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All developmental processes in metazoans require the establishment of different genetic programs to generate functionally specialised cells. Differential gene expression is also the basis for the alterations in the developmental potential of differentiating cells. However, the molecular details concerning how this is achieved are still poorly understood. The haematopoietic system has for many years served as an excellent model system to study how developmental processes are regulated at the epigenetic level. In this article we will summarise recent results from others and from our own laboratory that have yielded profound insights into the general principles of how cell-fate decisions are regulated in the cell nucleus. We summarise (1) how the interplay of sequence-specific transcription factors and chromatin components is responsible for the cell type and cell stage-specific activation of specific genes and (2) how these findings impact on current concepts of epigenetic regulation of developmental processes.

In mouse and most other mammalian species, the paternal and maternal genomes undergo parent-specific epigenetic reprogramming during preimplantation development. The paternal genome is actively demethylated within a few hours after fertilization in the mouse, rat, pig, bovine, and human zygote, whereas the maternal genome is passively demethylated by a replication-dependent mechanism after the two-cell embryo stage. These genome-wide demethylation waves may have a role in reprogramming of the genetically inactive sperm and egg chromatin for somatic development. Disturbances in this highly coordinated process may contribute to developmental failures and defects inmammals. The frequency and severity of abnormal phenotypes increase after interferingwith or bypassing essential steps of gametogenesis, early embryogenesis, or both. Nevertheless, it is plausible that normal fertilization, assisted reproduction, and embryo cloning are all susceptible to similar dysregulation of epigenetic components. Although themousemay be an excellentmodel for early human development, species and strain differences in the molecular and cellular events shortly after fertilization may have important implications for the efficiency of epigenetic reprogramming and the incidence of reprogramming defects. Some species, i.e., rabbit and sheep, do not require drastic genome-wide demethylation for early development, most likely because the transition from maternal to embryonic control occurs relatively late during preimplantation development. A better understanding of key reprogramming factors—in particular the demethylase activity in the fertilized egg—is crucial for improving human infertility treatment and the efficiency of mammalian embryo cloning.

Epigenetic regulation of gene transcription relies on molecular marks like DNA methylation or histone modifications. Here we review recent advances in our understanding of epigenetic regulation in the fruit fly . In the past, DNA methylation research has primarily utilized mammalian model systems. However, several recent landmark discoveries have been made in other organisms. For example, the interaction between DNA methylation and histone methylation was first described in the filamentous fungus . Another example is provided by the interaction between epigenetic modifications and the RNA interference (RNAi) machinery that was first reported in the fission yeast . Another organism with great experimental power is the fruit fly . Epigenetic regulation by chromatin has been extensively analyzed in the fly and several of the key components have been discovered in this organism. In this chapter, we will focus on three aspects that represent the complexity of epigenetic gene regulation. (1) We will discuss the available data about the DNA methylation system, (2) we will illuminate the interaction between DNA methylation and chromatin regulation, and (3) we will provide an overview over the Polycomb system of epigenetic chromatin modifiers that has proved to be an important paradigm for a chromatin system regulating epigenetic programming.

Epigenetics is the study of genes during development. Gene expression states are set by transcriptional activators and repressors and locked in by cell-heritable chromatin states. Inappropriate expression or repression of genes can change developmental trajectories and result in disease. Aberrant chromatin states leading to aberrant gene expression patterns (epimutations) have been detected in several recognizable syndromes as well as in cancer. They can occur secondary to a DNA mutation in a - or -acting factor, or as a “true” or primary epimutation in the absence of any DNA sequence change. Primary epimutations often occur after fertilization and lead to somatic mosaicism. It has been estimated that the rate of primary epimutations is one or two orders of magnitude greater than somatic DNA mutation. Therefore, the contribution of epimutations to human disease is probably underestimated.

Epigenotypes are modified cellular or viral genotypes which differ in transcriptional activity in spite of having an identical (or nearly identical) DNA sequence. Restricted expression of latent, episomal herpesvirus genomes is also due to epigenetic modifications. There is no virus production (lytic viral replication, associated with the expression of all viral genes) in tight latency. experiments demonstrated that DNA methylation could influence the activity of latent (and/or crucial lytic) promoters of prototype strains belonging to the three herpesvirus subfamilies (α-, β-, and γ-herpesviruses). , however, DNA methylation is not a major regulator of herpes simplex virus type 1 (HSV-1, a human α-herpesvirus) latent gene expression in neurons of infected mice. In these cells, the promoter/enhancer region of latency-associated transcripts (LATs) is enriched with acetyl histone H3, suggesting that histone modifications may control HSV-1 latency in terminally differentiated, quiescent neurons. Epstein-Barr virus (EBV, a human γ-herpesvirus) is associated with a series of neoplasms. Latent, episomal EBV genomes are subject to host cell-dependent epigenetic modifications (DNA methylation, binding of proteins and protein complexes, histone modifications). The distinct viral epigenotypes are associated with distinct EBV latency types, i.e., cell type-specific usage of latent EBV promoters controlling the expression of latent, growth transformation-associated EBV genes. The contribution of major epigenetic mechanisms to the regulation of latent EBV promoters is variable. DNA methylation contributes to silencing of Wp and Cp (alternative promoters for transcripts coding for the nuclear antigens EBNA 1–6) and LMP1p, LMP2Ap, and LMP2Bp (promoters for transcripts encoding transmembrane proteins). DNA methylation does not control, however, Qp (a promoter for EBNA1 transcripts only) in lymphoblastoid cell lines (LCLs), although methylated Qp-reporter gene constructs are silenced. The invariably unmethylated Qp is probably switched off by binding of a repressor protein in LCLs.

Despite significant effort, understanding the causes and mechanisms of complex non-Mendelian diseases remains a key challenge. Although numerous molecular genetic linkage and association studies have been conducted in order to explain the heritable predisposition to complex diseases, the resulting data are quite often inconsistent and even controversial. In a similar way, identification of environmental factors causal to a disease is difficult. In this article, a new interpretation of the paradigm of “genes plus environment” is presented in which the emphasis is shifted to epigenetic misregulation as a major etiopathogenic factor. Epigenetic mechanisms are consistent with various non-Mendelian irregularities of complex diseases, such as the existence of clinically indistinguishable sporadic and familial cases, sexual dimorphism, relatively late age of onset and peaks of susceptibility to some diseases, discordance of monozygotic twins and major fluctuations on the course of disease severity. It is also suggested that a substantial portion of phenotypic variance that traditionally has been attributed to environmental effects may result from stochastic epigenetic events in the cell. It is argued that epigenetic strategies, when applied in parallel with the traditional genetic ones, may significantly advance the discovery of etiopathogenic mechanisms of complex diseases. The second part of this chapter is dedicated to a review of laboratory methods for DNA methylation analysis, which may be useful in the study of complex diseases. In this context, epigenetic microarray technologies are emphasized, as it is evident that such technologies will significantly advance epigenetic analyses in complex diseases.

Epigenetics describes changes in genome function that occur without a change in the DNA sequence. Dosage compensation is a prime example of the regulation of gene expression by an epigenetic mechanism. Dosage compensation has evolved to balance the expression of sex-linked genes in males and females, which possess different numbers of sex chromosomes. However, the genetic sequence of the chromosomes is the same in both sexes. This mechanism therefore needs (1) to function in a sex-specific manner, (2) to target the sex chromosome from amongst the autosomes and (3) to establish and maintain through development a precise, equalised level of gene expression in one sex compared to the other. The process by which dosage compensation is orchestrated has been well characterised in fruit flies and mammals. Although each has evolved a specific dosage-compensation mechanism, these systems share some underlying themes; the molecular components that mediate dosage compensation in both include non-coding RNA molecules, which act as nucleation points for the compensation process. Both systems utilise chromatin-modifying enzymes to remodel large domains of a chromosome. This review will discuss the mechanism of dosage compensation in in light of recent developments that have brought into question the previous model of dosage compensation.

Tumor DNA contains valuable clues about the origin and pathogenesis of human cancers. Alterations in DNA methylation can lead to silencing of genes associated with distinct tumorigenic pathways. These pathway-specific DNA methylation changes help define tumor-specific DNA methylation profiles that can be used to further our understanding of tumor development, as well as provide tools for molecular diagnosis and early detection of cancer. Female sex hormones have been implicated in the etiology of several of the women’s cancers including breast, endometrial, ovarian, and proximal colon cancers. We have reviewed the DNA methylation profiles of these cancers to determine whether the hormonal regulation of these cancers results in specific DNA methylation alterations. Although subsets of tumors in each of these four types of cancers were found to share some DNA methylation alterations, we did not find evidence for global hormone-specific DNA methylation alterations, suggesting that female sex hormones may participate in different tumorigenic pathways that are associated with distinct DNA methylation-based molecular signatures. One such pathway may include methylation in the context of the CpG island methylator phenotype.

DNA methylation is an epigenetic modification of the DNA sequence and thus does not change the genetic code but affects chromosomal stability and gene expression. DNA methylation patterns are heritable and can be passed on to the daughter cell. In this review, we briefly summarize our current knowledge on normal DNA methylation patterns and move on to discuss the current state of the field with respect to altered DNA methylation in cancer. We make a special attempt to address current questions relating to genome-wide DNA methylation patterns. Since DNA methylation is used as a therapeutic target in clinical studies, it is of utmost importance to define potential target sequences that could be used as diagnostic or prognostic markers. We conclude the review by outlining possible scenarios that may explain tumor type-specific DNA methylation patterns described by assays evaluating genome-wide levels of DNA methylation.

Cancer cells that have a large number of aberrantly methylated CpG islands (CGIs) are known to have CpG island methylator phenotype (CIMP), and decreased fidelity in replicating methylation patters has been analyzed as an underlying mechanism. First we developed a method to analyze the number of errors in replicating CpG methylation patterns in a defined period. A single cell was expanded into 10 cells, and the number of errors during the culture was measured by counting the deviation from the original methylation patterns. It was shown that methylated status of a CpG site was more stably inherited than unmethylated status, suggesting that the genome is constantly exposed to methylation. Promoter CGIs showed higher fidelities than CGIs outside promoter regions. We then analyzed error rates in two gastric cancer cell lines without CIMP and two with CIMP for five promoter CGIs. Two CIMP(-) cell lines showed error rates smaller than 1.0×10 errors per site per generation (99.90%–100% fidelity) for all the five CGIs. In contrast, AGS cells showed significantly elevated error rates, mainly due to increased methylation, in three CGIs (1.6- to 3.2-fold), and KATOIII cells showed a significantly elevated error rate in one CGI (2.2-fold). Presence of densely methylated DNA molecules was observed only in KATOIII and AGS. These data demonstrated that some cancer cells have decreased fidelity in replicating CpG methylation patterns that underlie CIMP.