DNA Methylation

Approaches, Methods, and Applications

Manel Esteller

• Publisher: CRC Press
• Year: 2004
• ISBN:
  9781135491482
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Molecular Biology
• Index: Biological Sciences

DNA Methylation: Approaches, Methods and Applications describes the relation DNA methylation has to gene silencing in disease, and explores its promising role in treating cancer. Written by leaders in the field, this exceptional compilation of articles outlines the best techniques to use when addressing questions concerning the cytosine methylation

1

Impact of DNA Methylation on Health and Disease

Manel Esteller

THE RELEVANCE OF DNA METHYLATION TO HUMAN BIOLOGY, CLINICOPATHOLOGICAL SYNDROMES, AND EXPERIMENTAL MODELS

DNA METHYLATION IN PHYSIOLOGICAL CONDITIONS

We can lump within the scope of the enigmatic word of “epigenetics” all the heritable changes in gene expression patterns that are based on factors other than straightforward DNA sequences. The mechanisms controlling epigenetics are complex and we have only just begun to get our first glimpses of their nature. For example, chromatin structure, controlled by the patterns of acetylation and methylation of the histone proteins around the regulatory regions of genes (Jenuwein and Allis, 2001), is one critical layer of epigenetics [1]. However, at a deeper level still, the most “genetic” of all epigenetic modifications is DNA methylation [2].

In humans, the vast majority of DNA methylation occurs in the cytosine of the CpG dinucleotides. We need certain levels of methylcytosine in our genomes to be considered normal human beings. Endoparasitic sequences such as Alu elements or LINEs (long interspersed nuclear elements) refrain from jumping around thanks to the repression of DNA methylation; we keep our parental marks in our imprinted genes thanks to DNA methylation; and the need to silence one of each pair of X chromosomes in women is met by DNA methylation. At the same time, the distribution of the dinucleotide CpG in our genomes is not random. Most ubiquitously expressed genes have high concentrations of CpGs in their promoter-regulatory regions, from where the RNA transcript of the gene originates. These regions are called CpG islands. In a normal cell, they are unmethylated, and the gene is expressed if the required transcription factors are present.

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DNA METHYLATION IN CANCER

The perfect epigenetic equilibrium of DNA methylation previously described in the normal cell is dramatically transformed in the cancer cell. The DNA methylation aberrations observed can be considered as falling into one of two categories: transcriptional silencing of tumor suppressor genes by CpG island promoter hypermethylation and a massive global genomic hypomethylation. Let us briefly examine these two features.

Hypermethylation of Tumor Suppressor Genes

In human tumors, some CpG islands become hypermethylated with the result that the expression of the contiguous gene is shut down. If this aberration affects a tumor suppressor gene, it confers a selective advantage on that cell and is selected generation after generation. We and other researchers have contributed to the identification of a long list of hypermethylated genes in human neoplasias [3, 4], and this epigenetic alteration is now considered to be a common hallmark of all human cancers affecting all cellular pathways [5–7]. Extremely important genes in cancer biology, such as the cell-cycle inhibitor p16^INK4a; the p53-regulator p14^ARF; the DNA-repair genes hMLH1, BRCA1, and MGMT; the cell-adherence gene E-cadherin; and the estrogen and retinoid receptors, undergo methylation-associated silencing in cancer cells [6].

The profiles of CpG island hypermethylation are known to depend on the tumor type [3, 4]. Each tumor subtype can now be assigned a CpG island hypermethylation profile (methylotype) that almost completely defines that particular malignancy in a similar fashion, as do genetic and cytogenetic markers. Establishing a DNA methy1-fingerprint can be very useful for classifying these malignancies according to their aggressiveness or sensitivities to chemotherapy. Single-gene approaches can also be extremely useful. In gliomas, B-diffuse large cell lymphomas for example, we have demonstrated that hypermethylation of the DNA repair gene MGMT confers a good response to the chemotherapy regimens that include the alkylating drugs BCNU and cyclophosphamide [8–10].

Using DNA Hypermethylation in Cancer Management

DNA methylation can be exploited on three translational fronts for clinical purposes in cancer patients.

1. New lines of treatment based on DNA demethylation agents that reverse the CpG island hypermethylation of tumor suppressor genes. Unlike genetic changes in cancer, epigenetic changes are potentially reversible. For years, in cultured cancer cell lines, we have been able to reexpress genes that had been silenced by methylation by using demethylating agents such as 5-aza-2-deoxycytidine [11]. These compounds had previously been used in the clinic, but the doses administered at those times were quite toxic. Interestingly, we can reduce the doses by adding inhibitors of histone deacetylases, such as phenylbutyrate. Several Phase I and II clinical trials are underway to test this strategy. Chapters 12–14 are excellent guides to understanding this complex area.
2. Methylation as a molecular biomarker of cancer cells. The presence of CpG island hypermethylation of the tumor suppressor genes described is specific to transformed cells [5–7] and there is a particular profile of methylation for each tumor type [3, 12]. Methylation can, therefore, be used as an indicator for the presence of a particular malignancy. One of the best-accepted cases is the presence of hypermethylation of the glu-tathione-S-transferase P1 (GSTP1) gene in prostate cancer, as summarized in Chapter 2. Hypermethylation could also be used as a tool for detecting cancer cells in multiple biological fluids [2] or for monitoring hyperme-thylated promoter loci in serum DNA from cancer patients [13]. Chapters 2 and 5 provide useful information on this matter.
3. Gene promoter hypermethylation as a prognostic/predictive factor. Methylation is not only a positive marker, but also a qualitative one. We have recently provided compelling evidence about its strength: the methylation-associated silencing of MGMT (the DNA repair gene) in tumors indicates which patients will be sensitive to chemotherapy with certain alkylating agents [10]. Similar scenarios can now be outlined using the methylation status of hormone and growth factor receptor genes, and those encoding for DNA repair proteins, for many tumor types. The involvement of industry in developing some of these uses in a standardized manner is essential, and it is encouraging that some companies, such as Oncometh-ylome Sciences, are rising to the challenge.

Global Genomic Hypomethylation

At the same time the CpG islands become hypermethylated, the genome of the cancer cell undergoes global hypomethylation. The malignant cell can have 20 to 60% less genomic 5mC (5-methylcytosine) than its normal counterpart [14, 15]. The loss of methyl groups is accomplished mainly by hypomethylation of the “body” (coding region and introns) of genes, and through demethylation of repetitive DNA sequences, which account for 20 to 30% of the human genome. How does global DNA hypomethylation contribute to carcinogenesis? Three mechanisms can be invoked: chromosomal instability, reactivation of transposable elements, and loss of imprinting. Undermethylation of DNA might favor mitotic recombination, leading to loss of heterozygosity, as well as promoting karyotypically detectable rearrangements. Additionally, extensive demethylation in centromeric sequences is common in human tumors and may play a role in aneuploidy. As evidence of this, patients with germline mutations in DNA methyltransferase 3b (DNMT3b) are known to have numerous chromosome aberrations [16]. Hypomethylation of malignant cell DNA can also reactivate intragenomic parasitic DNA, such as L1 (LINE), and Alu (recombinogenic sequence) repeats [17]. These, and other previously silent transposons, may now be transcribed and even “moved” to other genomic regions, where they can disrupt normal cellular genes. The loss of methyl groups can affect imprinted genes and genes from the methylated X chromosome of women. The beststudied case is of the effects of the H19/IGF-2 locus on chromosome 11p15 in certain childhood tumors [18]. Chapter 3 reflects the relevance of DNA methylation to the control of gene imprinting and X inactivation.

In summary, the disruption of DNA methylation patterns is a major hallmark of cancer. Much is still unknown, but the unfolding scenario shows great promise for a better understanding of cancer biology and for improvement in the management of human tumors.

DNA METHYLATION IN IMMUNOLOGY

DNA methylation also occupies a place at the crossroads of many pathways in immunology, providing us with a clearer understanding of the molecular network of the immune system.

From the classical genetic standpoint, two immunodeficiency syndromes, the ICF (immunodeficiency, centromeric regions instability, facial anomalies) syndrome and ATR-X (X-linked form of syndromal retardation associated with alpha-thalassemia) syndrome, are caused by germline mutations in two epigenetic genes: the DNMT3b and the ATRX genes [19, 20]. DNMT3b is the putative de novo DNA methyltransferase (DNMT1 would be the maintenance DNA methyltransferase and DNMT3a the other de novo methyltransferase). In the rare ICF syndrome, characterized by DNA hypomethylation and chromosomal aberration at certain satellite regions, some lymphogenesis genes are expressed in a deregulated fashion [19]. On the other hand, the ATRX gene is a chromatin-remodeling gene that, when mutated, also causes DNA methylation changes, thereby revealing the intimate relationship between chromatin and methylation [20].

Autoimmunity and DNA methylation can also go hand in hand. Classical autoimmune diseases, such as systemic lupus erythematosus or rheumatoid arthritis, are characterized by massive genomic hypomethylation [21, 22]. This phenomenon is highly reminiscent of the global demethylation observed in the DNA of cancer cells compared with their normal-tissue counterparts [2, 15]. Other examples include the proposed epigenetic control of the histo-blood group ABO genes [23] and the silencing of human leukocyte antigen (HLA) class I antigens [24]. The melanoma antigen gene (MAGE) family of genes provides another illustrative example of immune response mediated by DNA methylation. These genes are not expressed in normal cells because their CpG islands are hypermethylated (contrary to the dogma). However, certain processes, such as malignant transformation, demethylate the island, causing the genes to be reexpressed, with the result that their products are recognized as tumor-specific antigens by cytolytic T lymphocytes [25].

DNA METHYLATION IN NEUROSCIENCES, CARDIOVASCULAR RESEARCH, METABOLIC DISEASES, IMPRINTING DISORDERS, DEVELOPMENT, AND CLONING

Aberrant DNA methylation patterns go beyond the fields of oncology and immunology to touch a wide range of biomedical and scientific knowledge. In neurology and autism research, for example, it was surprising to discover that germline mutations in the methyl-binding protein MeCP2 (a key element in the silencing of gene expression mediated by DNA methylation) causes the common neurodevelopmental disease known as Rett syndrome [26, 27]. This leads us to wonder how many DNA methylation alterations underlie other, more prevalent neurological pathologies, such as schizophrenia or Alzheimer’s disease. Beyond that, DNA methylation changes are also known to be involved in cardiovascular disease, the biggest killer in Western countries. For example, aberrant CpG island hypermethylation has been described in athe...

Table of contents

• Cover Page
• Title Page
• Copyright Page
• Preface
• Editor
• Contributors
• 1
• 2
• 3
• 4
• 5
• 6
• 7
• 8
• 9
• 10
• 11
• 12
• 13
• 14