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Epigenetic Effects of Smoking

• Pascal and Chris

Cigarette smoking has been found to affect global epigenetic regulation of transcription across tissue types. Many studies have shown differences in epigenetic markers like DNA methylation and miRNA expression between smokers and non-smokers. Similar differences exist in children whose mothers smoked during pregnancy. These epigenetic effects are thought to be linked to a number of negative health effects associated with smoking.

Health Impact

It has been well established that smoking cigarettes has a number of negative health impacts, including increased risk for cancer, cardiovascular disease, and chronic obstructive pulmonary disease (COPD). Children exposed prenatally to cigarette smoke demonstrate increased risk for fetal growth restriction, sudden infant death syndrome, and addictive behaviors later in life and a host of other secondary health effects. It is thought that epigenetic changes that arise from smoking cigarettes or exposure to cigarette smoke play a role in the development of these conditions.

Epigenetic modifications to the genome, including histone marking, DNA methylation, and expression regulating RNAi, are one of the primary ways vertebrates regulate the expression of genes. When the epigenome of an organism is altered by an environmental cue like smoking, gene expression changes accordingly. Changes in the regulation of critical genes can have disastrous consequences on health and quality of life. Irregular gene expression is one of the hallmarks of cancer, but is also found in a number of diseases and disorders.

Mechanisms for Changes in DNA Methylation

One of the most prominent and well studied epigenetic consequences of cigarette smoke is altered DNA methylation. Cigarette smoke acts through a number of mechanisms to effect this, cheif among these being smoke induced damage to the DNA and altered expression levels of proteins involved in DNA methylation.

Damage to DNA

Chemicals in smoke can damage DNA, which subsequently leads to changes in DNA methylation during the repair process. Damage typically comes in the form of double-stranded breaks that are linked to carcinogens like arsenic, chromium, formaldehyde, polycyclic aromatic hydrocarbons, and nitrosamines that are found in cigarette smoke. DNMT1 is an enzyme involved in the maintenance of DNA methylation marks. DNMT1 is recruited to DNA during its replication, or during DNA repair. As a new DNA strand is synthesized, unmethylated cytosines are incorporated into the sequence. This leads to hemimethylated DNA, where an older methylated strand is bound to a younger unmethylated one. DNMT1 is an enzyme which recognizes and corrects hemimethylation by transferring the appropriate methyl groups to the newly synthesized strand. Like all biological processes, DNMT1 based hemimethylation correction is not perfect. Mistakes in hemimethylation correction can occur, and are more likely to appear the more a DNA sequence is replicated or repaired. This is compounded by other effects which cigarette smoke has on the expression and activity of DNMT1. The end result is a decrease in the bodies ability to maintain proper methylation patterns, leading to miss-expression of genes.

Effects on DNA Methylating Proteins

Exposure to cigarette smoke impacts proteins involved in DNA methylation. These effects come from either hypoxia induced by the cigarette smoke, or the chemical consequences of nicotine. Inhaling cigarette smoke increases blood levels of carbon monoxide which negatively impacts oxygenation throughout the body leading to hypoxia [1]. One response to hypoxia is the upregulation in synthesis of the major methyl donor S-adenosyl methionine by methionine adenosyltransferase 2A. Upregulation of this methyl donor leads to increased DNA methylation, which can lead to the down-regulation of target genes. Nicotine, the principle component of cigarette smoke, binds to nicotinic acetylcholine receptors . This binding leads to an increase in calcium levels which in turn can activate a transcription factor called cAMP response element-binding protein (CREB). The most striking downstream effect of the upregulation of this transcription factor is the downregulation of the DNMT1 gene, which has a cAMP response element in it’s promoter. This down-regulation of DNMT1 can have serious consequences to DNA methylation, namely a failure to maintain normal methylation patterns during DNA replication and repair. The upregulation of DNMT3b has also been shown to occur as a result of cigarette exposure . DNMT3b is thought to be critical to de novo methylation, or the production of new methylation marks on DNA. This increased expression of DNMT3b and methionine adenosyltransferase 2A, taken together with the down-regulation of DNMT1, could result in myriad unintended epigenetic consequences.

Effects on Transcription Factors

Sp1, a transcription factor that plays a crucial role in early development, was shown to be expressed at higher rates in lung epithelial cells in the presence of cigarette-smoke condensate . This is relevant because Sp1 binds to GC-rich promoter regions which prevent the methylation of these regions during embryonic development. Increased Sp1 expression can lead to a global reduction in methylation, leading to a number of downstream health effects.

Consequences of Altered DNA methylation

Regardless of mechanism, several known differences in DNA methylation have been observed between smokers and non-smokers. An overall average decrease in DNA methylation is observed. This whole genome decrease in methylation can increase the expression of a number of genes, which could potentially lead to negative health effects. Several genes known to be affected by differential methylation are the CYP1A1 xenobiotic response element, AHRR, and F2RL3. CYP1A1 is critical to the detoxification of carcinogens, and is found to be hypomethylated in frequent smokers. F2RL3 is known to be involved in blood clotting and the inflammation response, and is also hypomethylated in frequent smokers. Effects on the regulation of this gene could be a link between epigenetic changes from smoking and increased risk of heart disease. Time specific changes in methylation of D4Z4 and NBL2 repeats, which are known factors in carcinogenesis, have also been observed .

Fetuses exposed in utero to cigarette smoke are also known to have some distinct epigenetic differences from smoke-free cohorts. CYP1A1 was similarly found to be less methylated in the placentas of fetuses prenatally exposed to cigarette smoke, along with the transposable element AluYB8 . Similar demethylation in a number of Alu elements has been observed, and could generally decrease genomic stability and increase the risk of cancer from deleterious insertion of a transposable element. Strikingly, BDNF appears to be hypomethylated in children who were exposed to smoke prenatally. BDNF is critical to long term memory formation and the upkeep of neurons. Downregulation of BDNF has also been linked to clinical depression .

Though smoking leads to an overall decrease in DNA methylation, several critical genes become hypermethylated. Two of the most noteworthy of these genes are p16 and p53. These genes critical to cell cycle regulation and were shown to have higher levels of methylation in smokers than in non smokers. The subsequent loss of function in these genes could potentially lead to dysregulation of the cell cycle, wherein cells are able to bypass normal growth impeding signals. Ultimately, uncontrolled cellular divisions and failure to properly regulate the cell cycle leads to cancer.

Effects on Histone Modifications

Histone modifications are another epigenetic phenomenon known to be affected by smoking. Cigarette smoke has been observed to globally alter histone modifications of promoter regions of pro-inflammatory genes, mainly through an overall increase in acetylation. Specifically in rat and mouse models, cigarette smoke was observed to increase acetylation of lysine 9 on H3 (H3K9) and lysine 12 on H4 (H4K12) but also phosphorylation of serine 10 on H3 (H3S10). These marks are associated with an increase in gene expression and prevent the accumulation of epigenetic marks that promote gene repression. Mechanistically, the increased frequency of these marks, especially the modifications on histone H3, is linked to the activation of IKK-α that directly phosphorylates histone H3 as a consequence of exposure to cigarette smoke. It is thought that this increase in acetylation of histones H3 and H4 in macrophages in the alveolus could potentially lead to the development of COPD.

Histone modifications are another epigenetic phenomenon known to be affected by smoking. Cigarette smoke has been observed to globally alter histone modifications of promoter regions of pro-inflammatory genes, mainly through an overall increase in acetylation. Specifically in rat and mouse models, cigarette smoke was observed to increase acetylation of lysine 9 on H3 (H3K9) and lysine 12 on H4 (H4K12) but also phosphorylation of serine 10 on H3 (H3S10). These marks are associated with an increase in gene expression and prevent the accumulation of epigenetic marks that promote gene repression. Mechanistically, the increased frequency of these marks, especially the modifications on histone H3, is linked to the activation of IKK-alpha that directly phosphorylates histone H3 as a consequence of exposure to cigarette smoke. It is thought that the accumulation of acetylation of histones H3 and H4 in macrophages in the alveolus could potentially lead to the development of COPD.

Cigarette smoke can alter histone acetylation by inducing the degradation of HDAC2, thereby preventing the removal of acetylation marks in affected cells. The degradation of the enzyme is likely to be caused by phosphorylation and subsequent ubiquitination, a process that is thought to be induced by cigarette smoke. The resulting reduction in the body's ability to demethylate histones prevents it from downregulating genes which normally require downregualtion. Abnormal overexpression of certain genes through this mechanism could potentially lead to cancer.

Effects on miRNA

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Micro-RNA, or miRNA, is known to be a major epigenetic regulator of gene expression in humans. These RNAs are short molecules which bind to mRNA through complementary base pairing. This impacts the expression of proteins encoded by those mRNAs by either inducing the cleavage of the mRNA, destabilizing the molecule, or limiting the efficiency of its translation. Unlike differences in DNA methylation, changes in miRNA activity induced by cigarette smoke are largely unknown. Research into how cigarettes impact miRNA and other forms of RNAi is still ongoing.

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