**3. DNA epigenetic modification**

demonstrate that "nurture" changes "nature" by modifying whether or not a given gene will be expressed. Understanding how one's environment (e.g., drug-taking behavior, stress, and learning) can alter gene expression in the brain may give insight into how drug addiction develops, how it may be passeddown into future generations, andperhaps, how it can be better

22 Recent Advances in Drug Addiction Research and Clinical Applications

While the DNA sequence of a gene can be modified directly (e.g., mutations, deletions, insertions, translocations, etc.) resulting in altered gene expression, epigenetics regulates gene expression by mechanisms other than changes to the DNA sequence. It has long been known that epigenetic mechanisms largely control cell differentiation by allowing some genes to be expressed and others to be silenced at various points in time during development. Indeed, even though all human cells possess the same DNA (with the exception of egg and sperm cells), what differentiates a given cell type from others (e.g., a neuron versus a liver cell) is the epigenetic mechanisms that permit or deny its genes to be transcribed and translated into cell type-specific functional proteins [3]. Beyond the hard-wire epigenetic programming of gene expression during development, epigenetic mechanisms also provide dynamic and heritable means of altering gene expression in response to environmental change. For example, either stressful life experiences or a history of chronic drug intake can invoke chemical modifications to either the DNA or the histone proteins that are involved in storing the DNA. Such epigenetic changes have an impact on how accessible the DNA is for gene transcription. Epigenetic changes can also be long lasting and passed down to future generations. In this way, not only does experience with stress and/or drugs place one's self at risk for SUDs, but also one's offspring due to heritable epigenetic modifications. Even in the more proximal time frame of an individual's lifespan, epigenetic mechanisms provide a "working memory" for gene expression changes that are involved in brain plasticity [4]. Brain plasticity changes resulting from drug exposure are thought to be the crux of the dysfunction underlying addiction [5]. An exciting implication of understanding the role of epigenetic changes in drug-induced brain

plasticity is that new strategies for therapeutic interventions may be discovered.

associated with drug addiction.

In this chapter, we review three epigenetic mechanisms that have been found to impact drug abuse-related behaviors in animal models: (1) chemical modifications to DNA, (2) chemical modifications to histones, and (3) the induction of noncoding RNAs that regulate gene expression. We will begin with a brief explanation of how drugs modify intracellular signaling pathways that propagate to the cell nucleus, leading to epigenetic changes. We will then provide a brief description of the epigenetic mechanisms listed above, followed by examples of how drugs of abuse invoke these mechanisms and how pharmacologically targeting the epigenome can alter drug-abuse-related behavior. Next, we will cover the latest developments in genetic tools that provide precise manipulation of epigenetic enzymes, further elucidating the roles of these specific molecules. We will also review literature supporting transgenera‐ tional inheritance of epigenetic changes associated with a history of drug intake. We conclude by discussing important future directions for research investigating epigenetic mechanisms

treated.

A given gene is composed of a sequence of nucleotide base pairs in the DNA that are unique to that gene. For coding genes, the DNA sequence of base pairs serves as the blueprint for making a particular protein. Given that proteins are the machinery for cell structure and function, gene expression changes in a neuron can alter cell protein composition and, in turn, change the way that the neuron functions and communicates with other neurons.

There are four different nucleotide bases that compose the sequence portion of the DNA molecule, including the pyrimidines cytosine (C) and thymine (T), and the purines adenine (A) and guanine (G). Due to the structures of these nucleotides, the chemical bond responsible for base pairing can only form between C and G or A and T, respectively. Cs followed by Gs in the DNA sequence (i.e., CpGs) can be modified by a reaction in which DNA methyltrans‐ ferases (DNMTs) add a methyl group (CH3) to the 5-position of the C to form 5mC. Intracellular signals may initiate newly synthesized *de novo* DNA methylation, which is mediated by DNMT subtypes DNMT3a and DNMT3b. Subtype DNMT1, on the other hand, maintains DNA methylation patterns across cell replication, such that the newly synthesized DNA has the exact methylation pattern that existed before DNA replication. In general, DNA methylation is correlated with a decrease in DNA accessibility and therefore is thought to be a mechanism of silencing gene expression. Methylated DNA can silence gene expression by interfering with the binding of transcriptional activators or by binding to proteins with a methyl-CpG-binding domain (MBD), such as methyl-CpG binding protein 2 (MECP2), that then form a complex with other proteins that together repress DNA accessibility [11].

Historically, DNA methylation was believed to be a permanent modification. However, demethylation of DNA can occur and also contributes to dynamic changes in gene expression. While passive demethylation in dividing cells may be due to malfunctioning of DNMT1, active demethylation occurs in both dividing and nondividing cells by enzymatic reactions. One reaction changes 5mC into a T, which is then recognized as a G/T mismatch. The mismatch activates a base excision repair (BER) pathway that utilizes thymine DNA glycosylase (TDG) and ultimately replaces T with a nonmethylated C [12]. Another reaction catalyzed by 10–11 translocation enzymes (TET) adds a hydroxyl (–OH) group to 5mC forming 5hmC. 5hmC itself has effects on gene expression and it can undergo further reactions that convert it back to a nonmethylated C [13]. Therefore, demethylation of DNA is generally correlated with an increase in DNA accessibility.
