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com/scitable/topicpage/The-Role-of-Methylation-in-Gene-Expression1070
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
Methylation
3 M cytidine (control) 0 100% 0.3 M Ara-C 0 127% 3 M 5-azacytidine 22,141 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); rather, only those analogs that impacted methylation resulted in such changes. These experiments opened the door for investigators to better understand exactly how methylation impacts gene expression and cellular differentiation.
Figure 1: DNA methylation and cancer. This diagram shows a representative region of genomic DNA in a normal cell. The region contains repeat-rich, hypermethylated pericentromeric heterochromatin and an actively transcribed tumor suppressor gene (TSG) associated with a hypomethylated CpG island (indicated in red). In tumor cells, repeat-rich heterochromatin becomes hypomethylated, and this contributes to genomic instability (a hallmark of tumor cells) through increased mitotic recombination events. De novo methylation of CpG islands also occurs in cancer cells, and it can result in the transcriptional silencing of growth-regulatory genes. These changes in methylation are early events in tumorigenesis. (Reproduced from Robertson, 2005.) Copyright 2005 Nature Publishing Group, Robertson, K., DNA methylation and human disease, Nature Reviews Genetics 6, 597-561
Summary
Within the past thirty years, researchers have discovered numerous details about the process of DNA methylation. For instance, scientists now know that methylation plays a critical role in the regulation of gene expression, and they have also determined that this process tends to occur at certain locations within the genomes of different species. Furthermore, DNA methylation has been shown to play a vital role in numerous cellular processes, and abnormal patterns of methylation have been liked to several human diseases. Nonetheless, as with other topics in the field of epigenetics, gaps remain in our knowledge of DNA methylation. As new laboratory techniques are developed and additional genomes are mapped, scientists will no doubt continue to uncover many of the unknowns of how, when, and where DNA is methylated, and for what purposes.
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