Nutrition in Epigenetics E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 872

Veröffentlichungsjahr: 2011

Beschreibung

The study of epigenetics, or how heritable changes in gene expression are regulated without modifying the coding DNA sequence, has become an increasingly important field of study in recent years. Rapid developments in our understanding of the way in which gene function is modulated by the environment has revolutionized the way we think about human development and health. Nutrition in Epigenetics reviews the latest research looking at the interaction between genes and nutrients and the role they play together in maintaining human health. Nutrition in Epigenetics is divided into two primary parts. The first part provides key principles such as epigenetic mechanisms, developmental epigenetics, and the role of epigenetics in disease. The second part looks specifically at the application of epigenetics to the field of human nutrition. Chapters review the role of specific nutrients in modulating epigenetic status and the effect on health and disease. Nutrition in Epigenetics is an indispensable resource for researchers, professionals and advanced students with an interest in human nutrition, epigenetics, and biomedical research.

Leseprobe

Contents

Cover

Title Page

Copyright

Contributors

Chapter 1: Introduction

1.1. ADAPTATION, AN EVOLVING CONCEPT

1.2. EPIGENETIC MECHANISMS AND THEIR ROLES

1.3. NUTRITION, EPIGENETICS, AND HEALTH

1.4. CONCLUSION

ACKNOWLEDGMENTS

REFERENCES

Part I: Fundamental Principles in Epigenetics

Section A: Epigenetic Mechanisms

Chapter 2: DNA Methylation

2.1. INTRODUCTION

2.2. MOLECULAR MECHANISMS OF DNA METHYLATION

2.3. DNA METHYLATION PATTERNS, PLURICELLULARITY, AND CELL MEMORY

2.4. THE ROLES OF DNA METHYLATION

2.5. ESTABLISHMENT, MAINTENANCE, AND REPROGRAMMING OF DNA METHYLATION PATTERNS

2.6. CONCLUSIONS

ACKNOWLEDGMENTS

REFERENCES

Chapter 3: Chromatin Modifications

3.1. INTRODUCTION ON CHROMATIN STRUCTURE

3.2. POSTTRANSLATIONAL HISTONE MODIFICATIONS

3.3. HISTONE VARIANTS

3.4. CHROMATIN REMODELING

3.5. CONCLUSIONS AND FUTURE PERSPECTIVES

ACKNOWLEDGMENTS

REFERENCES

Chapter 4: Roles of RNAi and Other Micro-RNAs in the Regulation of Epigenetic Processes

4.1. INTRODUCTION

4.2. BIOGENESIS AND PHYSIOLOGY OF siRNAs AND miRNAs

4.3. siRNA-DEPENDENT EPIGENETIC MODIFICATIONS

4.4. miRNAs AND EPIGENETIC REGULATION

4.5. CONCLUSION

REFERENCES

Chapter 5: Epigenetic Inheritance: Both Mitotic and Meiotic

5.1. INTRODUCTION

5.2. MITOTIC EPIGENETIC INHERITANCE

5.3. MEIOTIC EPIGENETIC INHERITANCE

5.4. TRANSGENERATIONAL EPIGENETIC EFFECTS

5.5. CONCLUSION

REFERENCES

Section B: Development Epigenetics

Chapter 6: Developmental Epigenetics: Roles in Embryonic Development

6.1. INTRODUCTION

6.2. GAMETOGENESIS IS A COMPLEX MATURATION PROCESS

6.3. TESTIS-SPECIFIC EPIGENETIC CODE

6.4. FROM GAMETES THROUGH FERTILIZATION TO THE EARLY EMBRYO

6.5. LIFELONG EPIGENETIC REPROGRAMMING AND DIET

6.6. CONCLUSION

REFERENCES

Section C: Epigenetic Mechanisms in Disease

Chapter 7: Epigenetics, Nutrition, and Cancer

7.1. INTRODUCTION

7.2. CELLULAR EPIGENETIC CHANGES AND CANCER

7.3. EPIGENETIC-BASED THERAPEUTIC STRATEGIES FOR CANCER

7.4. DIETARY METHYL GROUP INTAKE AND CANCER

7.5. CONCLUSION

REFERENCES

Chapter 8: Metabolic Syndrome, Obesity, and Diabetes

8.1. INTRODUCTION

8.2. METABOLIC SYNDROME

8.3. OBESITY

8.4. TYPE 2 DIABETES

8.5. CONCLUSIONS AND FUTURE DIRECTIONS

REFERENCES

Chapter 9: Autoimmunity

9.1. INTRODUCTION

9.2. ENVIRONMENTAL AGENTS, EPIGENETICS, AND AUTOIMMUNITY

9.3. EPIGENETICS IN DIFFERENT AUTOIMMUNE DISEASES

9.4. DIET, DNA METHYLATION, AND IMMUNE-MEDIATED DISEASES

9.5. CONCLUSIONS

REFERENCES

Chapter 10: Cardiovascular Diseases

10.1. INTRODUCTION

10.2. EPIGENETIC THEORY OF CARDIOVASCULAR RISK FACTORS

10.3. ROLE OF EPIGENETIC MODIFIERS IN THE PATHOGENESIS OF CARDIOVASCULAR DISEASES

10.4. CARDIOVASCULAR GENES REGULATED BY EPIGENETIC MECHANISMS

10.5. POTENTIAL OF EPIGENETIC THERAPY IN THE CONTROL OF CARDIOVASCULAR DISEASES

10.6. CONCLUSION

REFERENCES

Part II: Nutritional Status and Specific Nutrients

Section A: Maternal Nutrition and Fetal Development

Chapter 11: Maternal Nutrition and Developmental Outcomes

11.1. INTRODUCTION

11.2. EPIGENETICS PRIMER

11.3. EPIGENETICS AND EARLY DEVELOPMENT

11.4. EPIGENETICS AND THE AGOUTI MOUSE STORY

11.5. EPIGENETICS AND PROGRAMMING

11.6. IMPORTANCE OF EPIGENETICS AND ADULT HEALTH

11.7. EPIDEMIOLOGICAL EVIDENCE OF FETAL PROGRAMMING FROM MATERNAL DIET

11.8. EPIDEMIOLOGICAL EVIDENCE AND TIMING OF MALNUTRITION

11.9. ANIMAL MODEL STUDIES ON MATERNAL NUTRITION

11.10. OBESITY AND DYSLIPIDEMIA

11.11. IMPAIRED GLUCOSE HOMEOSTASIS AND INSULIN RESISTANCE

11.12. HYPERTENSION AND CARDIOVASCULAR DISEASE

11.13. SUMMARY AND FUTURE

ACKNOWLEDGMENTS

REFERENCES

Section B: Role of Specific Nutrients

Chapter 12: Folate, Vitamin B12, and Vitamin B6

12.1. INTRODUCTION

12.2. B VITAMINS AND EPIGENETICS

12.3. THE EFFECTS OF B VITAMINS ON THE CELLULAR METHYLATION POTENTIAL

12.4. MECHANISMS UNDERLYING EPIGENETIC PROGRAMMING BY ONE-CARBON METABOLISM

12.5. REGULATION OF METHYLTRANSFERASE EXPRESSION BY ONE-CARBON METABOLISM

12.6. KNOWLEDGE GAPS AND AREAS FOR FUTURE RESEARCH

ACKNOWLEDGMENT

REFERENCES

Chapter 13: Dietary Choline, Betaine, Methionine, and Epigenetic Mechanisms Influencing Brain Development

13.1. INTRODUCTION

13.2. CHOLINE, BETAINE, AND METHIONINE METABOLISM

13.3. DIETARY SOURCES OF CHOLINE, BETAINE, AND METHIONINE

13.4. EPIGENETICS

13.5. CHOLINE, BETAINE, METHIONINE, AND EPIGENETIC MARKS

13.6. SINGLE NUCLEOTIDE POLYMORPHISMS AND DIETARY REQUIREMENT FOR METHYL GROUPS

13.7. CONCLUSIONS

ACKNOWLEDGMENTS

REFERENCES

Chapter 14: Epigenetic Regulation by Retinoids

14.1. INTRODUCTION

14.2. RETINOIDS AND EPIGENETIC MECHANISMS

14.3. CONCLUSIONS, OTHER PERSPECTIVES, AND QUESTIONS

REFERENCES

Chapter 15: We Are What We Eat: How Nutritional Compounds Such As Isoflavones Shape Our Epigenome

15.1. INTRODUCTION

15.2. EPIGENETIC MECHANISMS OF GENE REGULATION

15.3. NUTRITION AND THE ENZYMATIC PROCESS OF DNA METHYLATION

15.4. DIETARY FLAVONOIDS AND EPIGENETIC CHANGES

15.5. A PROPOSED MECHANISM FOR ENDOCRINE-MEDIATED FLAVONOID ACTION ON EARLY MAMMALIAN EMBRYOS

15.6. EPIGENETICS AND NUTRITIONAL EPIDEMIOLOGIC STUDIES

15.7. DNA METHYLATION AND DISEASES

15.8. CONCLUDING REMARKS

ACKNOWLEDGMENTS

REFERENCES

Chapter 16: Isothiocyanates and Polyphenols

16.1. INTRODUCTION

16.2. HISTONE DEACETYLASE ACTIVITY OF DIALLYL DISULFIDE

16.3. HISTONE DEACETYLASE ACTIVITY OF SULFORAPHANE AND ITS METABOLITES

16.4. HDAC ACTIVITY OF OTHER ISOTHIOCYANATES

16.5. HDAC ACTIVITY OF PHYTOESTROGENS

16.6. PHYTOCHEMICALS AND CELL SIGNALING

16.7. CONCLUSION

REFERENCES

Section C: Macronutrient Intakes

Chapter 17: The Effect of Maternal Macronutrient Intake on Phenotype Induction and Epigenetic Gene Regulation

17.1. INTRODUCTION

17.2. MATERNAL NUTRITION AND PHENOTYPE INDUCTION

17.3. PHENOTYPE INDUCTION AND ALTERED TRANSCRIPTION

17.4. EPIGENETIC MECHANISMS AND REGULATION OF TRANSCRIPTION

17.5. ENVIRONMENTAL CHALLENGE IN EARLY LIFE AND EPIGENETIC REGULATION OF IMPRINTED GENES

17.6. PRENATAL NUTRITION AND ALTERED EPIGENETIC GENE REGULATION

17.7. TRANSGENERATIONAL TRANSMISSION OF ALTERED EPIGENETIC GENE REGULATION

17.8. REVERSAL OR PREVENTION OF ALTERED PHENOTYPE AND EPIGENOTYPE

17.9. MECHANISMS FOR INDUCED CHANGES IN THE EPIGENOME

17.10. CONCLUSION

REFERENCES

Section D: Environmental Exposures

Chapter 18: Epigenetic Manifestation of Environmental Exposures

18.1. INTRODUCTION

18.2. EPIGENETIC PHENOMENON SUBJECT TO ENVIRONMENTAL INFLUENCE

18.3. ENVIRONMENTAL EXPOSURES AFFECTING THE EPIGENOME

18.4. FUTURE PERSPECTIVES

ACKNOWLEDGMENTS

REFERENCES

Section E: Epidemiology of Nutritional Epigenetics

Chapter 19: Epigenetics, Nutrition, and Reproduction: Short- and Long-Term Consequences

19.1. EPIGENETICS

19.2. REPRODUCTIVE EPIGENETICS

19.3. DNA METHYLATION DEVELOPMENT

19.4. HUMAN EPIGENETIC VARIATION

19.5. NUTRITIONAL INFLUENCES ON DEVELOPMENTAL EPIGENETICS

19.6. EPIGENETICS AND HUMAN HEALTH

19.7. EPIGENETIC INHERITANCE AND HUMAN EVOLUTION

19.8. CONCLUSIONS

ACKNOWLEDGMENT

REFERENCES

Chapter 20: Nutrition, Epigenetics, and Cancer: An Epidemiological Perspective

20.1. INTRODUCTION

20.2. EPIGENETICS IN CANCER

20.3. NUTRITION AND CANCER

20.4. NUTRITION AND EPIGENETICS

20.5. CONCLUSION

REFERENCES

Index

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Nutrition in epigenetics / edited by Mihai D. Niculescu, Paul Haggarty. p. cm. Includes bibliographical references and index. ISBN 978-0-8138-1605-0 (hardcover : alk. paper) 1. Nutrition–Genetic aspects. 2. Epigenesis. 3. Genetic regulation. 4. Gene expression. I. Niculescu, Mihai D. II. Haggarty, Paul. QP144.G45N888 2011 612.3–dc22 2010047816

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Contributors

Arttu I. AholaDivision of Genetics and Population Health Queensland Institute of Medical Research Queensland Australia; Department of Biological and Environmental Sciences University of Helsinki Finland

Olivia S. AndersonDepartment of Environmental Health Sciences University of Michigan School of Public Health Ann Arbor, MI USA

Methode BacanamwoCardiovascular Research Institute Morehouse School of Medicine Atlanta, GA USA

Nigel J. BelshawInstitute of Food Research Norwich Research Park Colney, Norwich UK

Aurelian BidulescuCardiovascular Research Institute Morehouse School of Medicine Atlanta, GA USA

Graham C. BurdgeDOHaD Research Division University of Southampton Southampton UK

Liliana BurlibaaGenetics Department Bucharest University Romania

Susan J. ClarkEpigenetics Laboratory Cancer Program Garvan Institute of Medical Research Sydney Australia

Karen D. CorbinThe University of North Carolina at Chapel Hill Nutrition Research Institute Chapel Hill, NC USA

Natalia V. CucuEpigenetics Laboratory Department of Genetics Faculty of Biology University of Bucharest Romania

Dana C. DolinoyDepartment of Environmental Health Sciences University of Michigan School of Public Health Ann Arbor, MI USA

Muller FabbriDepartment of Molecular Virology, Immunology, and Medical Genetics The Ohio State University Columbus, OH USA

Lucian GavrilGenetics Department Bucharest University Romania

Carlos M. Guerrero-BosagnaCenter for Reproductive Biology School of Biological Sciences Washington State University Pullman, WA USA

Paul HaggartyNutrition & Epigenetics Group Rowett Institute of Nutrition & Health University of Aberdeen Aberdeen, Scotland UK

Sandra B. HakeAdolf-Butenandt Institute and Center for Integrated Protein Science Munich (CIPSM) Department of Molecular Biology Ludwig-Maximilians University Munich Germany

Mark A. HansonDOHaD Research Division University of Southampton Southampton UK

Shannon L. HaleyDivision of Neonatology University of Utah Salt Lake City, UT USA

Amy R. JohnsonDepartment of Nutrition University of North Carolina at Chapel Hill Chapel Hill, NC USA

Ian T. JohnsonInstitute of Food Research Norwich Research Park Colney, Norwich UK

Lisa A. Joss-MooreDivision of Neonatology University of Utah Salt Lake City, UT USA

Audrey JungDepartment of Epidemiology Biostatistics, and Health Technology Assessment Radboud University Nijmegen Medical Centre Nijmegen The Netherlands

Nina J. Kaminen-AholaDivision of Genetics and Population Health Queensland Institute of Medical Research Queensland Australia

Ellen KampmanDepartment of Epidemiology Biostatistics, and Health Technology Assessment Radboud University Nijmegen Medical Centre Nijmegen The Netherlands; Division of Human Nutrition Wageningen University Wageningen The Netherlands

Robert H. LaneDivision of Neonatology University of Utah Salt Lake City, UT USA

Karen A. LillycropSchool of Biological Sciences University of Southampton Southampton UK

Laurie J. Moyer-MileurDivision of Neonatology University of Utah Salt Lake City, UT USA

Mihai D. NiculescuDepartment of Nutrition and Nutrition Research Institute University of North Carolina at Chapel Hill Kannapolis, NC USA

Donna RayDepartment of Internal Medicine University of Michigan Ann Arbor, MI USA

Laura S. RozekDepartment of Environmental Health Sciences University of Michigan School of Public Health Ann Arbor, MI USA

Patrick J. StoverDivision of Nutritional Sciences Cornell University Ithaca, NY USA

Amandio VieiraNutrition and Metabolic RL Biomedical Physiology & Kinesiology Simon Fraser University Burnaby, BC Canada

Emma WhitelawDivision of Genetics and Population Health Queensland Institute of Medical Research Queensland Australia

Raymond YungDepartment of Internal Medicine University of Michigan Ann Arbor, MI USA

Steven H. ZeiselNutrition Research Institute and Departments of Nutrition and Pediatrics University of North Carolina at Chapel Hill Kannapolis, NC USA

Chapter 1

Introduction

Mihai D. Niculescu1 and Paul Haggarty2

1Department of Nutrition and Nutrition Research Institute, University of North Carolina at Chapel Hill, Kannapolis, NC, USA

2Nutrition & Epigenetics Group, Rowett Institute of Nutrition & Health, University of Aberdeen, Aberdeen, Scotland, UK

1.1. ADAPTATION, AN EVOLVING CONCEPT

The concept of adaptation was, and still is, considered one of the most important principles of biology. Related to the idea of transformation—epigenesis in Aristotle's words—adaptation exists as a means to better cope with environmental changes, whether on a long or a short term. In 1809, Jean-Baptiste Lamarck published his Philosophie zoologique ou exposition des considérations relatives à l’histoire naturelle des animaux, in which he argued that characteristics acquired during the life of an individual (because of exposure to various environmental influences) can be transmitted to the young during reproduction (soft inheritance). Since its publication, and until recently, Lamarck's theory of soft inheritance has been largely disregarded, while Darwin's theory of evolution became predominant in modern biology (Handel and Ramagopalan, 2010).

The resurrection of the soft inheritance concept manifested only recently, when it became obvious that environmental influences could trigger metabolic and phenotypic changes that could be transmitted to subsequent generations, even when such exposures were present only during the life of the first generation (Chmurzynska, 2010). Animal studies indicated that acquired characteristics could be inherited, and that practically any type of environmental changes might initiate such events (maternal nutrition, gestational exposure to endocrine disrupting chemicals, ionizing radiation, etc.) (Youngson and Whitelaw, 2008.

Potential mechanisms for the propagation of such influences from the parent to offspring are many, including poor maternal health (inducing similar phenotypes in the next generation), behavioral interactions (perpetuation of the same phenotype by either similar behavior or endocrine changes that are perpetuated between generations), postfertilization transfer of viruses or toxins, and epigenetic mechanisms (Youngson and Whitelaw, 2008). This book focuses, among other epigenetic mechanisms responsible for shaping gene–nutrient interactions, on epigenetic inheritance, which consists of the transmission of parental epigenetic patterns across the generations, and how nutrition may impact on this.

There are many definitions of epigenetics. Coined by Waddington in 1942 (Waddington, 1942), today epigenetics refers to the study of heritable patterns of gene expression that are not caused by changes in DNA sequence. The heritability of gene expression patterns refers to both cell division and transgenerational inheritance.

The epigenetic interaction between our genes and the environment allows a time-efficient process of adaptation that starts with the embryonic and fetal stages of development.

1.2. EPIGENETIC MECHANISMS AND THEIR ROLES

The epigenetic mechanisms consist of complex interactions between DNA and nuclear proteins (mainly histones), which define the pattern of gene expression in a given cell. These DNA–histone interactions, as well as gene expression, are also influenced by small, noncoding RNA (ncRNA) molecules, which further modulate the pattern of gene expression that defines a specific cellular phenotype (Figure 1.1).

Figure 1.1 Epigenetic processes (DNA methylation, histone modifications, and noncoding RNA involvement) contribute in shaping the gene expression profile and a specific cellular phenotype. Each of the three epigenetic components may cause changes to the other two components.

DNA methylation was first described as a natural chemical modification in 1950 (Wyatt, 1950), but its relationship with DNA activation remained unclear until 1971 when de Waard demonstrated that the biological activity of DNA was modulated by its methylation in vitro (de Waard, 1971). Soon it became clear that DNA methylation was a dynamic process that varied across different phases of the cell cycle, and that the amount of DNA methylation might be related to the active and inactive states of chromatin (Comings, 1972). The role of DNA methylation in regulating gene expression was clearly hypothesized by Venner and Reinert in 1973 (Venner and Reinert, 1973). The idea that DNA methylation could profoundly influence gene expression led to the hypothesis that the inactivation of chromosome X was epigenetic (Riggs, 1975), and the opposite relationship between DNA methylation and gene expression was established later (Christman et al., 1977). Since then, our understanding of the role that DNA methylation plays in gene expression and phenotype inheritance has increased exponentially.

Interestingly, the role of histone modifications was recognized earlier than that of DNA methylation. In 1964, both methylation and acetylation of histones were reported to have a role in RNA synthesis (Allfrey et al., 1964). However, because such histone modifications proved to be much more complex than DNA methylation, the progress was slower. Only within the last 10–15 years, the complex study of histone modifications became available due to the technological advances in detection of methylation/acetylation of various amino acids in these proteins.

The discovery of the role of ncRNA in modulating DNA methylation and histone acetylation/methylation is much recent. Chromatin remodeling by ncRNA plays an important role in gene silencing and holds the premise that epigenetic therapy is possible for targeting specific genes, in specific tissues, using targeted incorporation of silencing exogenous ncRNA (Malecova and Morris, 2010).

1.3. NUTRITION, EPIGENETICS, AND HEALTH

It is becoming clear that epigenetic regulation is a fundamental process that impinges on many areas of human biology relevant to nutrition and health. The challenge is to identify the causal connections between epigenetics and health and elucidate the way in which diet might influence these processes. Such work offers the hope of developing early epigenetic markers of disease, improving dietary and lifestyle advice to maintain health into old age and improving treatments through the elucidation of mechanisms.

Much is already known about epigenetic changes in cancer. A common observation in cancer is epigenetic change consisting of altered methylation of DNA and the histones associated with DNA. These changes occur early in the development of the disease and the pattern of methylation correlates with cancer stage (Szyf et al., 2004). More interesting is the possibility that widespread epigenetic change in normal cells may actually be causal in the transition to cancer and that nutrition may influence this process. The evidence in relation to this is discussed in the subsequent chapters. There is also growing interest in the role of epigenetic processes in the other major diseases such as cardiovascular disease and diabetes though much less is known about the role of epigenetics in these conditions. Epigenetics is also of growing interest in relation to normal and aberrant biological function in fields as diverse as cognition and reproduction. These are also discussed.

One of the problems encountered when attempting to establish causality between nutrition, epigenetics, and health is covariance between the markers of many of the candidate hypotheses (Figure 1.2). This is particularly problematic in human studies where much of the evidence arises from observational designs. Take the example of human vascular disease. A large number of observational studies have identified an inverse relationship between the dietary intake of folate and other B vitamins, and incidence of vascular disease (Rimm et al., 1998; He et al., 2004; Tavani et al., 2004). Further evidence for the role of B vitamins is provided by genetic association studies linking polymorphisms in the genes involved in B vitamin metabolism to the risk of coronary heart disease, cardiovascular disease, and stroke (Klerk et al., 2002; Wald et al., 2002; Casas et al., 2004). However, the mechanism by which B vitamin status and related genotype might influence cardiovascular health remains obscure. Until recently, the main hypothesis was that B vitamin status influenced vascular health via an effect on circulating homocysteine levels. However, despite a great deal of research in this field, a causal role for homocysteine has yet to be established (Brattstrom and Wilcken, 2000) and there is evidence that homocysteine may not be the causal link (Moat, 2004a, 2004b; Durga et al., 2005). Because both homocysteine and methyl groups are produced by the activity of the same folate/methylation pathway, it is possible that homocysteine may be acting as a proxy for health effects mediated by methylation and that the observational link between B vitamin status and health is mediated by an epigenetic mechanism.

Figure 1.2 An example of multiple possible mechanisms linking exposure to a nutrient to disease risk. In this specific example, the intake of nutrients comprising the vitamin B group could influence disease risk through a number of mechanisms including; direct effects on circulating homocysteine and related metabolites, limitation of DNA synthesis through precursor supply, and availability of methyl groups to support global and gene-specific DNA. The fact that many of these mechanisms may be correlated makes the task of establishing causality difficult in both observational studies and nutritional interventions.

The continuing uncertainty over B vitamin-related causal mechanisms partly arises because of the covariance of metabolite concentrations and biological functions, which depend on the folate/methylation cycle. Blood folate, homocysteine, and MTHFR C677T genotype all covary, and this linkage also appears to extend to global DNA methylation (Friso et al., 2005) with the level of methylation being correlated with plasma homocysteine (Castro et al., 2003). Furthermore, homocysteine is often correlated with the concentration of other folate/methylation cycle intermediates such as S-adenosyl-methionine and S-adenosyl-homocysteine (SAH) (James et al., 2002). The difficulty of interpreting this information is highlighted by the proposal that the often-reported association between homocysteine and disease may arise because homocysteine is acting as a proxy for a causal effect operating through DNA methylation since SAH is a potent inhibitor of the DNA methyltransferases and it changes in parallel with homocysteine concentration (James et al., 2002). Alcohol is another factor known to influence this cycle, disease risk, and methylation status. Animal models of chronic alcohol exposure result in altered DNA (Garro et al., 1991; Choi et al., 1999) and histone (Kim and Shukla, 2005) methylation. DNA methylation has also been shown to vary with alcohol exposure in humans (Bonsch et al., 2004, 2005). There is some evidence for epigenetic mediation of vascular disease. Direct evidence comes from studies in animals. Altered global DNA methylation has been observed in mouse and rabbit atherosclerotic lesions (Hiltunen et al., 2002). Studies in an atherogenic mouse model have shown that altered DNA methylation precedes the development of atherosclerosis (Lund et al., 2004) while proliferation of vascular smooth muscle cells is thought to be influenced by changes in DNA methylation (Post et al., 1999; Ying et al., 2000). Atherosclerosis in humans is also associated with altered DNA methylation compared with healthy controls (Castro, 2003). There are other ways in which nutrition can influence disease risk through epigenetic mechanisms, and these are covered in subsequent chapters, but the covariance often observed between multiple candidate processes illustrates the difficulty in establishing causal links between nutrition, epigenetics, and human health (Haggarty 2007).

1.4. CONCLUSION

The process of epigenetic marking is fundamental to homeostasis and the healthy functioning of cells. Modulation of epigenetic status can occur throughout the life course, it has been implicated in the etiology of disease, it is modifiable by diet and lifestyle, and it may even be passed between the generations. The promise of this emerging field is that the study of epigenetics and nutrition may help to elucidate the way in which nutrition interacts with the genome to influence human health.

ACKNOWLEDGMENTS

The author Mihai Niculescu acknowledges that this work was supported, in part, by funds awarded from an NIH grant (DK56350) to the University of North Carolina at Chapel Hill's Clinical Nutrition Research Unit, and by a grant from the UNC Center for Excellence in Children's Nutrition sponsored by Mead Johnson Nutrition.

The author Paul Haggarty acknowledges the support of the Scottish Government Rural and Environment Research and Analysis Directorate.

REFERENCES

Allfrey, V. G., Faulkner, R., Mirsky, A. E. (1964) Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc Natl Acad Sci U S A 51: 786–794.

Bønaa, K. H., Njølstad, I., Ueland, P. M., Schirmer, H., Tverdal, A., Steigen, T., Wang, H., Nordrehaug, J. E., Arnesen, E., Rasmussen, K. (2006) Homocysteine lowering and cardiovascular events after acute myocardial infarction. N Engl J Med 354(15): 1578–1588.

Bonsch, D., Lenz, B., Reulbach, U., Kornhuber, J., Bleich, S. (2004) Homocysteine associated genomic DNA hypermethylation in patients with chronic alcoholism. J Neural Transm 111(12): 1611–1616.

Bonsch, D., Lenz, B., Kornhuber, J., Bleich, S. (2005) DNA hypermethylation of the alpha synuclein promoter in patients with alcoholism. Neuroreport 16(2): 167–170.

Brattstrom, L., Wilcken, D. E. (2000) Homocysteine and cardiovascular disease: cause or effect? Am J Clin Nutr 72(2): 315–323.

British Heart Foundation. (2005) Heart Statisctics 2005. Available at: http://www.heartstats.org.

Casas, J. P., Bautista, L. E., Smeeth, L., Sharma, P., Hingorani, A. D. (2004) Homocysteine and stroke: evidence on a causal link from mendelian randomisation. Lancet 365(9455): 224–232.

Castro, R., Rivera, I., Struys, E. A., Jansen, E. E., Ravasco, P., Camilo, M. E., Blom, H. J., Jakobs, C., Tavares, de Almeida, I. (2003) Increased homocysteine and S-adenosylhomocysteine concentrations and DNA hypomethylation in vascular disease. Clin Chem 49(8): 1292–1296.

Chmurzynska, A. (2010) Fetal programming: link between early nutrition, DNA methylation, and complex diseases. Nutr Rev 68: 87–98.

Choi, S. W., Stickel, F., Baik, H. W., Kim, Y. I., Seitz, H. K., Mason, J. B. (1999) Chronic alcohol consumption induces genomic but not p53-specific DNA hypomethylation in rat colon. J Nutr 129(11): 1945–1950.

Christman, J. K., Price, P., Pedrinan, L., ACS, G. (1977) Correlation between hypomethylation of DNA and expression of globin genes in friend erythroleukemia cells. Eur J Biochem 81: 53–61.

Comings, D. E. (1972) Methylation of euchromatic and heterochromatic DNA. Exp Cell Res 74: 383–390.

Cooney, C. A., Dave, A. A., Wolff, G. L. (2002) Maternal methyl supplements in mice affect epigenetic variation and DNA methylation of offspring. J Nutr 132(8 Suppl): 2393S–2400S.

De Waard, A. (1971) Modification of biological activity of DNA in vitro by specific methylation. Proc K Ned Akad Wet C 74: 183–190.

Durga, J., Bots, M. L., Schouten, E. G., Kok, F. J., Verhoef, P. (2005) Low concentrations of folate, not hyperhomocysteinemia, are associated with carotid intima-media thickness. Atherosclerosis 179(2): 285–292.

Friso, S., Choi, S. W., Girelli, D., Mason, J. B., Dolnikowski, G. G., Bagley, P. J., Olivieri, O., Jacques, P. F., Rosenberg, I. H., Corrocher, R., Selhub, J. (2002) A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status. Proc Natl Acad Sci U S A 99(8): 5606–5611.

Friso, S., Girelli, D., Trabetti, E., Olivieri, O., Guarini, P., Pignatti, P. F., Corrocher, R., Choi, S. W. (2005) The MTHFR 1298A>C polymorphism and genomic DNA methylation in human lymphocytes. Cancer Epidemiol Biomarkers Prev 14: 938–943.

Garro, A. J., McBeth, D. L., Lima, V., Lieber, C. S. (1991) Ethanol consumption inhibits fetal DNA methylation in mice: implications for the fetal alcohol syndrome. Alcohol Clin Exp Res 15(3): 395–398.

Haggarty, P. (2007) B vitamins, genetics and causal links to disease. Proc Nutr Soc 66(4): 539–547.

Haggarty, P., McCallum, H., McBain, H., Andrews, K., Duthie, S., McNeill, G., Templeton, A., Haites, N., Campbell, D., Bhattacharya, S. (2006) Effect of B vitamins and genetics on success of in-vitro fertilisation: prospective cohort study. Lancet 367(9521): 1513–1519.

Handel, A. E., Ramagopalan, S. V. (2010) Is Lamarckian evolution relevant to medicine? BMC Med Genet 11: 73.

He, K., Merchant, A., Rimm, E. B., Rosner, B. A., Stampfer, M. J., Willett, W. C., Ascherio, A. (2004) Folate, vitamin B-6, and B-12 intakes in relation to risk of stroke among men. Stroke 35(1): 169–174.

Hiltunen, M. O., Turunen, M. P., Hakkinen, T. P., Rutanen, J., Hedman, M., Makinen, K., Turunen, A. M., Aalto-Setala, K., Yla-Herttuala, S. (2002) DNA hypomethylation and methyltransferase expression in atherosclerotic lesions. Vasc Med 7(1): 5–11.

James, S. J., Melnyk, S., Pogribna, M., Pogribny, I. P., Caudill, M. A. (2002) Elevation in S-adenosylhomocysteine and DNA hypomethylation: potential epigenetic mechanism for homocysteine-related pathology. J Nutr 132: 2361S–2366S.

Kim, J. S., Shukla, S. D. (2005) Histone H3 modifications in rate hepatic stellate cells by ethanol. Alcohol Alcohol 40(5): 367–372.

Klerk, M., Verhoef, P., Clarke, R., Blom, H. J., Kok, F. J., Schouten, E. G. (2002) MTHFR 677C–>T polymorphism and risk of coronary heart disease: a meta-analysis. JAMA 288(16): 2023–2031.

Locker, J., Reddy, T. V., Lombardi, B. (1986) DNA methylation and hepatocarcinogenesis in rats fed a choline-devoid diet. Carcinogenesis 7(8): 1309–1312.

Lund, G., Andersson, L., Lauria, M., Lindholm, M., Fraga, M. F., Villar-Garea, A., Ballestar, E., Esteller, M., Zaina, S. (2004) DNA methylation polymorphisms precede any histological sign of atherosclerosis in mice lacking apolipoprotein E. J Biol Chem 279(28): 29147–29154.

Malecova, B., Morris, K. V. (2010) Transcriptional gene silencing through epigenetic changes mediated by non-coding RNAs. Curr Opin Mol Ther 12: 214–222.

Moat, S. J., Doshi, S. N., Lang, D., McDowell, I. F. W., Lewis, M. J., Goodfellow, J. (2004a) Treatment of coronary heart disease with folic acid: is there a future? Am J Physiol Heart Circ Physiol 287(1): H1–H7.

Moat, S. J., Lang, D., McDowell, I. F. W., Clarke, Z. L., Madhavan, A. K., Lewis, M. J., Goodfellow, J. (2004b) Folate, homocysteine, endothelial function and cardiovascular disease. J Nutr Biochem 15(2): 64–79.

Post, W. S., Goldschmidt-Clermont, P. J., Wilhide, C. C., Heldman, A. W., Sussman, M. S., Ouyang, P., Milliken, E. E., Issa, J. P. (1999) Methylation of the estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system. Cardiovasc Res 43(4): 985–991.

Riggs, A. D. (1975) X inactivation, differentiation, and DNA methylation. Cytogenet Cell Genet 14: 9–25.

Rimm, E. B., Willett, W. C., Hu, F. B., Sampson, L., Colditz, G. A., Manson, J. E., Hennekens, C., Stampfer, M. J. (1998) Folate and vitamin B6 from diet and supplements in relation to risk of coronary heart disease among women. JAMA 279(5): 359–364.

Szyf, M., Pakneshan, P., Rabbani, S. A. (2004) DNA methylation and breast cancer. Biochem Pharmacol 68(6): 1187–1197.

Tavani, A., Pelucchi, C., Parpinel, M., Negri, E., La Vecchia, C. (2004) Folate and vitamin B-6 intake and risk of acute myocardial infarction in Italy. Eur J Clin Nutr 58(9): 1266–1272.

Venner, H., Reinert, H. (1973) Possible role of methylated DNA bases for the transcription of the genetic information. Z Allg Mikrobiol 13: 613–624.

Waddington, C. H. (1942) The epigenotype. Endeavour 1: 18–20.

Wald, D. S., Law, M., Morris, J. K. (2002) Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 325(7374): 1202.

Wyatt, G. R. (1950) Occurrence of 5-methylcytosine in nucleic acids. Nature 166: 237–238.

Ying, A. K., Hassanain, H. H., Roos, C. M., Smiraglia, D. J., Issa, J. J., Michler, R. E., Caligiuri, M., Plass, C., Goldschmidt-Clermont, P. J. (2000) Methylation of the estrogen receptor-alpha gene promoter is selectively increased in proliferating human aortic smooth muscle cells. Cardiovasc Res 46(1): 172–179.

Youngson, N. A., Whitelaw, E. (2008) Transgenerational epigenetic effects. Annu Rev Genom Hum Genet 9: 233–257.

Part I

Fundamental Principles in Epigenetics

Section A

Epigenetic Mechanisms

Chapter 2

DNA Methylation

Natalia V. Cucu

Epigenetics Laboratory, Department of Genetics, Faculty of Biology, University of Bucharest, Romania

2.1. INTRODUCTION

DNA methylation is a biological process that results in the addition of methyl groups to DNA. Methyl groups are “one-carbon” chemical groups and their covalent attachment to certain nucleotide residues in the DNA macromolecule is performed by a complex physiological process termed “DNA methylation.” In prokaryotes, DNA methylation occurs at both adenine and cytidine residues and is a part of their host restriction-based defense mechanism (Jeltsch, 2002, 2006; Ratel et al., 2006). In multicellular organisms (eukaryotes), the cytosine base is the major target for DNA methylation and the corresponding minor base, 5-methylcytosine, is presently recognized as the “fifth base” because of its particular role in the regulation of gene expression and genomic stability, which have important consequences in development, aging, and mechanisms of disease (Bird, 2003; Millar et al., 2003; Widschwendter, 2007; Turner, 2009).

DNA methylation is an epigenetic covalent modification as it does not alter the genetic information encoded in the primary DNA structure (represented by nucleotide sequence). Rather its role is to mark specific DNA fragments representing gene promoters, chromosomal fragments, or even entire chromosomes, such that methylated DNA interacts with other chromatin components. Thus, DNA methylation contributes to the epigenetic network (consisting of DNA methylation, histone modifications, and the involvement of noncoding RNA) that controls the gene expression and the chromosomal stability in eukaryotes during their development and later in life (Costello and Plass, 2001; Esteller and Almouzni, 2005; Vanyushin, 2006; Holliday, 2007).

DNA methylation was the first epigenetic code to be discovered, and it is extensively studied and used as a biomarker for gene expression variation and inheritance (Holliday, 2007). The discovery of 5-methylcytosine in prokaryotes (Hotchkiss, 1948) and in mammalian tissues (Chargaff et al., 1949; Wyatt, 1951) enlarged the scope of genetics by including epigenetics within the field of heredity (Watson and Goodman, 2002). DNA methylation's major role in the epigenetic control of gene expression has been recognized lately, during the 1990s, based on the distribution along DNA of 5MeC sites in specific DNA methylation patterns. This is a key feature of the additional level of heredity represented by epigenetics, as DNA methylation patterns determine the cell fate and memory that may be inherited by the daughter cells through mitotic or meiotic division, as the DNA sequence is replicated and kept unchanged (Holliday, 2007).

The DNA methylation process has long been considered as central to the field of epigenetics; hence, its suggestive “nickname” as the “prima donna of epigenetics” (Santos et al., 2005). It provided the most relevant way to explain the significance of the “epi” prefix to “genetics.” This is linked with the roles of chemical tags, the methyl groups, on the cytosine. Previously, such roles were neglected because neither methylated cytosines change the nucleotide sequence encoded as genetic information nor they alter the complementary pairing of DNA strands at guanidine (G) or 5-methyl cytidine (5MeC) sites, or impair the inheritance of DNA nucleotide sequence during replication. However, the study of heritable distribution on DNA methylation patterns determined the modern implementation of the concept of epigenetics: DNA methylation constitutes an additional heredity code to the genetic information encoded in the DNA sequence (Szyf, 1991).

As discussed in Chapter 3, chromatin components are specifically endowed with chemical tags through covalent modifications (DNA methylation and histone modifications) in order to actively participate in the complex interactions that contribute to the formation of specific transcriptional chromatin states. The methyl groups added to the pyrimidine ring of cytosine bases are the major DNA tags that emerge from the major groove, where further DNA–protein interactions occur, which control gene expression. The protein network reacting with these tags includes histone and nonhistone proteins that contribute, along with DNA methylation, to the establishment and remodeling of chromatin conformations. Together with noncoding RNA, DNA and histone covalent modifications represent the “three pillars of epigenetics” that orchestrate the extremely complex and subtle epigenetic gene expression process, and the developmental stages of eukaryotes (Wolffe and Matzke, 1999; Aufstatz et al., 2002; Bernstein et al., 2007; Turner, 2009).

A complex biochemical apparatus, which includes the activity of the effectors (enzymes) and readers (multiprotein complexes) of marked genome regions, is presently recognized as a DNA-sequence-independent process that controls gene expression through dynamical regulation of chromatin. Such chromatin changes are in fact the direct executors of the basic epigenetic instructions consisting of specificity, affinity recognition, and interaction and recruitment events (Jenuwein and Allis, 2001; Li and Bird, 2007).

However, far from being stable, DNA methylation may change under the influence of various signaling factors; thus, having a sensible and continuous conformational remodeling potential, while the DNA sequence or the genotype remains constant in each cell, regardless of the tissue type and the developmental stage. This epigenetic plasticity enables a given genotype to variably respond to endogenous factors during different developmental stages as well as to environmental triggers (such as diet, lifestyle, and pollution) (Reik et al., 2001; D’Alessio and Szyf, 2006; Goldberg et al., 2007; Feinberg, 2007; Zhang and Meaney, 2010).

This dynamic change of DNA methylation constitutes a reprogramming process that may result in either normal development or aberrant epigenome changes, such as neoplasic transformation in cancer, and other disease states (Baylin et al., 2001; Laird, 2003; Feinberg, 2004, 2007; Esteller, 2008). Moreover, specific developmental windows may present metastable states of covalently modified chromatin components, such as methylated cytosine bases, that may be inherited by subsequent generations, rendering such periods more susceptible to developmental defects. Presently, gene expression alterations such as epigenetic aberrant silencing or activation of critical genes and chromosomal instability are central to the new concept of pathogeny, besides the already-established genetic factors (Esteller et al., 2001a, 2001b; Esteller et al., 2002; Esteller, 2008, 2007; Esteller and Almouzni, 2005; Feinberg, 2007).

The relationship between epigenetics and heredity has been reinforced recently by the discovery of environmental effects on the epigenome and hence on the overall genome stability. Epigenetics is emerging as a basic concept in the new field of nutrigenomics, and its role is to act as an interface between environment and genome (Skinner, 2005; Wade and Archer, 2006). Environmental agents may influence the chromatin conformations around specific genes by triggering covalent modification of DNA through methylation and by not inducing mutations and yet these modifications are to be transmitted to the next generations (Zeisl, 2006; Jirtle and Skinner, 2007; Zhang and Meaney, 2010).

One key element of this concept, reversibility, represents an essential differentiating feature between genetic and epigenetic information (Szyf, 1991; Ramachandani et al., 1999). Although both information types are heritable, only the chemical tags acting at the epigenetic level may be added and removed from the DNA and from other chromatin components by specific enzymes. These processes actually control the erasure and the reset of methylation patterns; thus, being able to reprogram the gene expression settings in different cell lines, during specific developmental stages. Unlike genetic mutations, which are passed to succeeding generations along with the specific propensity toward a disease, versatile epigenetic modifications may be corrected by modulating the activity of enzymatic effectors, either directly by drugs or indirectly by environmental agents (Egger et al., 2004; Wade and Archer, 2006; Zeisel, 2006; Szyf, 2009).

2.2. MOLECULAR MECHANISMS OF DNA METHYLATION

DNA methylation is a biological process involving an endogenous enzymatic process (Bestor and Verdine, 1994; Bestor, 2000; Jeltsch, 2002). However, DNA methylation can be altered by other exogenous alkylating agents such as methylmethane sulfonate (MMS) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Such agents induce the addition of methyl groups to a number of nucleophilic sites on the DNA bases, thus producing N- and O-methyl adducts in DNA. These methylated bases have a carcinogenic potential as they can interfere with the repair pathways or be directly involved in producing strand breaks (Bignani et al., 2005; Wyatt and Pittman, 2006). Physiological DNA methylation is different from the promutagenic chemical methylation described above. First, the main product of the physiological reaction is 5-methylcytosine, which does not alter the complementary pairing process with guanine base. Second, the methyl group addition is the result of the catalysis performed by endogenous enzymes (called DNA methyltransferases, DNMTs) on specific DNA sites consisting of cytidine-guanine dinucleotide (CpG, where p stands for a phosphate group between the two nucleotides). Therefore, methyl groups are not provided by exogenous chemicals, instead an endogenous universal methyl donor, S-adenosyl-L-methionine (SAM) is used as cofactor for the enzyme activity catalyzing the methyl group addition to the cytosine base ring (Cheng et al., 1993; Bestor and Verdine, 1994; Bestor, 2000; Goll and Bestor, 2005).

2.2.1. Catalytic Mechanism of DNA (Cytosine-C5)-Methyltransferases

The enzymatic mechanism was originally proposed by Santi et al. (1983) and modified by Chen et al. (1991) and Erlanson et al. (1993). DNA methylation reaction involves several steps. First, it involves the covalent attachment of the enzyme to the substrate (cytosine ring at its C6 position), with its active functional domain containing cysteine (SH) thiolate residue. The catalytic mechanism is represented in Figure 2.1, where the succession of steps are as follows: (i) enzyme nucleophilic attack at position C6 of cytosine (I) that results in a covalent bond with the pyrymidine ring; (ii) subsequent transient protonation of the endocyclic N3 atom of the cytosine ring that produces the intermediate 4,5-enamine (II) and activates the C5 position; (iii) nucleophilic attack of the intermediate (II) through its C5 atom to the sulfonium-linked methyl group of the cofactor SAM and the second intermediate (III) formation; (iv) enzyme beta elimination by deprotonation at position C5, elimination of SH group from position C6, and the reestablishment of aromaticity in the pyrimidine ring. Finally, two products 5MeC and S-adenosyl-homocysteine (SAH) are obtained (Smith et al., 1992; Cheng et al., 1993; Kumar et al., 1994; Cheng, 1995; Jeltsch, 2002; Goll and Bestor, 2005).

Figure 2.1 Steps of catalysis performed by DNA (cytosine-C5-) methyltransferase during the DNA methylation reaction. (Modified after Goll et al., 2005.)

This reaction confers several features to the methylated DNA: modularity associated with the metastability of the methylated DNA, the mutagenicity of 5MeC residues, and the reversibility of DNA methylation. The enzymatic reaction, which has a Michaelis–Menten kinetics, proceeds according to a constant (Km) that is defined by the optimal concentration ratio of SAM to SAH, and depends upon DNMT activity. SAM concentrations lower than Km determines the enamine intermediates ((II) and (III)) needed to destabilize their exocyclic C4 amine group (Jeltsch, 2002) (Figure 2.2A). This state further increases the rate of oxidative deamination, which directly converts the cytosine ring (C) to uracyl (U) and the 5MeC ring to thymine (T), respectively (Kumar et al., 1994; Jeltsch, 2002; Goll and Bestor, 2005) (Figure 2.2B).

Figure 2.2 (A) Deoxycytidine residues may undergo spontaneous or DNMT3a/ DNMT3b-catalyzed deamination when SAM concentration is lower than its Km value. Repair enzymes are easily activated in order to excise the unnatural uracyl bases through glycosylase activity and providing the correct DNA substrate for the substitution of wrong base with cytidine. (B) The putative mechanism explaining 5-methyl cytidine mutagenicity: the deamination of 5MeC ring is catalyzed by the same de novo DNMT3a and DBNMT3b enzymes when intracellular SAM concentration is low.

This metastability of the reaction intermediaries explains the versatility of the enzymatic reaction involving the transition of the normal transferase activity toward the deaminase activity. These are key processes that contribute to the mutagenicity of the so-called “hot spots” represented by the 5MeC residues in high-density repetitive CpG genome regions. Therefore, such mutations may also explain the decreasing CpG density in the mammalian genomes throughout evolution of species (Mazin, 1994, 1995; Robertson and Jones, 1997).

The 5MeC residues in eukaryotic DNA may also promote the transition mutation to T (Figure 2.2B). Hydrolytic spontaneous deamination of C to U may also occur at a slow rate, with a half-life of ca 30,000 years, as compared with 5MeC that is deaminated two to four times more rapidly than C (Lindahl, 1974; Colondre et al., 1978; Shen et al., 1994) (Figure 2.2A). Moreover, the U/G mismatches, considered unnatural for the double-helix DNA, are rapidly recognized and repaired by a specific uracil–DNA glycosylase (Wood et al., 2001) (Figure 2.2A). In contrast, the T/G mismatches, which are not easily recognized as unusual in a DNA CG context, cannot activate a corresponding repair process and hence may accumulate. However, a thymine glycosylase activity has been detected in mammalian cells (Nedermann et al., 1996; Hardeland et al., 2001), in the proximity of and associated with the 5-methylcytosine binding protein 4 (MBD4) (Poole et al., 2001; Rai et al., 2008). Recently, numerous reports indicated a complex DNA demethylation process through DNA repair involving a deamination-mediated repair process (Metivier et al., 2008; Gehring et al., 2009; Niehs, 2009; Zhu, 2009) (Figure 2.3).

Figure 2.3 Cyclic demethylation/remethylation processes catalyzed by DNMT3a/DNMT3b enzymes through intermediary deamination and a MBD4 glycosylase-mediated repair steps.

Interestingly, the rate of cytosine and 5-methylcytosine deamination may be facilitated by the activity of DNMTs. As has been described in previous paragraphs, the intermediary compounds obtained during the methyl group transfer from the methyl donor, SAM, to cytosine, are unstable. Thus, the C ring may turn into U, and 5MeC into T, particularly when low SAM concentrations are present (Mazin, 1994; Shen et al., 1994).

As mentioned before, an interesting consequence of these methylation-mediated mutagenesis reactions in mammalian genomes consists in the depletion of CpG sequences during evolution (Cross et al., 1994). Presently, CpG dinucleotides are from 5- to 10-fold underrepresented, and only CpG islands contribute to the constant CpG content of the mammalian genome (DNA regions with higher than expected CpG sites, see below) (Jeltsch, 2002).

2.2.2. DNA Substrate

As mentioned before, the preferred substrate for DNA methylation are the palindromic repetitive CpG dinucleotides (Bender, 1998). These are important contributors to the DNA double-helix structure tensions, which determine, together with the enzyme activity, the cytosine flipping outside the rigid base DNA stacking arrangement (Klimasauskas et al., 1994; Roberts and Cheng, 1998; Cheng and Roberts, 2001). It has been demonstrated that much of our genome contains such repetitive DNA sequences, which are no more considered as “junk DNA” but rather have begun receiving attention from scientists investigating genomic stability and gene expression regulation through epigenetic factors (Bender, 1998; Pennisi, 2007).

Due to 5-methylcytosine's spontaneous or enzymatic conversion to thymine, the CpG sites are only 5–10% of their predicted frequency in mammalian genomes, as a result of its progressive elimination due to its high mutation rates (Gardiner-Garden and Frommer, 1987; Cross et al., 1994). About 70–80% of genomic CpG sites are methylated in most vertebrates, including humans (Ehrlich, 1982; Antequera and Bird, 1993, 1994; Bird, 1995; Mazin, 2009).

CpG sites may be identified within various types of DNA sequences (satellite DNA, repetitive elements including transposons and their ancestral representatives, nonrepetitive intergenic DNA, and regulatory DNA regions represented by promoters and exons of genes) (Antequera, 2003; Li and Bird, 2007).

One particular type of DNA sequences (CpG islands) is of special interest for the epigenetic regulation of gene expression. CpG islands are generally located within or in proximity to the promoter region of many genes and are defined as having a CG content of at least 55%, rendering them resistant to the activity of DNA methylases (Bestor et al., 1992; Caiafa and Zampiery, 2005). It was hypothesized that CpG islands normally present a protective chromatin structure determined by specific protein covalent modifications. These protective interactions may be altered in carcinogenesis where hypermethylation of CpG islands has been detected (Brandeis et al., 1994; Zardo and Caiafa, 1998; Zardo et al., 2003).

CpG islands are considered constant open chromatin configurations and hence are constantly exposed to the interaction between transcription factors and gene promoters (Cross and Bird, 1995). This is in contrast with CpG sites located outside CpG islands, which are prone to DNA methylation (Li and Bird, 2007). These sequences are methylated during early embryogenesis when the establishment of tissue-specific DNA methylation pattern occurs.

Due to the palindromic feature of the complementary sequence on the second DNA strand, CpG sites are symmetrically methylated on both strands, thus ensuring the inheritance of methylation patterns during DNA replication in the S-phase of the cell cycle (Bird, 1978; Bestor and Verdine, 1994; Chuang et al., 1997). Depending on their methylation state, CpG sites act as a preferential substrate for different DNA methylating enzymes (DNMTs) (Cheng and Roberts, 2001). The CpG sites may be fully methylated (on both strands) or unmethylated; that is, they may present methyl groups on both strands, or no methyl group on either C residue (Figure 2.4A). When the parental DNA is methylated, during the S-phase of cell cycle, the replicated double-stranded DNA comprises one methylated strand (corresponding to parental cell DNA) and one unmethylated, newly synthesized DNA strand, which represents the substrate for maintenance DNA methylation, according to the DNA methylation pattern present on the parental DNA strand (Robertson et al., 1999; Robertson, 2002; Goll and Bestor, 2005) (Figure 2.4B).

Figure 2.4 (A) DNA methylation reactions linked with differently methylated substrates: hemimethylated DNA is preferred by DNMT1 for maintenance methylation; de novo DNA methylases prefer nonmethylated substrates for establishing a new methylation pattern. (B) DNMT1 performs a postreplication maintenance methylation during the S-phase of the cell cycle, which explains the inheritance of the DNA methylation patterns.

This proves that DNA methylation is postreplicative and that the symmetry of the CpG substrates ensures the inheritance of the methylated parental template pattern during cell division. There are reports indicating that DNA methylation may also occur with lower frequency in mammalian genomes at substrates containing nonsymmetrical repetitive sequences, such as CpC, CpA, and CpT (Ramsahoye et al., 2000).

2.2.3. DNA Methyltransferases

The key players of the methylation process are the enzymes named epigenetic “effectors,” which perform the transfer of methyl group on the cytosine ring (Li and Bird, 2007). DNA methyltransferase activity is indexed as EC 2.1.1.37 (Bestor and Verdine, 1994; Fauman et al., 1999; Goll and Bestor, 2005; Jeltsch, 2006). Five types of methylating enzymes, namely, DNA (5-methylcytosine) methyltransferases (DNMTs), have been so far characterized having particular preference for the aforementioned DNA substrates (Figure 2.4A) (Bestor et al., 1988; Bestor and Verdine, 1994; Robertson et al., 1999; Robertson, 2002, 2005).

Among the five types of DNMTs, DNMT1 is the only enzyme and its preference for hemimethylated DNA substrate that results during DNA replication and repair processes (Bestor and Ingram, 1983; Pradhan et al., 1999) (Figure 2.4). Its vital importance has been demonstrated by Li and coworkers, who showed that targeted disruption of the murine Dnmt1 gene leads to genome-wide loss of DNA methylation and embryonic lethality in mice (Li et al., 1992). Numerous other studies later highlighted that artificial reduction of cellular DNMT1 levels by mutagenesis may affect mammalian development and genome stability (Gaudet et al., 2003), and, recently, the importance of DNMT1 maintenance activity for genome integrity and cell viability has been demonstrated in human cells (Egger et al., 2006; Brown and Robertson, 2007; Spada et al., 2007).

The preference of DNMT1 for hemimethylated DNA substrates is associated with its postreplicative activity: DNMT1 is the only enzyme acting immediately after DNA replication as is indicated by its association with PCNA during S-phase (Leonhardt et al., 1992; Chuang et al., 1997) (Figure 2.4A).

The DNMT1 protein structure explains its complex interaction and activity, and its preference for hemimethylated DNA (Goll and Bestor, 2005). It is a large protein comprising 1620 amino acids and having a molecular mass of 183 kDa (Jeltsch, 2002). The large N-terminal region controls numerous affinity interactions, especially because of its peculiar structure of the 1111 amino acids, connected to the catalytic C-terminal domain through a glycine–lysine repeat(GK)7 linker that provides conformational flexibility and explains many of the regulatory functions of this region (Figure 2.5) (Fellinger et al., 2009b).

Figure 2.5 DNMT protein structures. Functional domains of the C- and N-terminal parts are indicated with their corresponding targets for interaction. (Modified after Jeltsch, 2002.)

A special conformation of DNMT1, mediated by the flexibility of the two protein domains linked by the (GK) linker, may account for the enzyme's active and inactive forms. The C-terminal domain contains all the features of the bacterial Dnmt and is involved in catalysis. However, this domain is not able to perform this activity alone. Instead, specific interaction with the N-terminal domain is required (Fatemi et al., 2001). DNMT1 interacts with itself through this N-terminal regulatory domain, which recognizes the substrate (Fellinger et al., 2009a). This DNMT1 dimerization is supposed to facilitate its ability to discriminate the hemimethylated substrates from the unmethylated ones (Jeltsch, 2008).

DNMT1 performs its catalytic activity in concert with chromatin components; its one major interaction partner is Np95 (nuclear protein family, termed also as UHFR1), along with the proteins involved in chromatin modifications such as LSH, EZH2, and G9a (Hashimoto et al., 2008; Meilinger et al., 2009). However, the underlined mechanisms are still not entirely deciphered (Estève et al., 2006; Sharif et al., 2007; Arita et al., 2008).

Hemimethylated substrates are preferred by DNMT1, which suggests that DNMT1 is a maintenance methylase that is able to restore the methylation symmetry of the palindromic CpG sequences after their synthesis during DNA replication (Holliday and Pugh, 1975; Riggs, 1975; Riggs and Xiong, 2004) (Figure 2.4(B)). Such activity is vital for cytodifferentiation processes because it involves clonal inheritance of methylation patterns, which is necessary for the maintenance of tissue-specific gene expression profiles (cell memory) (Goll and Bestor, 2005; Hermann et al., 2004; Goyal et al., 2006) (Figure 2.6). A sex-specific DNMT1 isoform, Dnmt1o, is expressed during the early embryogenesis in oocyte, when its maintenance activity is transiently required (Reik et al., 2001).

Figure 2.6 Maintenance and de novo DNA methylation occur during specific developmental processes (such as cytodifferentiation and early embryogenesis), that is, when clonal inheritance of a cell type-defining pattern and the reprogramming of the cell type through the change of the methylation pattern, respectively, are required.

The second DNA methyltransferase family, namely, DNMT2, proved to have minimal methyltransferase activity in vitro (Bestor, 2000; Goll and Bestor, 2005); moreover, its absence did not affect the DNA methylation levels. DNMT2 enzyme was detected to be active in Drosophilas where its role is linked with non-CpG methylation activity (Lyko et al., 2000; Kunert et al., 2003; Tang et al., 2003). Recently, a tRNAAsp methyltransferase activity was suggested for DNMT2 in Mus, Drosophila, and Arabidopsis, its major substrate being cytosine 38 (Jeltsch et al., 2006; Jurkowski et al., 2008; Schaefer et al., 2009; Schaefer and Lyko, 2010). The human homolog of Dnmt2 is therefore suggested to be derived from RNA methyltransferases (Goll et al., 2006).

Another major type of DNMT activity, designated as “de novo DNA methyltransferase,” has been attributed to a third family of DNA methyltransferases (termed as DNMT3 family). They are proteins encoding related polypeptide sequences that, unlike DNMT1, prefer nonmethylated substrates, show no preferences for hemimethylated substrates and do not depend on the replication events (Okano et al., 1998, 1999) (Figures 2.4 and 2.6). Two representative enzymes have been described so far: DNMT3a and DNMT3b, which are important epigenetic effectors during early embryogenesis, when imprinting marks and tissue-specific patterns are established. During this developmental step, DNMT1 is not active in nucleus. Genetic studies revealed the major importance of DNMT3 enzymes: their inactivation resulted in postnatal lethality (Bestor, 2000; Gowher et al., 2006).

In contrast to the DNA methylation maintenance role of DNMT1, the role of de novo enzymes is the establishment of new DNA methylation patterns. Therefore, developmental stages, where normal expression and activation of de novo DNMT3 enzymes have been detected, consist of embryogenesis and gametogenesis, when X-chromosome inactivation and gene-specific imprinting are the central processes that determine the genome stability in the offspring (Morgan et al., 2005). During these vital early developmental processes, several waves of genomic demethylation and remethylation events occur, resulting in the erasure of the parental genome marks, subsequent offspring epigenome reset, when temporary nonmethylated substrates are released (Reik and Walter, 2001; Bird, 2002; Reik, 2007). A vital role of DNMT3b during early embryonic survival and in chromosomal pericentromere heterochromatin stability through satellite DNA methylation has also been revealed (Jeltsch, 2002; Goll and Bestor, 2005).

While DNMT1 targets the CpG sites in gene promoters (in a tissue-specific manner), additional replication-independent targets have been detected in the pericentromeric sites, which are especially preferred by a different DNA methyltransferase type, namely, the “de novo” DNMT3b enzyme (Easwaran et al., 2004). In the same way, CpG islands, which are normally nonmethylated substrates, are de novo DNMT targets in aberrant neoplasic transformation (Esteller, 2007, 2008; Gal-Yam et al., 2008).

DNA methyltransferase 3 (DNMT3L), although not a DNA methytransferase per se, interacts with DNMT1, DNMT3a, and DNMT3b in order to activate them, and contributes indirectly to the proper imprinting of parental alleles (Goll and Bestor, 2005; Hata et al., 2005; Kaneda et al. 2005). Its normal activity is associated with gametogenesis, while its aberrant activation has been recently suggested in somatic cells with induced carcinogenesis (Gokul et al., 2007). Recently, another role of DNMT3L has been revealed: a rare variant of this enzyme was not able to activate DNMT1 and DNMT3a enzymes and was associated with subtelomeric hypomethylation (El-Maarri et al., 2009).

Deaminase activity of DNMTs. Deamination and mutagenicity of methylated sites are closely linked, as it was previously presented. These are possible reaction pathways when SAM concentration drops below its Km concentration (50 mM) (Wyszynski et al., 1994). Moreover, SAH, the demethylated product of SAM, is a powerful DNMT inhibitor and also an important factor that is able to direct the DNMT catalyzed reaction toward demethylation or deamination. Its prevalence to SAM was correlated with high homocysteine content in blood and DNA hypomethylation (Duncan and Miller, 1980; Cantoni, 1985; James et al., 2002). Therefore, SAM concentration relative to its product SAH (SAM/SAH ratio) may change the reaction efficiency and this is a plausible explanation of the metastability of DNA methylation (Mazin, 1994, 1995; Macinyre et al., 2001). These deamination-mediated base transformations may also be induced by extreme conditions such as heat and the presence of an alkali (Wang et al., 1982). The SAM pool is therefore central to this concept as it is the type of DNMT involved. An intensive effort has been focused to investigate the link between SAM availability and its dietary sources, with endogenous genetic factors, as alterations in the SAM pool may influence DNMTs’ reaction kinetics. Numerous literature reports indicate a link between environment and gene expression that is mediated by the methyl group diet and cell pathways controlling the cell SAM pool (Niculescu and Zeisel, 2002).

Such deamination reactions, although present for DNMT1 in prokaryotes, have not been documented for the mammalian DNMT1 (Chan et al., 2001). However, recent reports are referring to DNMT3a and DNMT3b as de novo methylases that are able to perform a cyclic process including methylation, deamination, repair, and again remethylation reactions, independent of DNA replication (Metivier et al., 2008; Zhu, 2009) (Figure 2.3).

The discovery of deamination and of the mutagenic potential of DNA methyltransferases themselves (or other proteins) is linked with two other important epigenetic features: DNA demethylation and reversibility. The marks established on DNA by DNMTs may be depleted by processes controlled by the same enzymes during two distinct processes (Ramachandani et al., 1999; Ooi and Bestor, 2008). One is passive demethylation, involving DNA replication without DNMT activity. Thus, the lack of conversion of C to 5MeC will gradually decrease the DNA content in 5MeC (Figure 2.7A). Such hemimethylated DNA strands are obtained in the first round of replication. The subsequent rounds of replication result in further accumulation of the unmethylated DNA. Therefore, passive demethylation is a slow process that occurs during at least five rounds of replication and results in a slowdown in the DNA methylation process (Jeltsch, 2002; Zhu, 2009).

Figure 2.7 (A) Passive and active demethylation processes, respectively, are dependent and independent of replication. (B) Passive demethylation takes place when the DNMT inhibitor (I), 5-azacytidine, is incorporated in DNA strand during the first round of replication; the subsequent rounds proceed while DNMT is no more able to process the strands for detection and methylation of unmethylated symmetrical C sites.

Blocking of DNMT1 activity was initially achieved using 5-aza-cytidine (5azaC), a cytidine analog, which is considered one of the most powerful DNMT inhibitors (Jones and Taylor, 1980). It is able to demethylate DNA through a passive process that involves its analog incorporation (instead of C) during the first round of DNA replication, and irreversibly trapping the DNMT1 enzyme through its C6 enzyme covalent bond. The repressed processivity of DNMT1 is the cause of releasing more and more unmethylated DNA substrates during the subsequent replication rounds, as the enzyme is no more able to move along the hemimethylated DNA double helix in order to detect new methylable sites (Figure 2.7A) (Jones, 1985; Bestor, 2000).

An additional model of demethylation mechanism evidenced in mammalian cells involves the active demethylation process (Gehring et al., 2009; Zhu, 2009). The mechanism of active DNA demethylation is still challenging because it requires either the direct the disruption of the carbon–carbon bound between the methyl group and the cytosine ring, or an indirect base or nucleotide excision–repair mechanism (BER or, respectively, NER) that removes directly the minor base or the corresponding nucleotide or indirectly, an early deaminated form (thymine). A replication-independent active demethylation mechanism involving the enzyme intervention has been early proposed by Cedar and Verdine (1999). One potential mechanism was suggested also to occur through the action of a specific DNA glycosylase (such as 5-methylcytosine DNA glycosylase) (Jost et al., 1995) that is able to remove the methylated base, 5-methylcytosine, by splitting its N