Epigenetic Influences and Disease

DNA methylation is a chemical process that adds a methyl group to DNA. It is highly specific and always happens in a region in which a cytosine nucleotide is located next to a guanine nucleotide that is linked by a phosphate; this is called a CpG site (Egger et al., 2004; Jones & Baylin, 2002; Robertson, 2002). CpG sites are methylated by one of three enzymes called DNA methyltransferases (DNMTs) (Egger et al., 2004; Robertson, 2002). Inserting methyl groups changes the appearance and structure of DNA, modifying a gene's interactions with the machinery within a cell's nucleus that is needed for transcription. DNA methylation is used in some genes to differentiate which gene copy is inherited from the father and which gene copy is inherited from the mother, a phenomenon known as imprinting.

Histones are proteins that are the primary components of chromatin, which is the complex of DNA and proteins that makes up chromosomes. Histones act as a spool around which DNA can wind. When histones are modified after they are translated into protein (i.e., post-translation modification), they can influence how chromatin is arranged, which, in turn, can determine whether the associated chromosomal DNA will be transcribed. If chromatin is not in a compact form, it is active, and the associated DNA can be transcribed. Conversely, if chromatin is condensed (creating a complex called heterochromatin), then it is inactive, and DNA transcription does not occur.

There are two main ways histones can be modified: acetylation and methylation. These are chemical processes that add either an acetyl or methyl group, respectively, to the amino acid lysine that is located in the histone. Acetylation is usually associated with active chromatin, while deacetylation is generally associated with heterochromatin. On the other hand, histone methylation can be a marker for both active and inactive regions of chromatin. For example, methylation of a particular lysine (K9) on a specific histone (H3) that marks silent DNA is widely distributed throughout heterochromatin. This is the type of epigenetic change that is responsible for the inactivated X chromosome of females. In contrast, methylation of a different lysine (K4) on the same histone (H3) is a marker for active genes (Egger et al., 2004).

Genes can also be turned off by RNA when it is in the form of antisense transcripts, noncoding RNAs, or RNA interference. RNA might affect gene expression by causing heterochromatin to form, or by triggering histone modifications and DNA methylation (Egger et al., 2004).

The first human disease to be linked to epigenetics was cancer, in 1983. Researchers found that diseased tissue from patients with colorectal cancer had less DNA methylation than normal tissue from the same patients (Feinberg & Vogelstein, 1983). Because methylated genes are typically turned off, loss of DNA methylation can cause abnormally high gene activation by altering the arrangement of chromatin. On the other hand, too much methylation can undo the work of protective tumor suppressor genes.

As previously mentioned, DNA methylation occurs at CpG sites, and a majority of CpG cytosines are methylated in mammals. However, there are stretches of DNA near promoter regions that have higher concentrations of CpG sites (known as CpG islands) that are free of methylation in normal cells. These CpG islands become excessively methylated in cancer cells, thereby causing genes that should not be silenced to turn off. This abnormality is the trademark epigenetic change that occurs in tumors and happens early in the development of cancer (Egger et al., 2004; Robertson, 2002; Jones & Baylin, 2002). Hypermethylation of CpG islands can cause tumors by shutting off tumor-suppressor genes. In fact, these types of changes may be more common in human cancer than DNA sequence mutations (Figure 2).

Furthermore, although epigenetic changes do not alter the sequence of DNA, they can cause mutations. About half of the genes that cause familial or inherited forms of cancer are turned off by methylation. Most of these genes normally suppress tumor formation and help repair DNA, including O⁶-methylguanine-DNA methyltransferase (MGMT), MLH1 cyclin-dependent kinase inhibitor 2B (CDKN2B), and RASSF1A. For example, hypermethylation of the promoter of MGMT causes the number of G-to-A mutations to increase (Figure 2).

Hypermethylation can also lead to instability of microsatellites, which are repeated sequences of DNA. Microsatellites are common in normal individuals, and they usually consist of repeats of the dinucleotide CA. Too much methylation of the promoter of the DNA repair gene MLH1 can make a microsatellite unstable and lengthen or shorten it (Figure 2). Microsatellite instability has been linked to many cancers, including colorectal, endometrial, ovarian, and gastric cancers (Jones & Baylin, 2002).

Figure 2: Mechanism of action of nucleoside analogue inhibitors.

Deoxynucleoside analogues such as 5-aza-2-deoxycytidine (depicted by Z) are converted into the triphosphate inside S-phase cells and are incorporated in place of cytosine into DNA. Ribonucleosides such as 5-azacytidine or zebularine are reduced at the diphosphate level by ribonucleotide reductase for incorporation (not shown). Once in DNA, the fraudulent bases form covalent bonds with DNA methyltransferases (DNMTs), resulting in the depletion of active enzymes and the demethylation of DNA. Pink circles, methylated CpG; cream circles, unmethylated CpG.

© 2004 Nature Publishing Group Egger, G. et al. Epigenetics in human disease and prospects for epigenetic therapy. Nature 429, 460 (2004). All rights reserved.

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Figure 3

Fragile X syndrome is the most frequently inherited mental disability, particularly in males. Both sexes can be affected by this condition, but because males only have one X chromosome, one fragile X will impact them more severely. Indeed, fragile X syndrome occurs in approximately 1 in 4,000 males and 1 in 8,000 females. People with this syndrome have severe intellectual disabilities, delayed verbal development, and "autistic-like" behavior (Penagarikano et al., 2007).

Fragile X syndrome gets its name from the way the part of the X chromosome that contains the gene abnormality looks under a microscope; it usually appears as if it is hanging by a thread and easily breakable (Figure 3). The syndrome is caused by an abnormality in the FMR1 (fragile X mental retardation 1) gene. People who do not have fragile X syndrome have 6 to 50 repeats of the trinucleotide CGG in their FMR1 gene. However, individuals with over 200 repeats have a full mutation, and they usually show symptoms of the syndrome. Too many CGGs cause the CpG islands at the promoter region of the FMR1 gene to become methylated; normally, they are not. This methylation turns the gene off, stopping the FMR1 gene from producing an important protein called fragile X mental retardation protein. Loss of this specific protein causes fragile X syndrome. Although a lot of attention has been given to the CGG expansion mutation as the cause of fragile X, the epigenetic change associated with FMR1 methylation is the real syndrome culprit.

Fragile X syndrome is not the only disorder associated with mental retardation that involves epigenetic changes. Other such conditions include Rubenstein-Taybi, Coffin-Lowry, Prader-Willi, Angelman, Beckwith-Wiedemann, ATR-X, and Rett syndromes (Table 1).