ScienceWeek

MEDICAL BIOLOGY: GENE SILENCING AND CANCER

The following points are made by J.G. Herman and S.B. Baylin (New Engl. J. Med. 2003 349:2042):

1) It is abundantly clear that mutations, whether inherited through the germ line or, more commonly, arising in somatic tissues later in life,(1) can cause cancer. These mutations abnormally enhance the function of some genes, the oncogenes, or cause other genes, the tumor-suppressor genes, to lose function.(1) Students of the neoplastic process, however, have argued for decades about whether the initiation and progression of cancer are due only to mutations or, as well, to epigenetic changes that are not caused by alterations in the primary nucleotide sequence of DNA. Recent investigations have proved not only that both views are correct, but also that the two processes are intricately connected in driving the development of tumors from the earliest to the latest stages.

2) The term "epigenetic" refers to a heritable change in the pattern of gene expression that is mediated by mechanisms other than alterations in the primary nucleotide sequence of a gene.(2,3) As the evidence for genetic changes in cancer cells increased during the 1980s and early 1990s, interest in the contribution of epigenetic changes to neoplasia waned. The situation has changed dramatically in more recent years, however, because of convincing evidence of abnormal silencing of genes in cancer cells.(4,5) This change in gene expression involves the methylation of DNA in promoter regions of genes, the sites where transcription of DNA into RNA begins.

3) Transcription is the first major step in decoding DNA into a protein. An important aspect of the methylation mechanism is that it inactivates tumor-suppressor genes.(4,5) That DNA methylation could be an epigenetic mechanism of carcinogenesis was predicted years ago, but molecular evidence supporting the concept has emerged only recently. Current efforts to understand the mechanisms of gene silencing, and to find ways of exploiting the diagnostic and therapeutic implications of the abnormality, dovetail with, benefit from, and contribute to the explosion of investigations into the control of gene expression, one of the most vibrant areas of biologic research.(2)

4) Although only four bases -- adenine, guanine, cytosine, and thymine -- spell out the primary sequence of DNA, there is a covalent modification of postreplicative DNA (i.e., DNA that has replicated itself in a dividing cell) that produces a "fifth base". Reactions using S-adenosyl-methionine as a methyl donor and catalyzed by enzymes called DNA methyltransferases (DNMTs) add a methyl group to the cytosine ring to form methyl cytosine. In humans and other mammals, this modification is imposed only on cytosines that precede a guanosine in the DNA sequence (the CpG dinucleotide). The overall frequency of CpGs in the genome is substantially less than what would be mathematically predicted, probably because DNA methylation has progressively depleted the genome of CpG dinucleotides over the course of time. The mechanism of the depletion is related to the propensity of methylated cytosine to deaminate, thereby forming thymidine. If this mutation is not repaired, a cytosine-to-thymidine change remains. The depletion of CpG dinucleotides in the genome corresponds directly to sites of such nucleotide transitions, and this change is the most common type of genetic polymorphism (variation) in human populations.

References (abridged):

1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70

2. Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002;16:6-21

3. Russo VEA, Martienssen RA, Riggs AD, eds. Epigenetic mechanisms of gene regulation. Plainview, N.Y.: Cold Spring Harbor Laboratory Press, 1996.

4. Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet 1999;21:163-167

5. Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet

New Engl. J. Med. http://www.nejm.org

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DNA METHYLATION IN MAMMALIAN EPIGENETICS

The following points are made by P.A. Jones and D. Takai (Science 2001 293:1063):

1) DNA methylation is essential for the development of mammals (1,2), but despite 25 years of work, researchers still do not know exactly why. Recent advances have led to the cloning and preliminary characterization of the three known active DNA cytosine methyltransferases (DNMT1, -3a, and -3b) (3,4) and to a greater understanding of how the methylation signal is interpreted in mammalian cells.

2) The post-synthetic addition of methyl groups to the 5-position of cytosines alters the appearance of the major groove of DNA to which the DNA binding proteins bind. These epigenetic "markers" on DNA can be copied after DNA synthesis, resulting in heritable changes in chromatin structure. Methylation of CpG-rich promoters is used by mammals to prevent transcriptional initiation and to ensure the silencing of genes on the inactive X chromosome, imprinted genes, and parasitic DNAs. The potential role of methylation in tissue-specific gene expression or in the regulation of CpG-poor promoters is less well established. There is also tantalizing evidence that normal chromosome structure may be affected by methylation and that human diseases, including cancer, are caused and impacted by abnormal methylation.

3) CpG dinucleotides, the sites of almost all methylation in mammals, are underrepresented in DNA. Clusters of CpGs, called "CpG islands", are often found in association with genes, most often in the promoters and first exons but also in regions more toward the 3' end (5). The exact definition of a CpG island is evolving. The original suggestion by Gardiner-Garden and Frommer (1987) of a region greater than 200 base pairs (bp) with a high-GC content and an observed/expected ratio for the occurrence of CpG > 0.6, should probably be modified to slightly higher stringency in terms of length and GC content, thus excluding a substantial number of small exonic regions and repetitive parasitic DNAs. The salient property of a CpG island is that it is unmethylated in the germline (and indeed in most somatic tissues), thus ensuring its continued existence in the face of the strong mutagenic pressure of 5-methylcytosine deamination.

4) CpG islands often function as strong promoters and have also been proposed to function as replication origins. Even though these islands are generally not methylated, most investigations on the role of DNA methylation in mammals have focused on CpG islands rather than on the regions in which the majority of methylation is found.

5) In summary: Genes constitute only a small proportion of the total mammalian genome, and the precise control of their expression in the presence of an overwhelming background of noncoding DNA presents a substantial problem for their regulation. Noncoding DNA, containing introns, repetitive elements, and potentially active transposable elements, requires effective mechanisms for its long-term silencing. Mammals appear to have taken advantage of the possibilities afforded by cytosine methylation to provide a heritable mechanism for altering DNA-protein interactions to assist in such silencing. Genes can be transcribed from methylation-free promoters even though adjacent transcribed and nontranscribed regions are extensively methylated. Gene promoters can be used and regulated while keeping noncoding DNA, including transposable elements, suppressed. Methylation is also used for long-term epigenetic silencing of X-linked and imprinted genes and can either increase or decrease the level of transcription, depending on whether the methylation inactivates a positive or negative regulatory element.

References (abridged):

1. E. Li, T. H. Bestor, R. Jaenisch, Cell 69, 915 (1992)

2. M. Okano, D. W. Bell, D. A. Haber, E. Li, Cell 99, 247 (1999)

3. T. Bestor, A. Laudano, R. Mattaliano, V. Ingram, J. Mol. Biol. 203, 971 (1988)

4. M. Okano, S. Xie, E. Li, Nature Genet. 19, 219 (1998)

5. F. Larsen, G. Gundersen, R. Lopez, H. Prydz, Genomics 13, 1095 (1992)

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