By Ken Niemann
Important controls in the switching on and off of genes include but are not limited to: timing, gravity, spatial organization, environmental cues, and epigenetic mechanisms. It is the latter that we are exploring here. Epigenetics may be defined as changes in gene expression that don’t involve changes to the DNA sequence. It is the often heritable changes in gene activity and expression that occur without alteration to the sequence of DNA (the genetic code in each cell remains the same). In other words, epigenetics changes how the genes are expressed rather than changing the genes themselves. The epigenetic code is tissue and cell specific and responds differently to stress, nutrition, environmental toxins, behavior, and pharmacological stimuli. While the number of known epigenetic mechanisms now exist in the dozens, there is “robust evidence that repeated exposure to drugs of abuse induces changes within the brain’s reward regions in three major modes of epigenetic regulation—histone modifications such as acetylation and methylation, DNA methylation, and non-coding RNAs.” (Nestler, 2014). We will briefly look at each of these mechanisms.
DNA METHYLATION
A well-understood form of epigenetic signaling, called DNA methylation, involves the addition of a methyl group, in most cases, to the number 5 carbon of the cytosine pyrimidine ring within the promoters of genes The methylated base then interferes with the chemical signals that would put the gene into action and thus effectively either silences the gene or attenuates its expression as a dimmer switch. The addition of a methyl group is performed by one or more of several different enzymes in a class known as DNA methyltransferases (DNMTs). When the base pair is methylated by DNMT, it binds to a protein called MeCP2 (Methyl CpG binding protein 2). The MeCP2 has several roles: it may stop DNA transcription altogether by blocking the promotor, attract other proteins to assist in suppressing the gene, and promote the coiling of the DNA preventing access by transcription enzymes. (Carey, N., 2012).
CHROMATIN REMODELING & HISTONE MODIFICATION
Another form of epigenetic alteration blocks or unblocks access to the genes by histone modification. Here, the DNA in every cell is tightly wound around proteins known as histones and must be unwound to be transcribed. About 147 base pairs of DNA wrapped around a core histone octamer comprise the structural unit known as the nucleosome, the basic unit of chromatin. “Each octamer contains two copies each of the histones H2A, H2B, H3, and H4. Epigenetic mechanisms control the spacing of nucleosomes and the degree to which they are condensed, which thereby determines gene activity.”
“In simplified terms, chromatin exists in an inactivated, condensed state (heterochromatin), which does not allow transcription of genes, and in an activated, open state (euchromatin), which allows individual genes to be transcribed”. (Nestler, 2014) Chromatin may also exist in intermediate states that dampen or increase gene expression. Remodeling is accomplished a number of different ways including the post translational modification of the amino acids that make up histone protein via the acetylation of lysine residues, methylation of lysine and arginine, phosphorylation of serine and threonine, and the ubiquitination of lysine. Histone acetylation is accomplished by histone acetyl transferases (HATs) which adds acetyl groups to histone tails thereby reducing positive charge and weakening interaction of histones with DNA. This in turn facilitates transcription by making DNA more accessible to RNA polymerase II. Histone deacetylation, on the other hand, occurs through the action of histone deacetylases (HDACs) which removes acetyl groups from histone tails making it bind tighter to the DNA with resulting transcriptional repression.
Methylation of histones may result in either activation or repression of gene expression. For example, trimethylation of histone H3 at lysine 4 (H3K4) is signal for transcriptional activation and dimethylation of histone H3 at lysine 9 (H3K9) is a signal for transcriptional repression. (Nestler, 2014)
NONCODING RNAS
One of the outcomes of the Human Genome Project was the discovery of thousands of genes that are activated yet do not give rise to proteins. According to Nestler, “Such non-coding RNAs have been shown to play crucial regulatory roles in cell function. Most studied are microRNAs (miRNAs), typically 20–25 nt, which by binding to targeted mRNAs either inhibit their translation or induce their breakdown.” (Nestler 2014) In recent years, longer non-coding RNAs (lncRNAs), are also being identified as important regulators of gene transcription. “They modulate chromatin-modifying complexes through direct interactions with transcription factors and other nuclear proteins.” (Nestler, 2014)
Examples of Epigenetic Changes Following Cocaine Abuse
To complicate matters, different epigenetic modifiers (studied in animal models) such as the addition of an acetyl group, will have different effects depending on where in the octamer the group attaches. With different signals causing different outcomes in different places, how a drug affects gene expression and behavior quickly becomes a tangled web of information to decode. The process will take considerable time and effort to unfold and few conclusions as to how a stimulus leads to epigenetic changes which in turn affect behavior may be drawn at this point. However, it is instructive to follow Nestler (2014) as he offers an example of epigenetic changes influencing gene expression, activation and repression, resulting the upregulation of the reward pathways in the nucleus accumbens following chronic cocaine use. In this example we see how ΔFosB “functions as a type of sustained “molecular switch” that gradually converts acute drug responses into relatively stable adaptations that contribute to the long-term neural and behavioral plasticity that underlies addiction.” (Nestler, E. J., Barrot, M., Self, D., 2001)
“The figure is based on the mechanisms by which chronic cocaine, through ΔFosB, activates the Cdk5 gene (top) and represses the c-Fos gene (bottom). Top: ΔFosB binds to the Cdk5 gene and recruits several co-activators, including CBP (CREB binding protein) — a type of histone acetyltransferase (HAT) leading to increased histone acetylation, BRG1 (brahmarelated gene 1) — a type of chromatin remodeling factor — and SUG1 (proteasome 26SATPase subunit 5), another type of chromatin regulatory protein. ΔFosB also represses G9a expression, leading to reduced repressive histone methylation at the Cdk5 gene. The netresult is gene activation and increased CDK5 expression. Bottom: In contrast, ΔFosB binds to the c-Fos gene and recruits several co-repressors, including HDAC1 (histone deacetylase 1) and SIRT1 (sirtuin 1). The gene also shows increased G9a binding and repressive histone methylation (despite global decreases in these marks). The net result is c-Fos gene repression. As transcriptional regulatory complexes contain dozens or hundreds of proteins, much further work is needed to further define the activational and repressive complexes that cocaine recruits to particular genes to mediate their transcriptional regulation and to explore the range of distinct activational and repressive complexes involved in cocaine action.” (Nestler, EJ, 2014)
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