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The Role of Methylation in Gene Expression
By: Theresa Phillips, Ph.D. (Write Science Right) © 2008 Nature Education Citation: Phillips, T. (2008) The role of methylation in gene expression. Nature Education 1(1) Not all genes are active at all times. DNA methylation is one of several epigenetic mechanisms that cells use to control gene expression. There are many ways that gene expression is controlled in eukaryotes, but methylation of DNA (not to be confused with histone methylation) is a common epigenetic signaling tool that cells use to lock genes in the "off" position. In recent decades, researchers have learned a great deal about DNA methylation, including how it occurs and where it occurs, and they have also discovered that methylation is an important component in numerous cellular processes, including embryonic development, genomic imprinting, X-chromosome inactivation, and preservation of chromosome stability. Given the many processes in which methylation plays a part, it is perhaps not surprising that researchers have also linked errors in methylation to a variety of devastating consequences, including several human diseases.

5-azacytidine Experiments Provide Early Clues to the Role of Methylation in Gene Expression
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 betaglobin but methylated in other cell types (McGhee & Ginder, 1979). 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, a more direct experiment that examined the effects of inhibiting methylation on gene expression was performed using 5-azacytidine in mouse cells. 5-azacytidine is one of many chemical analogs for the nucleoside cytidine. When these analogs are integrated into growing DNA strands, some, including 5-azacytidine, severely inhibit the action of the DNA methyltransferase enzymes that normally methylate DNA. (Interestingly, other analogs, like Ara-C, do not negatively impact methylation.) Because most DNA methylation was known to occur on cytosine residues, scientists hypothesized that if they inhibited methylation by flooding cellular DNA with 5-azacytidine, then they could compare cells before and after treatment to see what impact the loss of methylation had on gene expression. Knowing that gene expression changes are responsible for cellular differentiation, these researchers used changes in cellular phenotypes as a proxy for gene expression changes (Table 1; Jones & Taylor, 1980). Table 1: Effect of Cytidine Analogs on Cell Differentiation and DNA Methylation

Chemical Added

Number of Differentiated Amount of Cells Measured

Methylation

Different members of the DNMT family of enzymes act either as de novo DNMTs.3 μM Ara-C 0 127% 3 μM 5-azacytidine 22. and in some species. How and Where Are Genes Methylated? Today. rather. However. The mechanism by which the transposons are recognized and methylated appears to involve small interfering RNA (siRNA). only those analogs that impacted methylation resulted in such changes. In other fungi. in Fungi. The roles and targets of DNA methylation vary among the kingdoms of organisms. On the other hand. distributed in definite CpG sequences throughout the entire genome. putting the initial pattern of methyl groups in place on a DNA sequence. The global pattern of methylation in mammals makes it difficult to determine whether methylation is targeted to certain gene sequences or is a default state. Currently. but the CpG islands tend to be near transcription start sites. As previously noted. with the exception of CpG islands. siRNA is involved in gene silencing. such as the silencing of tumor suppressor genes in cancer cells. which are converted to 5-methylcytosine by DNA methyltransferase (DNMT) enzymes. where regions of heavily methylated DNA are interspersed with nonmethylated regions. invertebrate animals generally have a "mosaic" pattern of methylation. such as fission yeast. copying the methylation from an existing DNA strand to its new partner after replication. or certain stretches (approximately 1 kilobase in length) where high CpG contents are found. .3 μM cytidine (control) 0 100% 0. The whole silencing mechanism invoking DNMTs could be a way for these organisms to defend themselves against viral infections. only repetitive DNA sequences are methylated. The altered cytosine residues are usually immediately adjacent to a guanine nucleotide. In mammals. Plantae are the most highly methylated eukaryotes.141 33% This straightforward experiment demonstrated that it was not the removal of cytidine residues alone that resulted in changes in cell differentiation (because Ara-C did not have an impact on differentiation). or it occurs on the DNA of transposable elements in the genome. mammals tend to have fairly globally distributed CpG methylation patterns. researchers know that DNA methylation occurs at the cytosine bases of eukaryotic DNA. Interestingly. with up to 50% of their cytosine residues exhibiting methylation. among Animalia. which could generate transposon-like sequences. methylation is found sparsely but globally. researchers have determined that some of these DNMTs are part of chromatin-remodeling complexes and serve to complete the remodeling process by performing on-the-spot DNA methylation to lock the closed shape of the chromatin in place. methylation is absent altogether. or as maintenance DNMTs. The methylation of these sequences can lead to inappropriate gene silencing. resulting in two methylated cytosine residues sitting diagonally to each other on opposing DNA strands. indicating that there is a recognition system in place. 2008). such as the centromeric DNA and the telomeric repeats at the chromosome ends. the mechanism by which de novo DNMT enzymes are directed to the sites that they are meant to silence is not well understood. but the targets include structural sequences of the chromosomes. although replicating them can still be a burden (Suzuki & Bird. Methylation can be observed by staining cells with an immunofluorescently labeled antibody for 5-methylcytosine. These experiments opened the door for investigators to better understand exactly how methylation impacts gene expression and cellular differentiation. Such sequences can do less harm to the organism if they are prevented from being expressed.

2005). however. 2008). stable conversion. results of immunoprecipitation studies using human cells suggest that DNA methylation and histone methylation work together during replication to ensure that specific methylation patterns are passed on to progeny cells (Sarraf & Stancheva. there are sequences in their genomes that are homologous to those that code for the DNMT enzymes). methylation has observed to play a role in mediating gene expression. there are significant differences in overall and specific methylation levels between different tissue types and between normal cells and cancer cells from the same tissue. when silencing of imprinted genes must be reversed. methylation of DNA is a fairly long-term.The Role of Methylation in Gene Expression For many years. For instance. Likewise.. In fact. However. . 2004). it appears that this process may be mediated by the removal of amino groups by DNA deaminases (Morgan et al. 2001) and Arabidopsis thaliana (Jackson et al." The exact mechanisms for demethylation are not entirely understood. After deamination. several studies have indicated that DNA methylation and histone methylation at certain positions are connected. 2008). causing it to become demethylated. while overall methylation levels and completeness of methylation of particular promoters are similar in individual humans. for example. proteins that bind to methylated DNA also form complexes with the proteins involved in deacetylation of histones. 2004). the DNA has a mismatch and is repaired. Also. the exact role of methylation in gene expression is unknown. such as the yeast Saccharomyces cerevisiae and the nematode worm Caenorhabditis elegans. resulting in compounded inhibitory effects on transcription. perhaps by blocking the promoters at which activating transcription factors should bind. Researchers have also determined that mice that lack a particular DNMT have reduced methylation levels and die early in development (Suzuki & Bird. Moreover. Evidence of this has been found in studies that show that methylation near gene promoters varies considerably depending on cell type. are thought to have no methylated DNA at all (although. allowing them to remain acetylated and more mobile. patterns of histone methylation can change rapidly during the course of the cell cycle. some organisms. but it appears that proper DNA methylation is essential for cell differentiation and embryonic development. Despite this difference. H3-K9 methylation (methylation of a specific lysine residue in the histone H3) is required in order for DNA methylation to take place. with more methylation of promoters correlating with low or no transcription (Suzuki & Bird. DNA Methylation and Histones Although patterns of DNA methylation appear to be relatively stable in somatic cells. Presently. thus promoting transcription. 2002). such as in germ cells. evidence has been presented that in some organisms. H3 methylation was reduced at a tumor suppressor gene in cells deficient in DNA methyltransferase (Martin & Zhang. nearby histones are deacetylated. in some cases. In most cases. This is not the case for all eukaryotes. but also in the removal of amino groups. demethylated DNA does not attract deacetylating enzymes to the histones. Therefore. demethylation can take place to allow for "epigenetic reprogramming. but in some cases. In an interestingly coordinated process. In one study. studies using inhibitors of one DNMT enzyme showed that this enzyme was involved in not only DNA methylation. exceptions have been observed in which the relationship is reversed. Indeed. such as Neurospora crassa (Tamaru & Selker. when DNA is methylated. at least in yeast.. however. methylation was believed to play a crucial role in repressing gene expression.

In tumor cells. hypermethylation is detectable early and might serve as a biomarker for the disease. and it can result in the transcriptional silencing of growth-regulatory genes. as well as for understanding and preventing complications that can arise during embryonic development due to abnormalities in X-chromosome methylation and gene imprinting. tumor cell invasion. hypermethylated pericentromeric heterochromatin and an actively transcribed tumor suppressor gene (TSG) associated with a hypomethylated CpG island (indicated in red). including various diseases.. The results of these studies will be invaluable for treating these disorders. Tumor suppressor genes are often silenced in cancer cells due to hypermethylation. medical scientists are currently studying the connections between methylation abnormalities and diseases such as cancer. 597-561 . Nature Reviews Genetics 6. it seems obvious that errors in methylation could give rise to a number of devastating consequences.DNA Methylation and Disease Given the critical role of DNA methylation in gene expression and cell differentiation. (Reproduced from Robertson. and this contributes to genomic instability (a hallmark of tumor cells) through increased mitotic recombination events. Figure 1: DNA methylation and cancer. K. and a range of birth defects that appear to be caused by defective imprinting mechanisms (Robertson. DNA methylation and human disease. The region contains repeat-rich. DNA repair. a large amount of research on DNA methylation and disease has focused on cancer and tumor suppressor genes. the genomes of cancer cells have been shown to be hypomethylated overall when compared to normal cells. In fact. with the exception of hypermethylation events at genes involved in cell cycle regulation. In contrast. Robertson. To date. such as that of the colon. These changes in methylation are early events in tumorigenesis. lupus. in certain cancers. Robertson. 2005). This diagram shows a representative region of genomic DNA in a normal cell. De novo methylation of CpG islands also occurs in cancer cells. and others events in which silencing propagates metastasis (Figure 1. Indeed. 2005).) Copyright 2005 Nature Publishing Group. 2005. muscular dystrophy. repeat-rich heterochromatin becomes hypomethylated.

Activation-induced cytidine deaminase deaminates 5-methylcytosine in DNA and is expressed in pluripotent tissues. 419–420 (1979) (link to article) Morgan. 556–560 (2002) doi:10.2004. H. J. & Selker. 597–610 (2005) doi:10. Nature Reviews Genetics 6. M.043 Suzuki. J. & Bird. For instance.1038/nrm1761 (link to article) McGhee. C. when. M.1038/nrg1655 (link to article) Sarraf. Control of CpNpG DNA methylation by the kryptonite histone H3 methyltransferase. 838–849 (2005) doi:10.. as with other topics in the field of epigenetics. Cell 20. A.molcel. Molecular Cell 15. & Zhang.. 595– 605 (2004) doi:10. A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. and DNA methylation. Nature 414. DNA methylation landscapes: Provocative insights from epigenomics. D. 277–283 (2001) doi:10. Nature Reviews Molecular Cell Biology 6. S. I. Nature Reviews Genetics 9. As new laboratory techniques are developed and additional genomes are mapped. 465–476 (2008) doi:10. et al. Nature 416. A.1016/j. Y. E.. DNA methylation has been shown to play a vital role in numerous cellular processes. gaps remain in our knowledge of DNA methylation. S.06. Specific DNA methylation sites in the vicinity of the chicken beta-globin genes. DNA methylation and human disease.1038/35104508 (link to article) . and for what purposes. The diverse functions of histone lysine methylation.. and they have also determined that this process tends to occur at certain locations within the genomes of different species. References and Recommended Reading Jackson.. scientists will no doubt continue to uncover many of the unknowns of how.1038/nrg2341 (link to article) Tamaru. and where DNA is methylated. & Taylor. et al. P. D. Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly. scientists now know that methylation plays a critical role in the regulation of gene expression. cytidine analogs.1074/jbc. 52353–52360 (2004) doi:10. and abnormal patterns of methylation have been liked to several human diseases. 85–93 (1980) Martin. G. Cellular differentiation.. Nature 280. researchers have discovered numerous details about the process of DNA methylation... & Stancheva. H.Summary Within the past thirty years. Journal of Biological Chemistry 279. K. Nonetheless. & Ginder.M407695200 Robertson.1038/nature731 (link to article) Jones. Furthermore.

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