The Role of Methylation in Gene Expression (2024)

Prior to 1980, there were a number of clues that suggested that methylation might play a role in the regulation of gene expression. For example, J. D. McGhee and G. D. Ginder compared the methylation status of the beta-globin locus in cells that did and did not express this gene. Using restriction enzymes that distinguished between methylated and unmethylated DNA, the duo showed that the beta-globin locus was essentially unmethylated in cells that expressed beta-globin but methylated in other cell types . This and other evidence of the time were indirect suggestions that methylation was somehow involved in gene expression.

Shortly after McGhee and Ginder published their results, amore direct experiment that examined the effects of inhibiting methylation ongene expression was performed using 5-azacytidine in mouse cells. 5-azacytidineis one of many chemical analogs for the nucleoside cytidine. When these analogsare integrated into growing DNA strands, some, including 5-azacytidine, severelyinhibit the action of the DNA methyltransferase enzymes that normally methylateDNA. (Interestingly, other analogs, like Ara-C, do not negatively impactmethylation.) Because most DNA methylation was known to occur on cytosineresidues, scientists hypothesized that if they inhibited methylation byflooding cellular DNA with 5-azacytidine, then they could compare cells beforeand after treatment to see what impact the loss of methylation had on geneexpression. Knowing that gene expression changes are responsible for cellulardifferentiation, these researchers used changes in cellular phenotypes as aproxy for gene expression changes (Table 1; Jones & Taylor, 1980).

Table 1: Effect of Cytidine Analogs on Cell Differentiation and DNA Methylation

This straightforward experiment demonstrated that it was notthe removal of cytidine residues alone that resulted in changes in cell differentiation(because Ara-C did not have an impact on differentiation); rather, only thoseanalogs that impacted methylation resulted in such changes. These experimentsopened the door for investigators to better understand exactly how methylationimpacts gene expression and cellular differentiation.

Today, researchers know that DNA methylation occurs at the cytosinebases of eukaryotic DNA, which are converted to 5-methylcytosine by DNAmethyltransferase (DNMT) enzymes. The altered cytosine residues are usuallyimmediately adjacent to a guanine nucleotide, resulting in two methylatedcytosine residues sitting diagonally to each other on opposing DNA strands. Differentmembers of the DNMT family of enzymes act either as de novo DNMTs, putting the initial pattern of methyl groups in placeon a DNA sequence, or as maintenance DNMTs, copying the methylation from an existingDNA strand to its new partner after replication. Methylation can be observed bystaining cells with an immunofluorescently labeled antibody for5-methylcytosine. In mammals, methylation is found sparsely but globally,distributed in definite CpG sequences throughout the entire genome, with theexception of CpG islands, or certain stretches (approximately 1 kilobase inlength) where high CpG contents are found. The methylation of these sequencescan lead to inappropriate gene silencing, such as the silencing of tumor suppressor genes incancer cells.

Currently, the mechanism by which de novo DNMT enzymes are directed to the sites that they are meantto silence is not well understood. However, researchers have determined that someof these DNMTs are part of chromatin-remodeling complexes and serve to complete the remodelingprocess by performing on-the-spot DNA methylation to lock the closed shape ofthe chromatin in place.

The roles andtargets of DNA methylation vary among the kingdoms of organisms. As previously noted,among Animalia, mammals tend to have fairly globally distributed CpG methylationpatterns. On the other hand, invertebrate animals generally have a "mosaic"pattern of methylation, where regions of heavily methylated DNA areinterspersed with nonmethylated regions. The global pattern of methylation inmammals makes it difficult to determine whether methylation is targeted tocertain gene sequences or is a default state, but the CpG islands tendto be near transcription start sites,indicating that there is a recognition system in place.

Plantae are the mosthighly methylated eukaryotes, with up to 50% of their cytosine residues exhibitingmethylation. Interestingly, in Fungi, only repetitive DNA sequences aremethylated, and in some species, methylation is absent altogether, or itoccurs on the DNA of transposable elements in the genome. The mechanism by which thetransposons are recognized and methylated appears to involve smallinterfering RNA (siRNA). The whole silencing mechanisminvoking DNMTs could be a way for these organisms to defend themselves againstviral infections, which could generate transposon-like sequences. Suchsequences can do less harm to the organism if they are prevented from beingexpressed, although replicating them can still be a burden (Suzuki & Bird,2008). In other fungi, such as fission yeast, siRNA is involved in genesilencing, but the targets include structural sequences of the chromosomes,such as the centromeric DNA and the telomeric repeats at the chromosome ends.

For many years,methylation was believed to play a crucial role in repressing gene expression,perhaps by blocking the promoters at which activating transcription factors shouldbind. Presently, the exact role of methylation in gene expression is unknown,but it appears that proper DNA methylation is essential for celldifferentiation and embryonic development. Moreover, in some cases, methylationhas observed to play a role in mediating gene expression. Evidence of this hasbeen found in studies that show that methylation near gene promotersvaries considerably depending on cell type, with more methylation of promoterscorrelating with low or no transcription (Suzuki & Bird, 2008). Also, while overallmethylation levels and completeness of methylation of particular promoters are similarin individual humans, there are significant differences in overall and specificmethylation levels between different tissue types and between normal cells andcancer cells from the same tissue.

Researchers havealso determined that mice that lack a particular DNMT have reduced methylationlevels and die early in development (Suzuki & Bird, 2008). This is not thecase for all eukaryotes, however; some organisms, such as the yeast Saccharomyces cerevisiae andthe nematodeworm Caenorhabditis elegans, arethought to have no methylated DNA at all (although, at least in yeast, thereare sequences in their genomes that are hom*ologous to those that code for theDNMT enzymes).

Although patterns of DNA methylation appear to be relativelystable in somatic cells, patterns of histone methylation can change rapidlyduring the course of the cell cycle. Despite this difference, several studieshave indicated that DNA methylation and histone methylation at certainpositions are connected. For instance, results of immunoprecipitation studies usinghuman cells suggest that DNA methylation and histone methylation work togetherduring replication to ensure that specific methylation patterns are passed on toprogeny cells (Sarraf & Stancheva, 2004). Indeed, evidence has beenpresented that in some organisms, such as Neurosporacrassa (Tamaru & Selker, 2001) and Arabidopsisthaliana (Jackson et al., 2002),H3-K9 methylation (methylation of a specific lysine residue in the histone H3)is required in order for DNA methylation to take place. However, exceptionshave been observed in which the relationship is reversed. In one study, forexample, H3 methylation was reduced at a tumor suppressor gene in cellsdeficient in DNA methyltransferase (Martin & Zhang, 2005).

In an interestingly coordinated process, proteins that bindto methylated DNA also form complexes with the proteins involved indeacetylation of histones. Therefore, when DNA is methylated, nearby histonesare deacetylated, resulting in compounded inhibitory effects on transcription.Likewise, demethylated DNA does not attract deacetylating enzymes to thehistones, allowing them to remain acetylated and more mobile, thus promotingtranscription.

In most cases, methylation of DNA is a fairly long-term,stable conversion, but in some cases, such as in germ cells, when silencing of imprintedgenes must be reversed, demethylation can takeplace to allow for "epigenetic reprogramming." The exact mechanisms fordemethylation are not entirely understood; however, it appears that thisprocess may be mediated by the removal of amino groups by DNA deaminases(Morgan et al., 2004). After deamination, the DNA has a mismatch andis repaired, causing it to become demethylated. In fact, studies using inhibitorsof one DNMT enzyme showed that this enzyme was involved in not only DNAmethylation, but also in the removal of amino groups.

Given the criticalrole of DNA methylation in gene expression and cell differentiation, it seemsobvious that errors in methylation could give rise to a number of devastatingconsequences, including various diseases. Indeed, medical scientists are currentlystudying the connections between methylation abnormalities and diseases such ascancer, lupus, muscular dystrophy, and a range of birth defects that appear tobe caused by defective imprinting mechanisms (Robertson, 2005). The results ofthese studies will be invaluable for treating these disorders, as well as forunderstanding and preventing complications that can arise during embryonicdevelopment due to abnormalities in X-chromosome methylation and geneimprinting.

To date, a largeamount of research on DNA methylation and disease has focused on cancer andtumor suppressor genes.Tumor suppressor genes are often silenced in cancer cells due to hypermethylation.In contrast, the genomes of cancer cells have been shown to be hypomethylated overallwhen compared to normal cells, with the exception of hypermethylation events atgenes involved in cell cycle regulation, tumor cell invasion, DNA repair, andothers events in which silencing propagates metastasis (Figure 1; Robertson, 2005). In fact, in certain cancers, such as thatof the colon, hypermethylation is detectable early and might serve as abiomarker for the disease.