1. DNA Methylation and the Mammalian Genome

Martina Paulsen and Sascha Tierling und Jörn Walter

In mammals, cytosine methylation at CpG positions of the DNA sequence is one of the hallmarks of epigenetic gene silencing. During evolution, CpG rich regions, so-called CpG islands, have been established as prominent features of promoter regions of genes. Whereas most regions of the genome are constantly methylated these elements are mainly kept free of methylation thereby facilitating the establishment of an open chromatin structure and of initiation of transcription. Besides its role in the regulation of genes, DNA methylation silences repetitive elements and appears to be important for the stability of the mammalian genome. Thus, DNA methylation influences the functional integrity of mammalian genome by shaping its overall structure and leaving its marks in the genomic DNA sequence during evolution.

2. DNA Methyltransferases and Methyl-CpG Binding Proteins as Multi-Functional Regulators of Chromatin Structure and Development in Mammalian Cells

Beth O. Van Emburgh and Keith D. Robertson

The epigenetic modification of DNA with 5-methylcytosine is an important regulatory event involved in chromatin structure, genomic imprinting, inactivation of the X chromosome, transcription, and retrotransposon silencing. This modification is catalyzed and maintained by the DNA methyltransferases and is interpreted by the methyl-CpG binding proteins. DNA methyltransferases are not limited to catalyzing DNA methylation, but also take part in the regulation of gene expression through interactions with other proteins that repress transcription and modify chromatin structure. The use of mouse models, as well as human diseases resulting from deficiencies in the methylation machinery, have been integral parts of understanding the role of these proteins in development and cellular homeostasis. More and more studies are reporting additional roles within the cell beyond their DNA methyltransferase and methyl-CpG binding properties. There is at this point, though, only limited understanding of how these enzymes and proteins are targeted to specific genomic regions. The methyltransferases that will be discussed in this review include DNMT1, DNMT2, and the DNMT3 family of enzymes as well as the methyl-CpG binding proteins MeCP2, MBD1, MBD2, MBD3, and MBD4. The function of these enzymes, as well as their interactions with other cellular proteins and each other, will be discussed along with the diseases attributed to aberrations in the DNA methylation machinery.

3. Methods for the Genome-Wide and Gene-Specific Analysis of DNA Methylation Levels and Patterns

Jörg Tost

DNA methylation has experienced a large increase in interest in the last years and the analysis of DNA methylation either on a genome-wide or gene-specific scale has come to center stage for many biological and medical questions. Realizing the importance of epigenetic changes in development and disease and the potential of DNA methylation as reliable biomarker, a large variety of techniques for the study of DNA methylation has been developed in the last years. No single method has emerged as the "gold" standard unifying quantitative accuracy and high sensitivity or possibilities for whole-genome analysis and precise investigation of individual CpG positions. In this review I summarize most of the developed methods that allow the discovery of a region with DNA methylation changes on a genome-wide scale, the methods to rapidly validate a region of interest, the available possibilities to fine-type the CpG positions in an identified target region and finally technologies that permit the sensitive detection of specific DNA methylation patterns in biological fluids. The choice of the method mainly depends on the desired application, the required sensitivity, the biological material and many studies will require a combination of several methods.

4. Histone Modifications and Epigenetics

Jennifer K. Sims, Tanya Magazinnik, Sabrina I. Houston, Shumin Wu and Judd C. Rice

While the DNA sequence in all cells of an organism is identical, each different cell type is largely defined by the specific sets of genes that are expressed and repressed in that particular cell type. Therefore, cell fate and identity are generally governed by gene expression patterns. One of the greatest challenges to modern research is the elucidation of how these gene expression/repression patterns are established and maintained. In the last decade it has become increasingly clear that the DNA-associated histone proteins play an important, yet enigmatic, role in gene regulation within the mammalian genome. In this chapter we discuss the various covalent chemical modifications of the histones and, by using histone methylation as a model, we illustrate the current paradigm of how histone modifications directly participate in various DNA-templated processes such as transcription.

5. Histone Variants and Nucleosome Occupancy

Sevinc Ercan and Michael J. Carrozza

The modification of chromatin structure is proving to be an important component of many processes involving DNA. The three recognized mechanisms for modifying chromatin are ATP dependent chromatin remodeling, covalent modification of histones and incorporation of histone sequence variants. This chapter focuses on the role of histone variants in several DNA metabolic processes that include transcription, DNA replication and DNA repair. Histone variants discussed in this chapter include H3.1, H3.2, H3.3, CenH3s, H2A.Z, gammaH2A.X, MacroH2A, H2A.Bbd, H1 variants and testis specific variants. Table 1 summarizes functions associated with these variants. This chapter also deals with the role of the above-mentioned chromatin-modifying mechanisms and DNA sequences in defining the range of nucleosome occupancy levels found throughout the eukaryotic genome.

6. Molecular Mechanisms of Polycomb Silencing

Yuri B. Schwartz and Vincenzo Pirrotta

Polycomb and Trithorax group proteins have long been known as important epigenetic regulators of homeotic genes. Recent advances in genome-wide mapping techniques have uncovered their broad role in cell differentiation, which is exerted through the direct control of hundreds of transcription factors as well as important signaling proteins and morphogens. Polycomb silencing, originally believed to result from stable packaging of chromatin is now viewed as dynamic process intimately dependent on histone modifications and balanced by antagonistic action of Trithorax proteins. Recruitment of Polycomb proteins (PcG) to chromatin is mediated by Polycomb Response Elements (PREs), DNA sequence elements found in the vicinity of PcG target genes. These elements are thought to contain a collection of binding sites for sequence-specific DNA binding proteins that assemble PcG complexes. PREs serve as binding hubs where Polycomb proteins remain localized but loop over to interact with the promoters and other parts of target genes.

7. Non-coding RNAs in Gene Regulation

Zhenbao Yu

Recent studies have revealed a surprisingly large number of RNAs transcribed in eukaryotic cells. The majority of them does not function as messenger, transfer or ribosomal RNAs, and are thus called non-coding RNAs (ncRNAs). Although the ncRNAs that have so far been characterized represent only the tip of iceberg, it is becoming increasingly evident that ncRNAs are functionally involved in many biological processes, such as proliferation, differentiation and development. NcRNAs function as regulators of gene expression on various levels, including chromatin modification, transcription, RNA modification, RNA splicing, RNA stability and translation. Among the best studied ncRNAs are small interfering RNAs (siRNAs) and microRNAs (miRNAs). Both of them regulate gene expression through the RNA interference (RNAi) pathway. It is currently estimated that miRNAs account for more than 1 % of predicted genes in higher eukaryotic genomes and up to 30 % of protein-encoding genes are estimated to be subjected to miRNA regulation. MiRNAs and their targets appear to form complex regulatory networks. In addition, miRNAs cooperate with transcription factors to control gene expression. The properties of gene regulatory networks such as feedback loops generated by the combinatorial action of TFs and miRNAs, which facilitate both sustained response and quick transition to stimulation, are beginning to be understood.

8. Plant Epigenetics: A Comparative Approach

Robert Grant-Downton

In this chapter, current knowledge of the epigenetic systems of plants is compared to those discovered in other eukaryotes. A broad overview of the components of epigenetic systems in plants will be made - covering small RNA pathways, DNA methylation and chromatin - and comparisons made to other organisms in respect to their regulation, organisation and function. In this way, both conserved elements and novel plant-specific innovations will be discussed. A particular theme is the comparative richness of the plant epigenetic machinery in small gene families, such as diversity in Argonaute and Polycomb-group proteins. The reason for the greater epigenetic complexity in plants is not simply their multicellular development but also their need to cope with an ever-changing environment due to their sessile lifestyle. Not only does this necessitate exceptional flexibility in gene expression and developmental programmes but also defence against invasive genomes such as viruses. All of these challenges are met by the various parts of their epigenetic systems.

9. Embryonic Stem Cell Epigenetics

Christine Powell and Brian Hendrich

Stem cells can both self-renew and produce multiple cell types. Unlike adult stem cells, which can give rise to either one differentiated cell type (unipotent) or multiple cell types (multipotent), embryonic stem (ES) cells are pluripotent, meaning they can contribute to any tissue type in the body. Each cell type, be it pluripotent or terminally differentiated, is defined by the genes that it expresses and represses, and control of gene expression is fundamental to the process of differentiation. A number of epigenetic processes, including histone modification, DNA methylation and chromatin remodelling, are vital for the ability of ES cells to differentiate correctly. The ES cell epigenome possesses certain features that are unique to these cell types and are involved in the regulation of pluripotency. It has been suggested that this unique epigenetic profile allows ES cells to both prevent transcription of genes associated with differentiation, but also to allow transcription should the correct developmental signals be received. Understanding transcriptional control in pluripotent and differentiating cells will be vitally important for ES cells fulfil their potential for regenerative medicine.

10. The Biology of Genomic Imprinting

Herry Herman and Robert Feil

'Genomic imprinting' refers to the epigenetic marking of the parental origin of certain chromosomal domains (i.e., depending whether they are inherited from the mother or the father). It takes place at a small subset of genes termed imprinted genes, where the epigenetic marking dictates parental allele-specific (imprinted) gene expression in somatic tissues. These marks take form as differential DNA methylation, at specialized sequence elements called 'imprinting control regions' (ICRs). Translating these methylation imprints into the appropriate patterns of gene expression is crucial for the development and growth of the embryo, and for postnatal well-being. This review focuses on the biology of genomic imprinting in mammals, discussed in two parts. The first part elaborates the 'reading' of the imprints; i.e., how tissues decode the imprints into imprinted expression. Included is a discussion on the related process, which assures the maintenance of these imprints in somatic tissues. Examples of various reading mechanisms are presented including the blocking of long-range promoter-enhancer interactions and the involvement of non-coding RNAs in chromatin repression. The second part addresses the mechanism involved in assuring the re-establishment of new imprints in the next generation. It discusses the processes which erase and re-establish the imprints in the male and female germ lines.

11. X Chromosome Inactivation

Aditya K. Sengupta, Tatsuya Ohhata and Anton Wutz

X inactivation in mammals achieves dosage compensation of X chromosomal genes between XX females and XY males. One of the two female X chromosomes becomes transcriptionally inactivated early in development, such that in both male and female embryos one X chromosome is active. A mechanism for counting and choice ensures that precisely one X chromosome remains active and all super numerous Xs are inactivated. X chromosome counting and regulation of choice are mediated by a single locus on the X - the X inactivation center (Xic). Chromosome-wide inactivation is initiated by and crucially depends on the expression of the long non-protein-coding Xist RNA. Xist RNA is transcribed from the Xic on the future inactive X (Xi), attaches to Xi chromatin and accumulates over the chromosome triggering transcriptional silencing. Thus, Xist is a powerful epigenetic regulator that is able to inactivate an entire chromosome. Xist is essential for initiation of X inactivation but the Xi is maintained independent of Xist by other epigenetic mechanisms. Therefore, X inactivation can be mechanistically separated into an initiation and a maintenance phase. The function of Xist RNA in initiation of silencing is strictly dependent on a particular cellular context. In differentiated cells Xist expression is not sufficient for initiating gene repression. X inactivation is a multistep process that comprises an ordered series of chromatin modifications that occur in a developmentally regulated manner. Recruitment of Polycomb group proteins (PcG), which are known to be required for maintaining the repression of Hox genes, to the Xi has been implicated in the transition from the initiation phase to the maintenance phase of X inactivation. Also factors with a role in chromatin and nuclear structure, such as scaffold attachment factor A (Saf-A) or the histone variant macroH2A, are recruited to the Xi and have been implicated in the stabilization of the inactive state. X inactivation, thus, provides a model for developmentally regulated formation of silent chromatin domains as similar mechanisms might regulate gene expression in a more general, albeit smaller, context.

12. Cancer Epigenetics

Joseph F. Costello and Romulo M. Brena

Genetic and epigenetic mechanisms contribute to the development of human tumors, yet the typical analysis of tumors is focused on only one or the other mechanism. This approach has led to a biased, primarily genetic view of human tumorigenesis. Epigenetic alterations such as aberrant DNA methylation are mitotically heritable, sufficient to induce tumor formation, and can modify the incidence and tumor type in genetic models of cancer. Complex epigenetic modification of histones, and genetic alterations of the genes encoding histone modifying genes also contribute to gene and chromosome dysfunction in tumorigenesis. These initial studies raise important questions about the degree to which genetic and epigenetic pathways cooperate in human tumorigenesis, the identity of the specific cooperating genes and how they interact functionally to determine the differing biological and clinical course of tumors. These gaps in our knowledge are, in part, due to the lack of methods for full-scale integrated genetic and epigenetic analyses. The ultimate comprehensive analysis will include sequencing relevant regions of the 3 billion nucleotide genome, determining the methylation status of the 28 million CpG dinucleotide methylome at single nucleotide resolution, and mapping relevant histone modifications genome-wide in different types of cancer. Here we discuss the fundamental differences between normal and cancer epigenomes, and the unique discovery potential of integrating cancer epigenomic and genomic data. We discuss the knowledge gained from single gene and large-scale epigenome analyses in the context of gene discovery, therapeutic application, and building a more widely applicable mechanism-based model of human tumorigenesis.

13. The Role of MicroRNAs in Human Cancer

Yoshimasa Saito, Jeffrey M. Friedman, and Peter A. Jones

MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous regulators of numerous target genes. Hundreds of human miRNAs have been identified in the human genome. They are expressed in a tissue specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development. Links between miRNAs and human diseases are increasingly apparent and aberrant expression of miRNAs may contribute to the development and progression of human cancer. Some miRNAs play roles as tumor suppressors or oncogenes. Recent studies have demonstrated that miRNA expression is regulated by different mechanisms including transcription factor binding, epigenetic alterations, and chromosomal abnormalities. In particular, epigenetic alterations induced by chromatin modifying drugs or by genetic disruption of key DNA methyltransferases cause distinct changes in miRNA expression profiles in cancer cells. miRNA expression profiling might be a powerful clinical tool for cancer diagnosis and regulation of miRNA expression could be a therapeutic strategy for human disease including cancer. In this chapter, we will review the current literature on the biological importance of miRNAs while focusing on the role of miRNAs during human carcinogenesis.

14. Longevity, Epigenetics and Cancer

Ruben Agrelo, Mario F. Fraga, and Manel Esteller

Aging is the main risk factor associated with cancer development. The accumulation of molecular lesions in cells from mature organisms during the aging proccess is perhaps the fact that drives cells to transformation. Molecular lessions can be of genetic or epigenetic nature. Cell epigenetics, in particular DNA methylation and histone modification, becomes altered in aging and cancer. Global hypomethylation and CpG island hypermethylation occur progressively during aging and lead to cell transformation. In particular, the Werner syndrome gene (WRN) promoter and lamin A/C promoter become hypermethylated during the human neoplastic process shedding light on the tight connection between aging and cancer with epigenetics as a link. Cellular modifications that control the length of telomeres and enzymes of the NAD+ dependant deacetylase family (sirtuins) show a progression in cellular aging that is totally reverted during cellular transformation. Here we explore the physiological significance of epigenetic modifications during cellular aging and transformation.

15. The Dynamic Epigenome: the Impact of the Environment on Epigenetic Regulation of Gene Expression and Developmental Programming

Ionel Sandovici, Noel H. Smith, Susan E. Ozanne and Miguel Constancia

Epigenetics refers to cellular mechanisms that confer stability of gene expression during development. The main molecular mechanisms that mediate these phenomena are DNA methylation and chromatin modifications. Recent research suggests that changes in the epigenome may underpin genetic-environmental interactions. Here, we review advances in the growing field of environmental epigenetics. External influences on epigenetic marking systems seen in diverse organisms from plants to animals may induce transient and long-lasting changes on epigenetic signatures. Environmental factors can therefore have long-term consequences for genome function. An effect of the environment on epigenetic programming in early life could underpin the phenomena known as developmental programming and explain the developmental origins of disease. Deficiencies in reprogramming of the germ line are likely to underlie environmentally-induced epigenetic transgenerational effects. The identification of mechanisms by which epigenetic "signaling" molecules are modulated by the environment will be instrumental in understanding these complex processes. This is an exciting area of future research, with many potential biomedical applications.

16. Transgenerational Epigenetic Inheritance

Timothy James Bruxner and Emma Whitelaw

Transgenerational epigenetic inheritance refers to the transfer of epigenetic information across generations, i.e. through meiosis. While it has always been recognized that it is not just DNA, but chromosomes, that are passed from the gametes to the zygote, there has been a general acceptance of the idea that the DNA sequence is the only component that carries information with respect to the offspring's ultimate phenotype. However, there is now strong evidence that this non-DNA sequence component, the epigenetic component, can play a role in the inheritance of phenotypes. This was first reported in plants and is now emerging as a common theme in many organisms, including Drosophila, yeast and mammals. Recent studies in humans have identified disease states that result from so-called epimutations, where the epigenetic state is disrupted, and in some cases these epimutations are seen in successive generations. Whether this is the direct result of the inheritance of epigenetic marks remains unclear, but the findings do rekindle interest in the area.