Methylstat, tolerated
GSKJ4, GSKJ1, QC6352, SP2509, KDOAM- Degenerate No Yes Yes
25, T-448, Daminozide, CPI-455, recognition
NCGC00244536, NCGC00247743, GSK-J2, Precision of Moderate High Moderate
Corin, GSK690, PBIT, S 2101, T-3775440 genome
hydrochloride editing
Proteins binding to methylated UNC669, UNC1215 Additional Yes Yes Yes
histones (PMHi) complex
Histone Acetyltransferase inhibitor Gallic acid, Garcinol, Anacardic acid, linkage
(HATi) Procyanidin, MB-3, CTK7A, Plumbagin, DNA No Yes Not known
Embelin methylation
Histone Deacetylase inhibitor Givinostat, AR-42, Entinostat, Apicidin, sensitivity
(HDACi) Pracinostat, Abexinostat, Resminostat, Design and Easy Labour intensive Complex and
CUDC-101, Toxoflavin, Inauhzin, Cambinol, assembly labour intensive
Salermide, Trichostatin A, CG-1521, OSU-
HDAC-42, HC-toxin, Plitidepsin,
Tasquinimod, Sodium butyrate, fusing FokI cleavage domain to DNA binding domain of zinc finger
Mocetinostat, Tefinostat, CHR-3996,
protein (ZFP), which can to recognise and cleave specific DNA se­
Curcumin, QUISINOSTAT, Sodium
phenylbutyrate, Pivanex, quences. Conjugated Cys2His2 motifs make up zinc fingers. Site speci­
Butyroyloxymethyl-diethyl phosphate, ficity is achieved through the 3–4 base pair recognition by DNA binding
Resveratrol, Dacinostat, Psammaplin A, ITF- domains in ZFPs which may be arranged in series for longer specific
A, ITF-B, OSU-HDAC-44, Ricolinostat,
locus, and this is the challenging aspect. ZFN dimers are required for
Tubastatin A, RGFP966, TMP195,
Fimepinostat, LMK-235, ACY-738, PCI-
double-strand DNA breaks. Their design involves a flanking pair of
34051, Nexturastat, CAY10603, ACY-775, unique sequence recognising ZFNs within which target spacer sequence
WT-161, MC1568, RGFP109, Citarinostat, of about 5–7 base pairs. ZFN cleavage induces DNA repair process by
Scriptaid, Tucidinostat (Chidamide), either non-homologous end-joining or homology-directed repair, during
Santacruzamate A, EDO-S101, Oxamflatin,
which, any desired target modification is effectively introduced [170].
HPOB, BML-210, Pomiferin, Domatinostat,
BG45, Bufexamac, Sinapinic acid, FT895, Zinc finger proteins can be clustered to recognize unique DNA sequences
CHDI-390576. of both strands in a target sequence. They can be fused with various
Proteins binding to acetylated CPI203, RVX-208, I-BET-726 epieffector molecules which can be activators or inhibitors of tran­
histones (PAHi) scription so as to form Zinc finger complexes.
Phosphorylation (Histone Kinase Ruxolitinib, KU-55933, VE-821
inhibitors)
Poly(ADP-ribose) Polymerase AMF-26, Talazoparib, Ilimaquinone,
inhibitors (PARPi) Veliparib, Olaparib, Niraparib, Rucaparib 5.2. Transcription activator-like effector nucleases (TALENs) / TALE-
Ubiquitin Signaling inhibitor PTC209, GW7647, PRT4165, ML323 complexes
Bromodomain & Extra terminal OTX15, JQ1, Thienodiazepine JQ1, I-BET762
motif proteins inhibitors (BETi) (GSK525762), I-BET151 (GSK1210151A), TALENs are gene editing protein complexes much like ZFNs. Typi­
GS-5829, CPI-0610, TEN-010, OTX-015,
BMS-986158, INCB057643 and ZEN003694
cally, a TALEN consists of a 12–28 repeat DNA binding domain, Fok1
Protein Arginine Deiminases (PADi) YW3–56, YW4–03, YW4–15, D-o-F-amidine, nuclease, a nuclear localization signal and a target gene transcription-
Cl-amidine, GSK484 activation domain. The DNA binding domains are proteins with a rela­
tively conserved 33–35 amino acid length, with polymorphisms at 12
and 13 positions called repeat variable diresidues (RVDs). Each RVD
(ZFNs) (represented in Fig. 4) have been exploited as targeted DNA
recognises a single base pair and determine nucleotide specificity. Thus
recognition or anchoring platforms, fused with epigenetic writer or
a chain of TALEs with aligned RVDs can recognise and effectively act on
eraser proteins (epieffectors) and delivered by transfection or trans­
a target sequence with precision. Fok1 dimerizes to generate double
duction methods.
strand breaks. TALEs can also be associated with various other proteins
or enzymes or epieffectors for better efficiency and precision. TALE-
5.1. Zinc finger nucleases (ZFNs) DNMT3ACD-3 L, TALE-CIB1; TALE/dCas9- KRAB, DNMT3ACD,
DNMT3L etc. are some remarkable targeted methylation tools with
Zinc finger nucleases (ZFNs) are engineered endonucleases made by varying efficacies. Similarly, TALE-TET1CD, TALE-TET1, etc. are

12
A.S. Koijam et al. Biomedicine & Pharmacotherapy 170 (2024) 115951

Fig. 4. Epigenetic editing tools. (A) A Zinc finger nuclease (ZFN) dimer bound to DNA. Endonuclease FokI is fused to a cluster of three Zinc finger proteins wherein
Fok1 dimerizes at target site (B) A Zinc finger complex. Zinc finger proteins recognising specific target nucleotide sequences is fused to one or more epieffector
molecules instead of Fok1. (C) Transcription activator-like effector nucleases (TALENs). Right and left strings of TALEs recognize specific DNA sequence targeting the
site for Fok1 cleavage. (D) Transcription activator-like effector complex. Right and left strings of TALEs fused with specific epieffectors recognize specific DNA
sequence for desired epigenetic alterations. (E) Clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). crRNA of guide RNA binds to target
DNA region. RuvB and HNH domains of Cas9 cleave DNA adjacent to PAM. (F) Clustered regularly interspaced short palindromic repeats/dCas9. Cleavage inac­
tivated dCas9 is fused to epieffectors depending on desired epigenetic alteration. crRNA provides the target DNA specificity.

demethylation tools employed for various studies [171]. TALE com­
plexes however pose delivery issues using viral systems due to its size
[172] and other alternatives need to be exploited.
5.3. Clustered regularly interspaced short palindromic repeats/ Cas9
(CRISPR/Cas 9)
CRISPR/Cas 9 is a highly efficient genome and epigenome editing
system comprised of guide RNA (gRNA) or single guide RNA (sg RNA)
and Cas 9 nuclease. The gRNA consists of two components viz. (i)
CRISPR RNA (crRNA) - of about 20 bp for binding to the target DNA
sequence and Protospacer Adjacent Motif (PAM) sequence required for
binding with Cas9, and (ii) Non coding Trans Activating crRNA
(tracrRNA) required for Cas9 cleavage. PAM is a 2–6 bp DNA sequence
adjacent to DNA cleavage target and required for cleavage by Cas
nuclease. Target DNA cleavage is processed by Cas9 through its domains
viz. RuvC and HNH causing double strand break (DSB). DSBs are
repaired by Homology directed repair (HDR) or Non-homologous end
joining (NHEJ). A slight variation of CRISPR/Cas9 system is the dCas9
complex system where catalytically dead Cas9 (dCas9) is fused with
various EpiEffector enzymes, and is reported to be highly efficient in
eukaryotic epigenome editing [173]. A dCas9 conserves DNA-binding
property while losing its cleavage activity. dCas9 epigenome editing
tools have evolved with three generations viz. (i) dCas9 fused to a
transcriptional activator or repressor (CRISPRa/i), e.g. dCas9-TET,
dCas9-DNMT (ii) dCas9 fused to transcriptional activators and multi­
ple epieffectors such as VPR, VP64, MS2, SAM, SunTag, scRNA etc. e.g.
dCas9-SUNTag-GCN4-VP64-scFv, and, (iii) the three system dCas9 tool,
consisting of coupling or dimerization system, receptor-coupled system
and split dCas9 system. In coupling or dimerization system, a light factor
(PhyB, PIF- activated by light) or chemical factor (ABI, PYL1- activated
by abscisic acid; GAI, GID- activated by gibberellins) fuse separately to
dCas9 and epieffector, and activate the later when coupled with their
drive. In Receptor-coupled system, epieffectors are activated by the
coupling of ligand and receptor. In Split dCas9 system, epieffectors are
activated by the connection of two cleft dCas9 parts in the target
sequence [173]. Overall, CRISPR-based epigenome editing tool requires
a target DNA binding protein, a gRNA sequence and an effector protein.
Site-specific DNA methylation manipulation in-vivo has been achieved
using deactivated Cas9 fused to enzymes which methylate or demeth­
ylate DNA. A few targeted methylation dCas9 complexes include dCas9-
DNMT3ACD, dCas9-DNMT3A, dCas9- DNMT3ACD3 L, dCas9-Sun
Tag-DNMT3A, dCas9-Sun TagDNMT3ACD, dCas9- MQ1, dCas9-split
M.Sss etc. while targeted demethylation dCas9 complexes include
dCas9- TET1CD, dCas9- TET1CD, MS2-TET1CD, dCasTET1CD,
dCas9-Sun Tag-TET1CD, Gal4- ROS1 CD [171]. For instance, Shayevitch
et al., [174] studied by co-transfecting cells with dCas9-DNMT3A-3 L
and single guide RNA (sgRNA) expression plasmids or dCas9-TET1 and
sgRNA expression plasmids. In this, sgRNA guides dCas9 fusion protein
to targeted site of genome allowing DNMT3A-3 L or TET1 for
site-specific methylation or demethylation respectively. The promising
aspect of CRISPR based genome and epigenome editing has the need to
overcome the basic hurdles of crossing the blood brain barrier,
non-specific cell targeting and in-vivo instability. Study [169] providing

13
A.S. Koijam et al.
remarkable evidence towards the role of epigenetic remodeling of gene
and subsequent control of stress response and reward, was demonstrated
through targeting epigenetic marks at histone H3 viz H3 lysine 9/14
acetylation (H3K9/14ac) and H3 lysine 9 dimethylation (H3K9me2) of
Cdk5 locus in NAc, in- vivo, employing engineered zinc finger proteins
and viral-mediated delivery. Resilience to social stress and
cocaine-induced locomotor behaviour increased with Cdk5-targeted
H3K9/14ac while Cdk5-targeted H3K9me2 impaired both conditioned
place preference and locomotor behaviour, but stress-induced social
avoidance behaviour was unaffected [169]. Targeted epigenetic editing
of H3K36me3 at Srsf11 in mice using dCas9 complex has been suc­
cessfully achieved in establishing its alternative splicing and cocaine
rewarding behaviour [175]. Experimental evidence of suppression of
cocaine-induced behaviour by Nr4a1 through epigenetic regulation was
demonstrated using dCas9 system fused to VP64 transcriptional acti­
vation domain (dCas9-VP64), driven by a neuron specific promoter and
a single-guide RNA (sgRNA) targeting the Nr4a1 promoter [176]. A
modified CRISPR/dCas9 approach was also used by Teague and col­
leagues to demonstrate the counteractive reinforcement effects of
cocaine by CREB-Zfp189 interaction within the NAc Drd2 + neurons
[177]. The repertoire of prospective CRISPR/Cas target sequences has
recently been greatly expanded by the advanced engineered PAMs. With
web-based resources predicting the possible off-targets and efficacy of
gRNA, the crucial step of target sequence and corresponding gRNA se­
lection has become easier [178]. Interesting advancements in
CRISPR-Cas9 delivery at targeted cells including brain, are gradually in
the horizon. Zou et al., [179] has reported unique nanocapsule-based
CRISPR-Cas9 delivery system that is non-invasive, facilitates BBB
penetration and safe for glioblastoma gene therapy.
Epigenetic therapy has also been approached from various aspects
including agents with multiple high affinity epigenetic targets wherein
one is an epigenetic enzyme and others being unrelated cellular targets
[180]. This approach reduces adverse drug reactions, potential drug
resistance and simplifies treatment [181]. In the light of such de­
velopments, advancement in targeted epigenetic remodelling or DNA
cleavage techniques with fused or engineered epigenetic enzymes,
small-molecule epigenetic modulators, nanoparticle-based, vir­
al-mediated, peptide-based, and receptor-based drug delivery systems
with ability to cross blood-brain barrier is expected for addiction
treatment. Limitations in understanding addiction-associated epigenetic
changes include differential epigenetic alterations by different drugs,
non-uniformity in study designs, obtaining participants with similar age,
sex and shared environmental factors, high quality post-mortem brain
tissues, uncertain epigenetic modifications, lack of clarity in time
duration that epigenetic changes can persist, and limited knowledge on
how epigenetic mechanisms and landscape works. Deeper studies
addressing these limitations and determination of whether the variety of
addiction-induced or epidrug-induced modifications and epimarks are
intergenerational or transgenerational are required. It would be
worthwhile to study individual and co-ordinated effects of various
environmental factors, genetic predisposition, protein or enzymatic in­
teractions, microRNAs, gene expressions, genetic penetrance of risk
gene/ alleles/ variants, vulnerable epigenetic marks and epigenetic
penetrance of the epimarks for achieving effective addiction treatment
strategies.
6. Concluding remarks
The complex nature of drug addiction with multiple factors and their
varied combinatorial effects has been an unresolved loop. Proper iden­
tification of epigenetic drug targets, effective epigenetic alterations,
correction or prevention of epigenetic marks, precision delivery and
minimal interaction with unintended regions are some key character­
istics that are expected from epidrugs. At present, epidrugs for addiction
treatment is at its infancy that needs to be proven against various con­
cerns and future studies should be directed towards the collective
14
Biomedicine & Pharmacotherapy 170 (2024) 115951
understanding of the different factors of addiction. With the continuous
advancement in epigenetic understanding, epidrug developments, pre­
cision epigenome editing complexes, and safe and efficient delivery
systems, the control of epigenetic switch of addiction is in the next leap.
The prevention and reversal of addiction phenotypes with appropriate
epidrugs and combinatorial treatment is expected soon.
Author statement
A.S.K., R.H. and Y.R. conceptualized the study; A.S.K. performed
literature searches and wrote the first draft, A.S.K., K.D.S., B.S.N., R.H.
and Y.R reviewed and edited the manuscript in its final form. All authors
contributed to and have approved the final manuscript.
CRediT authorship contribution statement
Koijam Arunkumar Singh: Conceptualization, Data curation,
Formal analysis, Investigation, Validation, Writing – original draft.
Nameirakpam Bunindro Singh: Formal analysis, Software, Validation,
Visualization. Singh Kabrambam Dasanta: Data curation, Formal
analysis, Resources, Software, Writing – original draft. Haobam Reena:
Funding acquisition, Supervision, Writing – review & editing. Raja­
shekar Yallappa: Conceptualization, Funding acquisition, Project
administration, Supervision, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data Availability
Data will be made available on request.
Acknowledgements
Financial support from the DBT-RA Program in Biotechnology and
Life Sciences to A.S.K. is gratefully acknowledged. We thank the Di­
rector, Institute of Bioresources and Sustainable Development, Imphal,
for supporting this study. The IBSD manuscript number is IBSD/MS/
2020/01/089.
References
[1] G.F. Koob, N.D. Volkow, Neurocircuitry of addiction, Neuropsychopharmacology
35 (2010) 217–238, https://doi.org/10.1038/npp.2009.110.
[2] L. Strathearn, C.E. Mertens, L. Mayes, H. Rutherford, P. Rajhans, G. Xu, M.
N. Potenza, S. Kim, Pathways relating the neurobiology of attachment to drug
addiction, Front. Psychiatry 10 (2019), 737, https://doi.org/10.3389/
fpsyt.2019.00737.
[3] N. Hiroi, S. Agatsuma, Genetic susceptibility to substance dependence, Mol.
Psychiatry 10 (2005) 336–344, https://doi.org/10.1038/sj.mp.4001622.
[4] S.H. Rhee, J.K. Hewitt, S.E. Young, R.P. Corley, T.J. Crowley, M.C. Stallings,
Genetic and environmental influences on substance initiation, use and problem
use in adolescents, Arch. Gen. Psychiatry 60 (2003) 1256–1264, https://doi.org/
10.1001/archpsyc.60.12.1256.
[5] K.S. Kendler, E. Schmitt, S.H. Aggen, C.A. Prescott, Genetic and environmental
influences on alcohol, caffeine, cannabis, and nicotine use from early adolescence
to middle adulthood, Arch. Gen. Psychiatry 65 (2008) 674–682, https://doi.org/
10.1001/archpsyc.65.6.674.
[6] M. Whitesell, A. Bachand, J. Peel, M. Brown, Familial, social and individual
factors contributing to risk for adolescent substance use, J. Addict. (2013),
579310, https://doi.org/10.1155/2013/579310.
[7] J.C. Anthony, L.A. Warner, R.C. Kessler, Comparative epidemiology of
dependence on tobacco, alcohol, controlled substances and inhalants: Basic
findings from the National Comorbidity Survey, Exp. Clin. Psychopharmacol. 2
(1994) 244–268, https://doi.org/10.1037/1064-1297.2.3.244.
[8] E.E. Zumbrun, J.M. Sido, P.S. Nagarkatti, M. Nagarkatti, Epigenetic regulation of
immunological alterations following prenatal exposure to marijuana
cannabinoids and its long term consequences in offspring, J. Neuroimmune
Pharmacol. 10 (2015) 245–254, https://doi.org/10.1007/s11481-015-9586-0.
A.S. Koijam et al.
[9] M.L. Gerra, M.C. Gerra, L. Tadonio, P. Pellegrini, C. Marchesi, E. Mattfeld,
G. Gerra, P. Ossola, Early parent-child interactions and substance use disorder: An
attachment perspective on a biopsychosocial entanglement, Neurosci. Biobehav.
Rev. 131 (2021) 560–580, https://doi.org/10.1016/j.neubiorev.2021.09.052.
[10] R.G. Lewis, E. Florio, D. Punzo, E. Borrelli, The brain’s reward system in health
and disease, Adv. Exp. Med. Biol. 1344 (2021) 57–69, https://doi.org/10.1007/
978-3-030-81147-1_4.
[11] I. Maze, E.J. Nestler, The epigenetic landscape of addiction, Ann. N. Y. Acad. Sci.
1216 (2011) 99–113, https://doi.org/10.1111/j.1749-6632.2010.05893.x.
[12] S. Gupta, P. Kulhara, Cellular and molecular mechanisms of drug dependence: An
overview and update, Indian J. Psychiatry 49 (2007) 85–90, https://doi.org/
10.4103/0019-5545.33253.
[13] N.D. Volkow, M. Michaelides, R. Baler, The neuroscience of drug reward and
addiction, Physiol. Rev. 99 (2019) 2115–2140, https://doi.org/10.1152/
physrev.00014.2018.
[14] K.M. Lattal, M.A. Wood, Epigenetics and persistent memory: implications for
reconsolidation and silent extinction beyond the zero, Nat. Neurosci. 16 (2013)
124–129, https://doi.org/10.1038/nn.3302.
[15] C.C. Wong, J. Mill, C. Fernandes, Drugs and addiction: an introduction to
epigenetics, Addiction 106 (2011) 480–489, https://doi.org/10.1111/j.1360-
0443.2010.03321.x.
[16] R.M. Bastle, J.L. Neisewander, Epigenetics and drug abuse (editors), in: W.
M. Meil, C.L. Ruby (Eds.), Recent advances in drug addiction research and clinical
applications, IntechOpen, 2016, pp. 21–45, https://doi.org/10.5772/63952
(editors).
[17] H.W. Shen, S. Toda, K. Moussawi, A. Bouknight, D.S. Zahm, P.W. Kalivas, Altered
dendritic spine plasticity in cocaine-withdrawn rats, J. Neurosci. 29 (2009)
2876–2884, https://doi.org/10.1523/JNEUROSCI.5638-08.2009.
[18] T.E. Robinson, B. Kolb, Structural plasticity associated with exposure to drugs of
abuse, Neuropharmacology 47 (Suppl 1) (2004) 33–46, https://doi.org/10.1016/
j.neuropharm.2004.06.025.
[19] J.L. Cadet, C. Brannock, S. Jayanthi, I.N. Krasnova, Transcriptional and
epigenetic substrates of methamphetamine addiction and withdrawal: evidence
from a long-access self-administration model in the rat, Mol. Neurobiol. 51 (2015)
696–717, https://doi.org/10.1007/s12035-014-8776-8.
[20] Y.H. Huang, O.M. Schluter, Y. Dong, Silent synapses speak up, updates of the
neural rejuvenation hypothesis of drug addiction, Neuroscientist 21 (2015)
451–459, https://doi.org/10.1177/1073858415579405.
[21] H. Marie, W. Morishita, X. Yu, N. Calakos, R.C. Malenka, Generation of silent
synapses by acute in vivo expression of CaMKIV and CREB, Neuron 45 (2005)
741–752, https://doi.org/10.1016/j.neuron.2005.01.039.
[22] C. Engelmann, R. Haenold, Transcriptional control of synaptic plasticity by
transcription factor NF-kappaB, Neural Plast. 2016 (2016), 7027949, https://doi.
org/10.1155/2016/7027949.
[23] A. Eagle, B. Al Masraf, A.J. Robison, Transcriptional and epigenetic regulation of
reward circuitry in drug addiction, in: T. Mary (Ed.), Neural mechanisms of
addiction, Cambridge: Academic Press, 2019, pp. 23–34, https://doi.org/
10.1016/B978-0-12-812202-0.00003-8.
[24] A. Bošković, O.J. Rando, Transgenerational Epigenetic Inheritance, Ann. Rev.
Genet. 52 (2018) 21–41, https://doi.org/10.1146/annurev-genet-120417-
031404.
[25] P.R. Nielsen, D. Nietlispach, H.R. Mott, J. Callaghan, A. Bannister, T. Kouzarides,
A.G. Murzin, N.V. Murzina, E.D. Laue, Structure of the HP1 chromodomain bound
to histone H3 methylated at lysine 9, Nature 416 (2002) 103–107, https://doi.
org/10.1038/nature722.
[26] E. Li, Chromatin modification and epigenetic reprogramming in mammalian
development, Nat. Rev. Genet. 3 (2002) 662–673, https://doi.org/10.1038/
nrg887.
[27] F.M. Vassoler, S.L. White, H.D. Schmidt, G. Sadri-Vakili, R.C. Pierce, Epigenetic
inheritance of a cocaine-resistance phenotype, Nat. Neurosci. 16 (2013) 42–47,
https://doi.org/10.1038/nn.3280.
[28] S.L. White, F.M. Vassoler, H.D. Schmidt, R.C. Pierce, M.E. Wimmer, Enhanced
anxiety in the male offspring of sires that self-administered cocaine, Addict. Biol.
21 (2016) 802–810, https://doi.org/10.1111/adb.12258.
[29] M.E. Wimmer, L.A. Briand, B. Fant, L.A. Guercio, A.C. Arreola, H.D. Schmidt,
S. Sidoli, Y. Han, B.A. Garcia, R.C. Pierce, Paternal cocaine taking elicits
epigenetic remodeling and memory deficits in male progeny, Mol. Psychiatry 22
(2017) 1641–1650, https://doi.org/10.1038/mp.2017.8.
[30] V. López, A.F. Fernández, M.F. Fraga, The role of 5-hydroxymethylcytosine in
development, aging and age-related diseases, Ageing Res. Rev. 37 (2017) 28–38,
https://doi.org/10.1016/j.arr.2017.05.002.
[31] A. Majchrzak-Celińska, A. Warych, M. Szoszkiewicz, Novel approaches to
epigenetic therapies: From drug combinations to epigenetic editing, Genes 12
(2021) 208, https://doi.org/10.3390/genes12020208.
[32] R.A. Irizarry, C. Ladd-Acosta, B. Wen, Z. Wu, C. Montano, P. Onyango, H. Cui,
K. Gabo, M. Rongione, M. Webster, H. Ji, J. Potash, S. Sabunciyan, A.P. Feinberg,
The human colon cancer methylome shows similar hypo- and hypermethylation
at conserved tissue-specific CpG island shores, Nat. Genet. 41 (2009) 178–186,
https://doi.org/10.1038/ng.298.
[33] L. Moore, T. Le, G. Fan, DNA Methylation and its basic function,
Neuropsychopharmacology 38 (2013) 23–38, https://doi.org/10.1038/
npp.2012.112.
[34] Y. Yin, E. Morgunova, A. Jolma, E. Kaasinen, B. Sahu, S. Khund-Sayeed, P.K. Das,
T. Kivioja, K. Dave, F. Zhong, K.R. Nitta, M. Taipale, A. Popov, P.A. Ginno,
S. Domcke, J. Yan, D. Schübeler, C. Vinson, J. Taipale, Impact of cytosine
15
Biomedicine & Pharmacotherapy 170 (2024) 115951
methylation on DNA binding specificities of human transcription factors, Sci. (N.
Y., N. Y. ) 356 (2017), eaaj2239, https://doi.org/10.1126/science.aaj2239.
[35] A. de Mendoza, D. Poppe, S. Buckberry, J. Pflueger, C.B. Albertin, T. Daish,
S. Bertrand, E. de la Calle-Mustienes, J.L. Gómez-Skarmeta, J.R. Nery, J.R. Ecker,
B. Baer, C.W. Ragsdale, F. Grützner, H. Escriva, B. Venkatesh, O. Bogdanovic,
R. Lister, The emergence of the brain non-CpG methylation system in vertebrates,
Nat. Ecol. Evol. 5 (2021) 369–378, https://doi.org/10.1038/s41559-020-01371-
2.
[36] M. Doke, V. Jeganathan, J.P. McLaughlin, T. Samikkannu, HIV-1 Tat and cocaine
impact mitochondrial epigenetics: effects on DNA methylation, Epigenetics 16
(2021) 980–999, https://doi.org/10.1080/15592294.2020.1834919.
[37] N.T. Tavares, S. Gumauskaitė, J. Lobo, C. Jerónimo, R. Henrique, DNA
methylation biomarkers for prediction of response to platinum-based
chemotherapy: where do we stand? Cancers 14 (2022) 2918, https://doi.org/
10.3390/cancers14122918.
[38] M. Ehrlich, M.A. Gama-Sosa, L.H. Huang, R.M. Midgett, K.C. Kuo, R.A. McCune,
C. Gehrke, Amount and distribution of 5-methylcytosine in human DNA from
different types of tissues of cells, Nucleic Acids Res 10 (1982) 2709–2721,
https://doi.org/10.1093/nar/10.8.2709.
[39] R. Lister, E.A. Mukamel, J.R. Nery, M. Urich, C.A. Puddifoot, N.D. Johnson,
J. Lucero, Y. Huang, A.J. Dwork, M.D. Schultz, M. Yu, J. Tonti-Filippini, H. Heyn,
S. Hu, J.C. Wu, A. Rao, M. Esteller, C. He, F.G. Haghighi, T.J. Sejnowski, M.
M. Behrens, J.R. Ecker, Global epigenomic reconfiguration during mammalian
brain development, Science 341 (2013), 1237905, https://doi.org/10.1126/
science.1237905.
[40] L.F. Rizzardi, P.F. Hickey, V. Rodriguez DiBlasi, R. Tryggvadóttir, C.M. Callahan,
A. Idrizi, K.D. Hansen, A.P. Feinberg, Neuronal brain-region-specific DNA
methylation and chromatin accessibility are associated with neuropsychiatric
trait heritability, Nat. Neurosci. 22 (2019) 307–316, https://doi.org/10.1038/
s41593-018-0297-8.
[41] E. Lax, G. Warhaftig, D. Ohana, R. Maayan, Y. Delayahu, P. Roska, A.
M. Ponizovsky, A. Weizman, G. Yadid, M. Szyf, A DNA methylation signature of
addiction in t cells and its reversal with DHEA intervention, Front. Mol. Neurosci.
11 (2018), 322, https://doi.org/10.3389/fnmol.2018.00322.
[42] E. Lax, M. Szyf, The role of dna methylation in drug addiction: implications for
diagnostic and therapeutics, Prog. Mol. Biol. Transl. Sci. 157 (2018) 93–104,
https://doi.org/10.1016/bs.pmbts.2018.01.003.
[43] C. Camilo, M. Maschietto, H.C. Vieira, A.C. Tahira, G.R. Gouveia, A.C. Feio Dos
Santos, A.B. Negrão, M. Ribeiro, R. Laranjeira, H. Vallada, H. Brentani, Genome-
wide DNA methylation profile in the peripheral blood of cocaine and crack
dependents, Rev. Bras. De. Psiquiatr. 41 (2019) 485–493, https://doi.org/
10.1590/1516-4446-2018-0092.
[44] J. Liao, R. Karnik, H. Gu, M.J. Ziller, K. Clement, A.M. Tsankov, V. Akopian, C.
A. Gifford, J. Donaghey, C. Galonska, R. Pop, D. Reyon, S.Q. Tsai, W. Mallard, J.
K. Joung, J.L. Rinn, A. Gnirke, A. Meissner, Targeted disruption of DNMT1,
DNMT3A and DNMT3B in human embryonic stem cells, Nat. Genet. 47 (2015)
469–478, https://doi.org/10.1038/ng.3258.
[45] M. Gagliardi, M. Strazzullo, M.R. Matarazzo, DNMT3B Functions: Novel insights
from human disease, Front. Cell. Dev. Biol. 6 (2018), 140, https://doi.org/
10.3389/fcell.2018.00140.
[46] Q. Du, P.L. Luu, C. Stirzaker, S.J. Clark, Methyl-CpG-binding domain proteins:
readers of the epigenome, Epigenomics 7 (2015) 1051–1073, https://doi.org/
10.2217/epi.15.39.
[47] S. Pol Bodetto, D. Carouge, M. Fonteneau, J.B. Dietrich, J. Zwiller, P. Anglard,
Cocaine represses protein phosphatase-1Cβ through DNA methylation and
Methyl-CpG Binding Protein-2 recruitment in adult rat brain,
Neuropharmacology 73 (2013) 31–40, https://doi.org/10.1016/j.
neuropharm.2013.05.005.
[48] N. Cannella, A.M.M. Oliveira, T. Hemstedt, T. Lissek, E. Buechler, H. Bading,
R. Spanagel, Dnmt3a2 in the nucleus accumbens shell is required for
reinstatement of cocaine seeking, J. Neurosci. 38 (2018) 7516–7528, https://doi.
org/10.1523/JNEUROSCI.0600-18.2018.
[49] H. Wu, Y. Zhang, Reversing DNA methylation: Mechanisms genomics and
biological functions, Cell 156 (2014) 45–68, https://doi.org/10.1016/j.
cell.2013.12.019.
[50] K.D. Rasmussen, K. Helin, Role of TET enzymes in DNA methylation,
development, and cancer, Genes Dev. 30 (2016) 733–750, https://doi.org/
10.1101/gad.276568.115.
[51] F.Z. Jiang, W. Zheng, C. Wu, Y. Li, F. Shen, J. Liang, M. Li, J.J. Zhang, N. Sui,
Double dissociation of inhibitory effects between the hippocampal TET1 and
TET3 in the acquisition of morphine self-administration in rats, Addict. Biol. 26
(2021), e12875, https://doi.org/10.1111/adb.12875.
[52] J. Feng, N. Shao, K.E. Szulwach, V. Vialou, J. Huynh, C. Zhong, T. Le,
D. Ferguson, M.E. Cahill, Y. Li, J.W. Koo, E. Ribeiro, B. Labonte, B.M. Laitman,
D. Estey, V. Stockman, P. Kennedy, T. Couroussé, I. Mensah, G. Turecki, K.
F. Faull, G. Ming, H. Song, G. Fan, P. Casaccia, L. Shen, P. Jin, E.J. Nestler, Role of
Tet1 and 5-hydroxymethylcytosine in cocaine action, Nat. Neurosci. 18 (2015)
536–544, https://doi.org/10.1038/nn.3976.
[53] M.A. Surani, K. Hayashi, P. Hajkova, Genetic and epigenetic regulators of
pluripotency, Cell 128 (2007) 747–762, https://doi.org/10.1016/j.
cell.2007.02.010.
[54] S. Seisenberger, J.R. Peat, T.A. Hore, F. Santos, W. Dean, W. Reik,
Reprogramming DNA methylation in the mammalian life cycle: Building and
breaking epigenetic barriers, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 368 (2013)
20110330, https://doi.org/10.1098/rstb.2011.0330.
A.S. Koijam et al.
[55] Y.A. Medvedeva, A.M. Khamis, I.V. Kulakovskiy, W. Ba-Alawi, M.S.I. Bhuyan,
H. Kawaji, T. Lassmann, M. Harbers, A.R.R. Forrest, V.B. Bajic, Effects of cytosine
methylation on transcription factor binding sites, B. M. C. Genom. 15 (2014),
119, https://doi.org/10.1186/1471-2164-15-119.
[56] T. Bartke, M. Vermeulen, B. Xhemalce, S.C. Robson, M. Mann, T. Kouzarides,
Nucleosome interacting proteins regulated by DNA and histone methylation, Cell
143 (2010) 470–484, https://doi.org/10.1016/j.cell.2010.10.012.
[57] X. Shi, T. Hong, K.L. Walter, M. Ewalt, E. Michishita, T. Hung, D. Carney, P. Peña,
F. Lan, M.R. Kaadige, N. Lacoste, C. Cayrou, F. Davrazou, A. Saha, B.R. Cairns, D.
E. Ayer, T.G. Kutateladze, Y. Shi, J. Côté, K.F. Chua, O. Gozani, ING2 PHD domain
links histone H3 lysine 4 methylation to active gene repression, Nature 442
(2006) 96–99, https://doi.org/10.1038/nature04835.
[58] J. Cheng, R. Blum, C. Bowman, D. Hu, A. Shilatifard, S. Shen, B.D. Dynlacht,
A role for H3K4 monomethylation in gene repression and partitioning of
chromatin readers, Mol. Cell 53 (2014) 979–992, https://doi.org/10.1016/j.
molcel.2014.02.032.
[59] A. la Torre, F. Lo Vecchio, A. Greco, Epigenetic mechanisms of aging and aging-
associated diseases, Cells 12 (2023) 1163, https://doi.org/10.3390/
cells12081163.
[60] S.J.D. Tjalsma, M. Hori, Y. Sato, A. Bousard, A. Ohi, A.C. Raposo, J. Roensch,
A. Le Saux, J. Nogami, K. Maehara, T. Kujirai, T. Handa, S. Bagés-Arnal,
Y. Ohkawa, H. Kurumizaka, S.T. da Rocha, J.J. Żylicz, H. Kimura, E. Heard,
H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X
chromosome but dispensable for inducing gene silencing, EMBO Rep. 22 (2021),
e51989, https://doi.org/10.15252/embr.202051989.
[61] M. Shoaib, Q. Chen, X. Shi, N. Nair, C. Prasanna, R. Yang, D. Walter, K.
S. Frederiksen, H. Einarsson, J.P. Svensson, C.F. Liu, K. Ekwall, M. Lerdrup,
L. Nordenskiöld, C.S. Sørensen, Histone H4 lysine 20 mono-methylation directly
facilitates chromatin openness and promotes transcription of housekeeping genes,
Nat. Commun. 12 (2021), 4800, https://doi.org/10.1038/s41467-021-25051-2.
[62] E.L. Greer, Y. Shi, Histone methylation: a dynamic mark in health disease and
inheritance, Nat. Rev. Genet. 13 (2012) 343–357, https://doi.org/10.1038/
nrg3173.
[63] S. Gupta, S.Y. Kim, S. Artis, D.L. Molfese, A. Schumacher, J.D. Sweatt, R.E. Paylor,
F.D. Lubin, Histone methylation regulates memory formation, J. Neurosci. 30
(2010) 3589–3599, https://doi.org/10.1523/JNEUROSCI.3732-09.2010.
[64] D. Ikegami, M. Narita, S. Imai, K. Miyashita, R. Tamura, M. Narita, S. Takagi,
A. Yokomizo, H. Takeshima, T. Ando, K. Igarashi, J. Kanno, N. Kuzumaki,
T. Ushijima, T. Suzuki, Epigenetic modulation at the CCR2 gene correlates with
the maintenance of behavioral sensitization to methamphetamine, Addict. Biol.
15 (2010) 358–361, https://doi.org/10.1111/j.1369-1600.2010.00219.x.
[65] A. Aguilar-Valles, T. Vaissière, E.M. Griggs, M.A. Mikaelsson, I.F. Takács, E.
J. Young, G. Rumbaugh, C.A. Miller, Methamphetamine-associated memory is
regulated by a writer and an eraser of permissive histone methylation, Biol.
Psychiatry 76 (2014) 57–65, https://doi.org/10.1016/j.biopsych.2013.09.014.
[66] S. Heerboth, K. Lapinska, N. Snyder, M. Leary, S. Rollinson, S. Sarkar, Use of
epigenetic drugs in disease: an overview, Genet. Epigenet. 6 (2014) 9–19, https://
doi.org/10.4137/GEG.S12270.
[67] N. Chessum, K. Jones, E. Pasqua, M. Tucker, Recent advances in cancer
therapeutics, in: G. Lawton, D.R. Witty (Eds.), Progress in Medicinal Chemistry,
Vol. 54, Elsevier, 2015, pp. 1–63, https://doi.org/10.1016/bs.
pmch.2014.11.002-.
[68] M. Tan, H. Luo, S. Lee, F. Jin, J.S. Yang, E. Montellier, T. Buchou, Z. Cheng,
S. Rousseaux, N. Rajagopal, Z. Lu, Z. Ye, Q. Zhu, J. Wysocka, Y. Ye, S. Khochbin,
B. Ren, Y. Zhao, Identification of 67 histone marks and histone lysine
crotonylation as a new type of histone modification, Cell 146 (2011) 1016–1028,
https://doi.org/10.1016/j.cell.2011.08.008.
[69] G. Jiang, C. Li, M. Lu, K. Lu, H. Li, Protein lysine crotonylation: past, present,
perspective, Cell Death Dis. 12 (2021), 703, https://doi.org/10.1038/s41419-
021-03987-z.
[70] A. Kumar, K.H. Choi, W. Renthal, N.M. Tsankova, D.E. Theobald, H.T. Truong, S.
J. Russo, Q. LaPlant, T.S. Sasaki, K.N. Whistler, R.L. Neve, D.W. Self, E.J. Nestler,
Chromatin remodeling is a key mechanism underlying cocaine-induced plasticity
in striatum, Neuron 48 (2005) 303–314, https://doi.org/10.1016/j.
neuron.2005.09.023.
[71] L. Wang, Z. Lv, Z. Hu, J. Sheng, B. Hui, J. Sun, L. Ma, Chronic cocaine-induced H3
acetylation and transcriptional activation of CaMKIIalpha in the nucleus
accumbens is critical for motivation for drug reinforcement,
Neuropsychopharmacology 35 (2010) 913–928, https://doi.org/10.1038/
npp.2009.193.
[72] I. Maze, H.E. Covington, 3rd, D.M. Dietz, Q. LaPlant, W. Renthal, S.J. Russo,
M. Mechanic, E. Mouzon, R.L. Neve, S.J. Haggarty, Y. Ren, S.C. Sampath, Y.
L. Hurd, P. Greengard, A. Tarakhovsky, A. Schaefer, E.J. Nestler, Essential role of
the histone methyltransferase G9a in cocaine-induced plasticity, Science 327
(2010) 213–216, https://doi.org/10.1126/science.1179438.
[73] M. Malvaez, E. Mhillaj, D.P. Matheos, M. Palmery, M.A. Wood, CBP in the nucleus
accumbens regulates cocaine-induced histone acetylation and is critical for
cocaine-associated behaviors, J. Neurosci. 31 (2011) 16941–16948, https://doi.
org/10.1523/JNEUROSCI.2747-11.2011.
[74] W. Renthal, A. Kumar, G. Xiao, M. Wilkinson, H.E. Covington, 3rd, I. Maze,
D. Sikder, A.J. Robison, Q. LaPlant, D.M. Dietz, S.J. Russo, V. Vialou,
S. Chakravarty, T.J. Kodadek, A. Stack, M. Kabbaj, E.J. Nestler, Genome-wide
analysis of chromatin regulation by cocaine reveals a novel role for sirtuins,
Neuron 62 (2009) 335–348, https://doi.org/10.1016/j.neuron.2009.03.026.
[75] Z. Wang, Y. Zhao, N. Xu, S. Zhang, S. Wang, Y. Mao, Y. Zhu, B. Li, Y. Jiang,
Y. Tan, W. Xie, B.B. Yang, Y. Zhang, NEAT1 regulates neuroglial cell mediating A
16
Biomedicine & Pharmacotherapy 170 (2024) 115951
β clearance via the epigenetic regulation of endocytosis -related genes expression,
Cell Mol. Life Sci. (15) (2019) 3005–3018, https://doi.org/10.1007/s00018-019-
03074-9.
[76] T. Maurice, F. Duclot, J. Meunier, G. Naert, L. Givalois, J. Meffre, A. Célérier,
C. Jacquet, V. Copois, N. Mechti, K. Ozato, C. Gongora, Altered memory
capacities and response to stress in p300/CBP-associated factor (PCAF) histone
acetylase knockout mice, Neuropsychopharmacol 33 (2008) 1584–1602, https://
doi.org/10.1038/sj.npp.1301551.
[77] T.J. Gould, Addiction and cognition, Addict. Sci. Clin. Pract. 5 (2010) 4–14.
[78] J. Li, Y. Guo, F.A. Schroeder, R.M. Youngs, T.W. Schmidt, C. Ferris, C. Konradi,
S. Akbarian, Dopamine D2-like antagonists induce chromatin remodeling in
striatal neurons through cyclic AMP-protein kinase A and NMDA receptor
signaling, J. Neurochem 90 (2004) 1117–1131, https://doi.org/10.1111/j.1471-
4159.2004.02569.x.
[79] L. Ni, L. Wang, C. Yao, Z. Ni, F. Liu, C. Gong, X. Zhu, X. Yan, S.S. Watowich, D.
A. Lee, S. Zhu, The histone deacetylase inhibitor valproic acid inhibits NKG2D
expression in natural killer cells through suppression of STAT3 and HDAC3, Sci.
Rep. 7 (2017), 45266, https://doi.org/10.1038/srep45266.
[80] D. Ferguson, J.W. Koo, J. Feng, E. Heller, J. Rabkin, M. Heshmati, W. Renthal,
R. Neve, X. Liu, N. Shao, V. Sartorelli, L. Shen, E.J. Nestler, Essential role of SIRT1
signaling in the nucleus accumbens in cocaine and morphine action, J. Neurosci.
33 (2013) 16088–16098, https://doi.org/10.1523/JNEUROSCI.1284-13.2013.
[81] J.L. Cadet, R. Patel, S. Jayanthi, Compulsive methamphetamine taking and
abstinence in the presence of adverse consequences: Epigenetic and
transcriptional consequences in the rat brain, Pharmacol. Biochem. Behav. 179
(2019) 98–108, https://doi.org/10.1016/j.pbb.2019.02.009.
[82] J.L. Cadet, B. Ladenheim, I. Krasnova, S. Jayanthi, Differential expression of
mRNAs coding for histone deacetylases (HDACs) in the nucleus accumbens of
compulsive methamphetamine takers and abstinent rats, J. Drug Alcohol Res. 5
(2016) 1–9, https://doi.org/10.4303/jdar/235998.
[83] T.G. Bredfeldt, C.L. Walker, Epigenetics, in: C.A. McQueen (Ed.), Comprehensive
Toxicology (2nd edn), Elsevier, 2010, pp. 335–358, https://doi.org/10.1016/
B978-0-08-046884-6.00219-0.
[84] T. Banerjee, D. Chakravarti, A peek into the complex realm of histone
phosphorylation, Mol. Cell. Biol. 31 (2011) 4858–4873, https://doi.org/10.1128/
MCB.05631-11.
[85] D. Rossetto, N. Avvakumov, J. Côté, Histone phosphorylation: a chromatin
modification involved in diverse nuclear events, Epigenetics 7 (2012) 1098–1108,
https://doi.org/10.4161/epi.21975.
[86] Y. Wang, A. Singh, G. Li, S. Yue, K. Hertel, Z.J. Wang, Opioid induces increased
DNA damage in prefrontal cortex and nucleus accumbens, Pharmacol. Biochem.
Behav. 224 (2023), 173535, https://doi.org/10.1016/j.pbb.2023.173535.
[87] J.V. Deng, Y. Wan, X. Wang, S. Cohen, W.C. Wetsel, M.E. Greenberg, P.J. Kenny,
N. Calakos, A.E. West, MeCP2 phosphorylation limits psychostimulant-induced
behavioral and neuronal plasticity, J. Neurosci. 34 (2014) 4519–4527, https://
doi.org/10.1523/JNEUROSCI.2821-13.2014.
[88] B.A. Alhamwe, R. Khalaila, J. Wolf, V. von Bülow, H. Harb, F. Alhamdan, C.S. Hii,
S.L. Prescott, A. Ferrante, H. Renz, H. Garn, D.P. Potaczek, Histone modifications
and their role in epigenetics of atopy and allergic diseases, Allergy Asthma Clin.
Immunol. 14 (2018), 39, https://doi.org/10.1186/s13223-018-0259-4.
[89] J.J. Chen, D. Stermer, J.C. Tanny, Decoding histone ubiquitylation, Front. Cell
Dev. Biol. 10 (2022), 968398, https://doi.org/10.3389/fcell.2022.968398.
[90] A.E. Hare, J.D. Parvin, Processes that regulate the ubiquitination of chromatin
and chromatin-associated proteins, in: M. Summers (Ed.), Ubiquitin Proteasome
System - Current insights into mechanism cellular regulation and disease,
Intechopen, 2019, pp. 1–22, https://doi.org/10.5772/intechopen.82567.
[91] P. Krumova, J.H. Weishaupt, Sumoylation in neurodegenerative diseases, Cell.
Mol. Life Sci. 70 (2013) 2123–2138, https://doi.org/10.1007/s00018-012-1158-
3.
[92] N.G. Simonet, G. Rasti, A. Vaquero, The histone code and disease:
Posttranslational modification as potential prognostic factors for clinical
diagnosis, in: J.L. García-Giménez (Ed.), Epigenetic biomarkers and diagnostics,
Academic Press, Cambridge, 2016, pp. 417–445, https://doi.org/10.1016/B978-
0-12-801899-6.00021-8.
[93] M.D. Rubio, K. Wood, V. Haroutunian, J.H. Meador-Woodruff, Dysfunction of the
ubiquitin proteasome and ubiquitin-like systems in schizophrenia,
Neuropsychopharmacology 38 (2013) 1910–1920, https://doi.org/10.1038/
npp.2013.84.
[94] G. Manco, G. Lacerra, E. Porzio, G. Catara, ADP-Ribosylation post-translational
modification: An overview with a focus on rna biology and new pharmacological
perspectives, Biomolecules 12 (2022) 443, https://doi.org/10.3390/
biom12030443.
[95] K.N. Scobie, D. Damez-Werno, H. Sun, N. Shao, A. Gancarz, C.H. Panganiban,
C. Dias, J. Koo, P. Caiafa, L. Kaufman, R.L. Neve, D.M. Dietz, L. Shen, E.J. Nestler,
Essential role of polyADP-ribosylation in cocaine action, Proc. Natl. Acad. Sci. U.
S. A. 111 (2014) 2005–2010, https://doi.org/10.1073/pnas.1319703111.
[96] C.D. Striplin, P.W. Kalivas, Correlation between behavioral sensitization to
cocaine and G protein ADP-ribosylation in the ventral tegmental area, Brain Res
579 (1992) 181–186, https://doi.org/10.1016/0006-8993(92)90049-f.
[97] E. Lax, A. Friedman, R. Massart, R. Barnea, L. Abraham, D. Cheishvili, M. Zada,
H. Ahdoot, T. Bareli, G. Warhaftig, L. Visochek, M. Suderman, M. Cohen-Armon,
M. Szyf, G. Yadid, PARP-1 is required for retrieval of cocaine-associated memory
by binding to the promoter of a novel gene encoding a putative transposase
inhibitor, Mol. Psychiatry 22 (2017) 570–579, https://doi.org/10.1038/
mp.2016.119.
A.S. Koijam et al.
[98] S. Dash, M. Balasubramaniam, T. Rana, A. Godino, E.G. Peck, J.S. Goodwin,
F. Villalta, E.S. Calipari, E.J. Nestler, C. Dash, J. Pandhare, Poly (ADP-ribose)
polymerase-1 (PARP-1) induction by cocaine is post-transcriptionally regulated
by miR-125b, eNeuro 4 (2017), ENEURO.0089-17.2017, https://doi.org/
10.1523/ENEURO.0089-17.2017.
[99] S. Dash, M. Balasubramaniam, F.J. Martínez-Rivera, A. Godino, E.G. Peck,
S. Patnaik, M. Suar, E.S. Calipari, E.J. Nestler, F. Villalta, C. Dash, J. Pandhare,
Cocaine-regulated microRNA miR-124 controls poly (ADP-ribose) polymerase-1
expression in neuronal cells, Sci. Rep. 10 (2020), 11197, https://doi.org/
10.1038/s41598-020-68144-6.
[100] S. Dash, C. Dash, J. Pandhare, Activation of proline metabolism maintains ATP
levels during cocaine-induced polyADP-ribosylation, Amino Acids 53 (2021)
1903–1915, https://doi.org/10.1007/s00726-021-03065-w.
[101] Y. Ke, J. Zhang, X. Lv, X. Zeng, X. Ba, Novel insights into PARPs in gene
expression: regulation of RNA metabolism, Cell. Mol. Life Sci. 76 (2019)
3283–3299, https://doi.org/10.1007/s00018-019-03120-6.
[102] R. Gupte, Z. Liu, W.L. Kraus, PARPs and ADP-ribosylation: recent advances
linking molecular functions to biological outcomes, Genes Dev. 31 (2017)
101–126, https://doi.org/10.1101/gad.291518.116.
[103] M.P. Marjanović, K. Crawford, I. Ahel, PARP, transcription and chromatin
modeling, Semin. Cell Dev. Biol. 63 (2017) 102–113, https://doi.org/10.1016/j.
semcdb.2016.09.014.
[104] R. Krishnakumar, W.L. Kraus, PARP-1 regulates chromatin structure and
transcription through a KDM5B-dependent pathway, Mol. Cell. 39 (2010)
736–749, https://doi.org/10.1016/j.molcel.2010.08.014.
[105] L.A. Farrelly, R.E. Thompson, S. Zhao, A.E. Lepack, Y. Lyu, N.V. Bhanu, B. Zhang,
Y.E. Loh, A. Ramakrishnan, K.C. Vadodaria, K.J. Heard, G. Erikson, T. Nakadai, R.
M. Bastle, B.J. Lukasak, H. Zebroski 3rd, N. Alenina, M. Bader, O. Berton, R.
G. Roeder, H. Molina, F.H. Gage, L. Shen, B.A. Garcia, H. Li, T.W. Muir, I. Maze,
Histone serotonylation is a permissive modification that enhances TFIID binding
to H3K4me3, Nature 567 (2019) 535–539, https://doi.org/10.1038/s41586-019-
1024-7.
[106] J. Zhao, W. Chen, Y. Pan, Y. Zhang, H. Sun, H. Wang, F. Yang, Y. Liu, N. Shen,
X. Zhang, X. Mo, J. Zang, Structural insights into the recognition of histone H3Q5
serotonylation by WDR5, Sci. Adv. 7 (2021), eabf4291, https://doi.org/10.1126/
sciadv.abf4291.
[107] A.E. Lepack, C.T. Werner, A.F. Stewart, S.L. Fulton, P. Zhong, L.A. Farrelly, A.C.
W. Smith, A. Ramakrishnan, Y. Lyu, R.M. Bastle, J.A. Martin, S. Mitra, R.
M. O’Connor, Z.J. Wang, H. Molina, G. Turecki, L. Shen, Z. Yan, E.S. Calipari, D.
M. Dietz, P.J. Kenny, I. Maze, Dopaminylation of histone H3 in ventral tegmental
area regulates cocaine seeking, Science 368 (2020) 197–201, https://doi.org/
10.1126/science.aaw8806.
[108] K. Blum, M.S. Gold, J.L. Cadet, D. Baron, A. Bowirrat, P.K. Thanos, R. Brewer, R.
D. Badgaiyan, M.C. Gondré-Lewis, Dopaminylation in psychostimulant use
disorder protects against psychostimulant seeking behavior by normalizing
Nucleus Accumbens (NAc) Dopamine expression, Curr. Psychopharmacol. 11
(2022) 11–17, https://doi.org/10.2174/2211556009666210108112737.
[109] A. Ntorla, J.R. Burgoyne, The regulation and function of histone crotonylation,
Front. Cell Dev. Biol. 9 (2021), 624914, https://doi.org/10.3389/
fcell.2021.624914.
[110] E. Montellier, F. Boussouar, S. Rousseaux, K. Zhang, T. Buchou, F. Fenaille,
H. Shiota, A. Debernardi, P. Héry, S. Curtet, M. Jamshidikia, S. Barral, H. Holota,
A. Bergon, F. Lopez, P. Guardiola, K. Pernet, J. Imbert, C. Petosa, M. Tan, Y. Zhao,
M. Gérard, S. Khochbin, Chromatin-to-nucleoprotamine transition is controlled
by the histone H2B variant TH2B, Genes Dev. 27 (2013) 1680–1692, https://doi.
org/10.1101/gad.220095.113.
[111] K. Li, Z. Wang, Histone crotonylation-centric gene regulation, Epigenetics
Chromatin 14 (2021), 10, https://doi.org/10.1186/s13072-021-00385-9.
[112] X. Xiong, T. Panchenko, S. Yang, S. Zhao, P. Yan, W. Zhang, W. Xie, Y. Li, Y. Zhao,
C.D. Allis, H. Li, Selective recognition of histone crotonylation by double PHD
fingers of MOZ and DPF2, Nat. Chem. Biol. 12 (2016) 1111–1118, https://doi.
org/10.1038/nchembio.2218.
[113] Y. Zou, X.H. Bai, L.C. Kong, F.F. Xu, T.Y. Ding, P.F. Zhang, F.L. Dong, Y.J. Ling, B.
C. Jiang, Involvement of histone lysine crotonylation in the regulation of nerve-
injury-induced neuropathic pain, Front. Immunol. 13 (2022), 885685, https://
doi.org/10.3389/fimmu.2022.885685.
[114] C.M. Cahill, A.M. Taylor, Neuroinflammation-a co-occurring phenomenon linking
chronic pain and opioid dependence, Curr. Opin. Behav. Sci. 13 (2017) 171–177,
https://doi.org/10.1016/j.cobeha.2016.12.003.
[115] G.C. Sartor, G. St Laurent 3rd, C. Wahlestedt, The emerging role of non-coding
RNAs in drug addiction, Front. Genet. 3 (2012) 106, https://doi.org/10.3389/
fgene.2012.00106.
[116] C. Wang, L. Wang, Y. Ding, X. Lu, G. Zhang, J. Yang, H. Zheng, H. Wang, Y. Jiang,
L. Xu, LncRNA structural characteristics in epigenetic regulation, Int. J. Mol. Sci.
18 (2017) 2659, https://doi.org/10.3390/ijms18122659.
[117] D.N. Albertson, C.J. Schmidt, G. Kapatos, M.J. Bannon, Distinctive profiles of
gene expression in the human nucleus accumbens associated with cocaine and
heroin abuse, Neuropsychopharmacology 31 (2006) 2304–2312, https://doi.org/
10.1038/sj.npp.1301089.
[118] S.K. Michelhaugh, L. Lipovich, J. Blythe, H. Jia, G. Kapatos, M.J. Bannon, Mining
Affymetrix microarray data for long non-coding RNAs: altered expression in the
nucleus accumbens of heroin abusers, J. Neurochem. 116 (2011) 459–466,
https://doi.org/10.1111/j.1471-4159.2010.07126.x.
[119] D.A. Nielsen, A. Utrankar, J.A. Reyes, D.D. Simons, T.R. Kosten, Epigenetics of
drug abuse: predisposition or response, Pharmacogenomics 13 (2012)
1149–1160, https://doi.org/10.2217/pgs.12.94.
17
Biomedicine & Pharmacotherapy 170 (2024) 115951
[120] J.C. Peng, H. Lin, Beyond transposons: The epigenetic and somatic functions of
the Piwi-piRNA mechanism, Curr. Opin. Cell Biol. 25 (2013) 190–194, https://
doi.org/10.1016/j.ceb.2013.01.010.
[121] D. Pezic, S.A. Manakov, R. Sachidanandam, A.A. Aravin, piRNA pathway targets
active LINE1 elements to establish the repressive H3K9me3 mark in germ cells,
Genes Dev. 28 (2014) 1410–1428, https://doi.org/10.1101/gad.240895.114.
[122] M. Litwin, A. Szczepańska-Buda, A. Piotrowska, P. Dzięgiel, W. Witkiewicz, The
meaning of PIWI proteins in cancer development, Oncol. Lett. 13 (2017)
3354–3362, https://doi.org/10.3892/ol.2017.5932.
[123] I.R. Henderson, S.E. Jacobsen, Epigenetic inheritance in plants, Nature 447
(2007) 418–424, https://doi.org/10.1038/nature05917.
[124] K. Zhang, G. Ji, M. Zhao, Y. Wang, Candidate l-methionine target piRNA
regulatory networks analysis response to cocaine-conditioned place preference in
mice, Brain Behav. 11 (2021), e2272, https://doi.org/10.1002/brb3.2272.
[125] J.Y. Zhong, R.R. Cui, X. Lin, F. Xu, T. Zhu, F. Li, F. Wu, E. Zhou, L. Yi, L.Q. Yuan,
Aberrant DNA methylation of synaptophysin is involved in adrenal cortisol-
producing adenoma, Aging 11 (2019) 5232–5245, https://doi.org/10.18632/
aging.102119.
[126] E. Dai, X. Yu, Y. Zhang, F. Meng, S. Wang, X. Liu, D. Liu, J. Wang, X. Li, W. Jiang,
EpimiR: a database of curated mutual regulation between miRNAs and epigenetic
modifications, Database (2014) bau023, https://doi.org/10.1093/database/
bau023.
[127] J.A. Hollander, H.I. Im, A.L. Amelio, J. Kocerha, P. Bali, Q. Lu, D. Willoughby,
C. Wahlestedt, M.D. Conkright, P.J. Kenny, Striatal microRNA controls cocaine
intake through CREB signalling, Nature 466 (2010) 197–202, https://doi.org/
10.1038/nature09202.
[128] H.I. Im, J.A. Hollander, P. Bali, P.J. Kenny, MeCP2 controls BDNF expression and
cocaine intake through homeostatic interactions with microRNA-212, Nat.
Neurosci. 13 (2010) 1120–1127, https://doi.org/10.1038/nn.2615.
[129] A. Schaefer, H.I. Im, M.T. Venø, C.D. Fowler, A. Min, A. Intrator, J. Kjems, P.
J. Kenny, D. O’Carroll, P. Greengard, Argonaute 2 in dopamine 2 receptor-
expressing neurons regulates cocaine addiction, J. Exp. Med. 207 (2010)
1843–1851, https://doi.org/10.1084/jem.20100451.
[130] V. Chandrasekar, J.L. Dreyer, microRNAs miR-124, let-7d and miR-181a regulate
cocaine-induced plasticity, Mol. Cell Neurosci. 42 (2009) 350–362, https://doi.
org/10.1016/j.mcn.2009.08.009.
[131] R. Saba, P.H. Störchel, A. Aksoy-Aksel, F. Kepura, G. Lippi, T.D. Plant, G.
M. Schratt, Dopamine-regulated microRNA MiR-181a controls GluA2 surface
expression in hippocampal neurons, Mol. Cell Biol. 32 (2012) 619–632, https://
doi.org/10.1128/MCB.05896-11.
[132] J.E. Eipper-Mains, D.D. Kiraly, D. Palakodeti, R.E. Mains, B.A. Eipper, B.
R. Graveley, MicroRNA-Seq reveals cocaine-regulated expression of striatal
microRNAs, RNA 17 (2011) 1529–1543, https://doi.org/10.1261/rna.2775511.
[133] F.M. Sanchez-Simon, X.X. Zhang, H.H. Loh, P.Y. Law, R.E. Rodriguez, Morphine
regulates dopaminergic neuron differentiation via miR-133b, Mol. Pharmacol. 78
(2010) 935–942, https://doi.org/10.1124/mol.110.066837.
[134] R.R. Kanherkar, N. Bhatia-Dey, A.B. Csoka, Epigenetics across the human lifespan,
Front. Cell. Dev. Biol. 2 (2014) 49, https://doi.org/10.3389/fcell.2014.00049.
[135] V.S. Knopik, M.A. Maccani, S. Francazio, J.E. McGeary, The epigenetics of
maternal cigarette smoking during pregnancy and effects on child development,
Dev. Psychopathol. 24 (2012) 1377–1390, https://doi.org/10.1017/
S0954579412000776.
[136] C.R. Zhang, M.F. Ho, M.C.S. Vega, T.H. Burne, S. Chong, Prenatal ethanol
exposure alters adult hippocampal VGLUT2 expression with concomitant changes
in promoter DNA methylation H3K4 trimethylation and miR-467b-5p levels,
Epigenetics Chromatin 8 (2015), 40, https://doi.org/10.1186/s13072-015-0032-
6.
[137] M. Suter, A. Abramovici, L. Showalter, L, M. Hu, C.D. Shope, M. Varner,
K. Aagaard-Tillery, In utero tobacco exposure epigenetically modifies placental
CYP1A1 expression, Metabolism 59 (2010) 1481–1490, https://doi.org/10.1016/
j.metabol.2010.01.013.
[138] P. Biliński, A. Wojtyła, L. Kapka-Skrzypczak, R. Chwedorowicz, M. Cyranka,
T. Studziński, Epigenetic regulation in drug addiction, Ann. Agric. Environ. Med.
19 (2012) 491–496.
[139] R.R. Campbell, E.A. Kramár, L. Pham, J.H. Beardwood, A.S. Augustynski, A.
J. López, O.S. Chitnis, G. Delima, J. Banihani, D.P. Matheos, M.A. Wood, HDAC3
Activity within the nucleus accumbens regulates cocaine-induced plasticity and
behavior in a cell-type-specific manner, J. Neurosci. 41 (2021) 2814–2827,
https://doi.org/10.1523/JNEUROSCI.2829-20.2021.
[140] L. Host, J.B. Dietrich, D. Carouge, D. Aunis, J. Zwiller, Cocaine self-administration
alters the expression of chromatin-remodelling proteins; modulation by histone
deacetylase inhibition, J. Psychopharmacol. 25 (2011) 222–229, https://doi.org/
10.1177/0269881109348173.
[141] J.M. Levenson, T.L. Roth, F.D. Lubin, C.A. Miller, I.C. Huang, P. Desai, L.
M. Malone, J.D. Sweatt, Evidence that DNA (cytosine-5) methyltransferase
regulates synaptic plasticity in the hippocampus, J. Biol. Chem. 281 (2006)
15763–15773, https://doi.org/10.1074/jbc.M511767200.
[142] A. Finegersh, G.E. Homanics, Acute ethanol alters multiple histone modifications
at model gene promoters in the cerebral cortex, Alcohol. Clin. Exp. Res. 38 (2014)
1865–1873, https://doi.org/10.1111/acer.12465.
[143] S. Zakhari, Alcohol metabolism and epigenetics changes, Alcohol Res 35 (2013)
6–16.
[144] J.C. Peng, H. Lin, Beyond transposons: The epigenetic and somatic functions of
the Piwi-piRNA mechanism, Curr. Opin. Cell Biol. 25 (2013) 190–194, https://
doi.org/10.1016/j.ceb.2013.01.010.
A.S. Koijam et al.
[145] D. Bonsch, B. Lenz, U. Reulbach, J. Kornhuber, S. Bleich, Homocysteine
associated genomic DNA hypermethylation in patients with chronic alcoholism,
J. Neural Transm. 111 (2004) 1611–1616, https://doi.org/10.1007/s00702-004-
0232-x.
[146] B. Ruggeri, C. Nymberg, E. Vuoksimaa, A. Lourdusamy, C.P. Wong, F.
M. Carvalho, T. Jia, A. Cattrell, C. Macare, T. Banaschewski, G.J. Barker, A.
L. Bokde, U. Bromberg, C. Büchel, P.J. Conrod, M. Fauth-Bühler, H. Flor,
V. Frouin, J. Gallinat, H. Garavan, P. Gowland, A. Heinz, B. Ittermann, J.
L. Martinot, F. Nees, Z. Pausova, T. Paus, M. Rietschel, T. Robbins, M.N. Smolka,
R. Spanagel, G. Bakalkin, J. Mill, W.H. Sommer, R.J. Rose, J. Yan, F. Aliev,
D. Dick, J. Kaprio, S. Desrivières, G. Schumann, IMAGEN Consortium, Association
of protein phosphatase PPM1G with alcohol use disorder and brain activity
during behavioral control in a genome-wide methylation analysis, 543-452, Am.
J. Psychiatry 172 (2015) (2015), https://doi.org/10.1176/appi.
ajp.2014.14030382.
[147] R.A. Philibert, T.D. Gunter, S.R. Beach, G.H. Brody, A. Madan, MAOA methylation
is associated with nicotine and alcohol dependence in women, Am. J. Med. Genet.
B Neuropsychiatr. Genet. 147B (2008) 565–570, https://doi.org/10.1002/ajmg.
b.30778.
[148] H. Liu, Y. Zhou, S.E. Boggs, S.A. Belinsky, J. Liu, Cigarette smoke induces
demethylation of prometastatic oncogene synuclein-gamma in lung cancer cells
by downregulation of DNMT3B, Oncogene 26 (2007) 5900–5910, https://doi.
org/10.1038/sj.onc.1210400.
[149] A. Godino, S. Jayanthi, J.L. Cadet, Epigenetic landscape of amphetamine and
methamphetamine addiction in rodents, Epigenetics 10 (2015) 574–580, https://
doi.org/10.1080/15592294.2015.1055441.
[150] S. Jayanthi, M.T. McCoy, B. Chen, J.P. Britt, S. Kourrich, H.J. Yau, B. Ladenheim,
I.N. Krasnova, A. Bonci, J.L. Cadet, Methamphetamine downregulates striatal
glutamate receptors via diverse epigenetic mechanisms, Biol. Psychiatry 76
(2014) 47–56, https://doi.org/10.1016/j.biopsych.2013.09.034.
[151] D. De Sa Nogueira, K. Merienne, K. Befort, Neuroepigenetics and addictive
behaviors: Where do we stand? Neurosci. Biobehav. Rev. 106 (2019) 58–72,
https://doi.org/10.1016/j.neubiorev.2018.08.018.
[152] D. Govorko, R.A. Bekdash, C. Zhang, D.K. Sarkar, Male germline transmits fetal
alcohol adverse effect on hypothalamic proopiomelanocortin gene across
generations, Biol. Psychiatry 72 (2012) 378–388, https://doi.org/10.1016/j.
biopsych.2012.04.006.
[153] F.M. Vassoler, D.J. Oliver, C. Wyse, A. Blau, M. Shtutman, J.R. Turner, E.
M. Byrnes, Transgenerational attenuation of opioid self-administration as a
consequence of adolescent morphine exposure, Neuropharmacology 113 (2017)
271–280, https://doi.org/10.1016/j.neuropharm.2016.10.006.
[154] NIDA, Treatment and Recovery. https://nida.nih.gov/publications/drugs-brains-
behavior-science-addiction/treatment-recovery, 2023, (accessed on November 12
2023).
[155] A.T. McLellan, D.C. Lewis, C.P. O’Brien, H.D. Kleber, Drug dependence, a chronic
medical illness: implications for treatment, insurance, and outcomes evaluation,
JAMA 284 (2000) 1689–1695, https://doi.org/10.1001/jama.284.13.1689.
[156] R. Massart, R. Barnea, Y. Dikshtein, M. Suderman, O. Meir, M. Hallett,
P. Kennedy, E.J. Nestler, M. Szyf, G. Yadid, Role of DNA methylation in the
nucleus accumbens in incubation of cocaine craving, J. Neurosci. 35 (2015)
8042–8058, https://doi.org/10.1523/JNEUROSCI.3053-14.2015.
[157] W. Tian, J. Wang, K. Zhang, H. Teng, C. Li, M. Szyf, Z.S. Sun, M. Zhao,
Demethylation of c-MYB binding site mediates upregulation of Bdnf IV in cocaine-
conditioned place preference, Sci. Rep. 6 (2016), 22087, https://doi.org/
10.1038/srep22087.
[158] K.N. Wright, F. Hollis, F. Duclot, A.M. Dossat, C.E. Strong, T.C. Francis, R. Mercer,
J. Feng, D.M. Dietz, M.K. Lobo, E.J. Nestler, M. Kabbaj, Methyl supplementation
attenuates cocaine-seeking behaviors and cocaine-induced c-Fos activation in a
DNA methylation-dependent manner, J. Neurosci. 35 (2015) 8948–8958, https://
doi.org/10.1523/JNEUROSCI.5227-14.2015.
[159] M. Montalvo-Casimiro, R. González-Barrios, M.A. Meraz-Rodriguez, V.T. Juárez-
González, C. Arriaga-Canon, L.A. Herrera, Epidrug repurposing: Discovering new
faces of old acquaintances in cancer therapy, Front. Oncol. 10 (2020), 605386,
https://doi.org/10.3389/fonc.2020.605386.
[160] E. Dong, M. Nelson, D.R. Grayson, E. Costa, A. Guidotti, Clozapine and sulpiride
but not haloperidol or olanzapine activate brain DNA demethylation, Proc. Natl.
Acad. Sci. U. S. A. 105 (2008) 13614–13619, https://doi.org/10.1073/
pnas.0805493105.
[161] V. Warnault, E. Darcq, A. Levine, S. Barak, D. Ron, Chromatin remodeling-a novel
strategy to control excessive alcohol drinking, Transl. Psychiatry 3 (2013), e231,
https://doi.org/10.1038/tp.2013.4.
[162] P. Romieu, L. Host, S. Gobaille, G. Sandner, D. Aunis, J. Zwiller, Histone
deacetylase inhibitors decrease cocaine but not sucrose self-administration in rats,
J. Neurosci. 28 (2008) 9342–9348, https://doi.org/10.1523/JNEUROSCI.0379-
08.2008.
[163] M.A. Dos Santos, S.S. Júnior, Escudeiro, G.S. Vasconcelos, N.C.B. Matos, M.R.
M. de Souza, M.C.A. Patrocínio, L.P. Dantas, D. Macêdo, S.M.M. Vasconcelos, The
interplay between ventro striatal BDNF levels and the effects of valproic acid on
the acquisition of ethanol-induced conditioned place preference in mice,
Neurosci. Lett. 660 (2017) 86–89, https://doi.org/10.1016/j.neulet.2017.09.009.
18
Biomedicine & Pharmacotherapy 170 (2024) 115951
[164] M.S. Reid, V. Thakkar, Valproate treatment and cocaine cue reactivity in cocaine
dependent individuals, Drug Alcohol Depend. 102 (2009) 144–150, https://doi.
org/10.1016/j.drugalcdep.2009.02.010.
[165] R. Coccurello, A. Caprioli, O. Ghirardi, A. Virmani, Valproate and acetyl-L-
carnitine prevent methamphetamine-induced behavioral sensitization in mice,
Ann. N. Y. Acad. Sci. (2007) 260–275, https://doi.org/10.1196/annals.1403.019.
[166] M. Malvaez, S.C. McQuown, G.A. Rogge, M. Astarabadi, V. Jacques, S. Carreiro, J.
R. Rusche, M.A. Wood, HDAC3-selective inhibitor enhances extinction of cocaine-
seeking behavior in a persistent manner, Proc. Natl. Acad. Sci. U. S. A. 110 (2013)
2647–2652, https://doi.org/10.1073/pnas.1213364110.
[167] C.J. Peter, S. Akbarian, Balancing histone methylation activities in psychiatric
disorders, Trends Mol. Med. 17 (2011) 372–379, https://doi.org/10.1016/j.
molmed.2011.02.003.
[168] D. Kringel, S. Malkusch, J. Lötsch, Drugs and epigenetic molecular functions. A
pharmacological data scientometric analysis, Int. J. Mol. Sci. 22 (2021) 7250,
https://doi.org/10.3390/ijms22147250.
[169] E.A. Heller, P.J. Hamilton, D.D. Burek, S.I. Lombroso, C.J. Pena, R.L. Neve, E.
J. Nestler, Targeted epigenetic remodeling of the Cdk5 gene in nucleus
accumbens regulates cocaine- and stress-evoked behaviour, J. Neurosci. 36
(2016) 4690–4697, https://doi.org/10.1523/JNEUROSCI.0013-16.2016.
[170] T. Gaj, C.A. Gersbach, C.F. Barbas 3rd, ZFN, TALEN, and CRISPR/Cas-based
methods for genome engineering, Trends Biotechnol. 31 (2013) 397–405, https://
doi.org/10.1016/j.tibtech.2013.04.004.
[171] Y. Lei, Y.H. Huang, M.A. Goodell, DNA methylation and de-methylation using
hybrid site-targeting proteins, Genome Biol. 19 (2018), 187, https://doi.org/
10.1186/s13059-018-1566-2.
[172] M. Holkers, I. Maggio, J. Liu, J.M. Janssen, F. Miselli, C. Mussolino, A. Recchia,
T. Cathomen, M.A. Gonçalves, Differential integrity of TALE nuclease genes
following adenoviral and lentiviral vector gene transfer into human cells, Nucleic
Acids Res 41 (2013), e63, https://doi.org/10.1093/nar/gks1446.
[173] F. Maroufi, A. Maali, M. Abdollahpour-Alitappeh, M.H. Ahmadi, M. Azad,
CRISPR-mediated modification of DNA methylation pattern in the new era of
cancer therapy, Epigenomics 12 (2020) 1845–1859, https://doi.org/10.2217/epi-
2020-0110.
[174] R. Shayevitch, D. Askayo, I. Keydar, G. Ast, The importance of DNA methylation
of exons on alternative splicing, RNA 24 (2018) 1351–1362, https://doi.org/
10.1261/rna.064865.117.
[175] S.J. Xu, S.I. Lombroso, D.K. Fischer, M.D. Carpenter, D.M. Marchione, P.
J. Hamilton, C.J. Lim, R.L. Neve, B.A. Garcia, M.E. Wimmer, R.C. Pierce, E.
A. Heller, Chromatin-mediated alternative splicing regulates cocaine-reward
behaviour, Neuron 109 (2021) 2943–2966.e8, https://doi.org/10.1016/j.
neuron.2021.08.008.
[176] M.D. Carpenter, Q. Hu, A.M. Bond, S.I. Lombroso, K.S. Czarnecki, C.J. Lim,
H. Song, M.E. Wimmer, R.C. Pierce, E.A. Heller, Nr4a1 suppresses cocaine-
induced behavior via epigenetic regulation of homeostatic target genes, Nat.
Commun. 11 (2020), 504, https://doi.org/10.1038/s41467-020-14331-y.
[177] C.D. Teague, J.A. Picone, W.J. Wright, C.J. Browne, G.M. Silva, R. Futamura,
A. Minier-Toribio, M.E. Estill, A. Ramakrishnan, F.J. Martinez-Rivera, A. Godino,
E.M. Parise, K.H. Schmidt, N.V. Pulido, Z.S. Lorsch, J.H. Kim, L. Shen, R.L. Neve,
Y. Dong, E.J. Nestler, P.J. Hamilton, CREB binding at the Zfp189 promoter within
medium spiny neuron subtypes differentially regulates behavioral and
physiological adaptations over the course of cocaine use, Biol. Psychiatry 93
(2023) 502–511, https://doi.org/10.1016/j.biopsych.2022.07.022.
[178] J.G. Doench, N. Fusi, M. Sullender, M. Hegde, E.W. Vaimberg, K.F. Donovan,
I. Smith, Z. Tothova, C. Wilen, R. Orchard, H.W. Virgin, J. Listgarten, D.E. Root,
Optimized sgRNA design to maximize activity and minimize off-target effects of
CRISPR-Cas9, Nat. Biotechnol. 34 (2016) 184–191, https://doi.org/10.1038/
nbt.3437.
[179] Y. Zou, X. Sun, Q. Yang, M. Zheng, O. Shimoni, W. Ruan, Y. Wang, D. Zhang,
J. Yin, X. Huang, W. Tao, J.B. Park, X.J. Liang, K.W. Leong, B. Shi, Blood-brain
barrier-penetrating single CRISPR-Cas9 nanocapsules for effective and safe
glioblastoma gene therapy, Sci. Adv. 8 (2022), eabm8011, https://doi.org/
10.1126/sciadv.abm8011.
[180] A.R. De Lera, A. Ganesan, Two-hit wonders: the expanding universe of
multitargeting epigenetic agents, Curr. Opin. Chem. Biol. 57 (2020) 135–154,
https://doi.org/10.1016/j.cbpa.2020.05.009.
[181] D. Tomaselli, A. Lucidi, D. Rotili, A. Mai, Epigenetic polypharmacology: a new
frontier for epi-drug discovery, Med. Res. Rev. 40 (2020) 190–244, https://doi.
org/10.1002/med.21600.
[182] S.G. Shah, T. Mandloi, P. Kunte, A. Natu, M. Rashid, D. Reddy, N. Gadewal,
S. Gupta, HISTome2: a database of histone proteins, modifiers for multiple
organisms and epidrugs, Epigenetics Chromatin 13 (2020), 31, https://doi.org/
10.1186/s13072-020-00354-8.
[183] R. Guo, J. Li, J. Hu, Q. Fu, Y. Yan, S. Xu, X. Wang, F. Jiao, Combination of
epidrugs with immune checkpoint inhibitors in cancer immunotherapy: From
theory to therapy, Int. Immunopharmacol. (2023), 110417, https://doi.org/
10.1016/j.intimp.2023.110417.
[184] M.G. Gladkova, E. Leidmaa, E.A. Anderzhanova, Epidrugs in the therapy of
central nervous system disorders: a way to drive on? Cells 12 (2023) 1464,
https://doi.org/10.3390/cells12111464.