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Specialist Review Epigenetic variation: amount, causes, and consequences Elena de la Casa-Esperón University of Texas at Arlington, Arlington, TX, US Carmen Sapienza Temple University, Philadelphia, PA, US 1. Introduction The diversity of human phenotypes that we observe is the result of genetic and epigenetic variation and the interaction of these “biological” variables with environmental factors. Both large-scale and small-scale genome sequencing projects, as well as more recent efforts to define structural variation (copy number variation and subkaryotypic insertions, deletions and rearrangements), have resulted in an important initial description of the amount and type of genetic variation in the human genome. On the other hand, the scale of epigenetic variation in the human population is only beginning to be investigated. Epigenetic variation may arise by diverse mechanisms but, at the molecular level, it reflects differences in the spatial configuration of chromatin and its interactions and function. Multiple biochemical processes (DNA methylation, histone methylation, acetylation, phosphorylation, sumoylation, etc.) are associated with these differences. One important consequence of this variability is the resultant variation in gene expression, although many other effects have also been described (see the following text). In the same way that somatic mutations can be transmitted through successive cell divisions, epigenetic marks can change during the lifespan of an organism and also be transmitted somatically through subsequent cell divisions. In fact, the normal phenotypic diversity found between the different cell types of an organism is, with a few notable exceptions in the immune system, epigenetically controlled. Interestingly, traits that result from particular patterns of epigenetic modification can also be transmitted between generations in some circumstances. The term “epialleles” has been coined to describe such different epigenetic states (Article 36, Variable expressivity and epigenetics, Volume 1). However, unlike DNA sequence changes, epigenetic modifications are often reversible at much higher frequencies than the mutation rate. This is an important characteristic, because epigenetic marks can be reset between generations and they can change in response 2 Epigenetics to the environment. Because epigenetic variation can also be genetically controlled, it constitutes a potentially important link between environmental and genetic factors (Cui et al ., 1998; Nakagawa et al ., 2001; Sandovici et al ., 2003). Such a response to the environment could be mediated by metabolic changes that result in epigenetic modifications (Paldi, 2003; Waterland and Jirtle, 2004; Wolff et al ., 1998). Consequently, epigenetic variability is not only a source of phenotypic plasticity in response to the environment, but these epigenetic alterations can also, potentially, be transmitted between generations, with very important implications in evolution (Rutherford and Henikoff, 2003; Sollars et al ., 2003). To better understand the relevance of epigenetic variation, we will discuss the extent (how much?), the origin (what are the causes?), and the implications (what are the consequences?) of this important source of phenotypic variation. 2. Epigenetic variation: how much? 2.1. Epigenetic variation arises from multiple mechanisms It is difficult to estimate the precise extent of epigenetic variation because it occurs at multiple levels and as a result of multiple processes. The epigenetic variation resulting from inactivation of X chromosome provides a classic example of how multiple and distinct processes can give rise to very large fluctuation in phenotype among genetically similar or identical (Fraga et al ., 2005) individuals. In human females (as in other female mammals), one of the two X chromosomes is inactivated by epigenetic means. Once one of the two X chromosomes is chosen for inactivation early in development, and the same X chromosome remains inactive in all descendants of that cell (Article 41, Initiation of X-chromosome inactivation, Volume 1). The inactive X chromosome becomes a cytologically visible heterochromatic body. This cytological manifestation of femaleness (the Barr body) is to a large extent (but not completely (Disteche, 1995)) transcriptionally inert. This means that each single cell expresses only one allele of most (approximately 85%) (Carrel and Willard, 2005) X-linked genes. If both X chromosomes have the same probability of being inactivated, the “average” women will have the paternal X chromosome inactive in 50% of her cells and the maternal X inactive in the remaining 50% of her cells. However, because the process of choosing the X chromosome for inactivation has a large stochastic component (Article 41, Initiation of X-chromosome inactivation, Volume 1), individual women will have different patterns of X-inactivation (Figure 1a). In fact, there is a minor fraction of females in whom >90% of the cells have the same X chromosome inactivated (Figure 1a). These females will have highly preferential expression of either maternal or paternal alleles of all X-linked genes affected by the inactivation process. In addition to this partly stochastic, partly genetic variability in the fraction of cells in which a particular X chromosome remains active (see next section), there is also population-level and intraindividual variability in the extent of X-inactivation. It has been documented that a fraction of X-linked genes have escaped inactivation’ (reviewed in Disteche, 1995). Interestingly, some genes are Specialist Review Stochastic Environmental IX-inactivation time 1 X-inactivation time 2I /10 0.35 0.3 0.25 90−100 80−90 70−80 60−70 50−60 0.2 0.15 0.1 0.05 0 (a) 3 0.1 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 0 Percentage of cells with same X active (b) 20 40 60 Age at first determination 80 Genetic 0 (c) 10 20 30 40 50 60 70 80 90 100 Percentage of cells with active Xce −carryingX-chromosomes a Figure 1 Origin of X-inactivation variation. (a) Much of the variation in the human results from the stochastic component of the X-inactivation choice process. Y-axis represents the fraction of women with the indicated percent of cells with the same X chromosome active (Naumova et al ., 1996 and our unpublished data). Approximately one-third of women have either X chromosomes inactivated in one-half of their of cells (purple bar) and approximately 60% of women (purple bar plus green bar) have X-inactivation ratios between 50:50 and 70:30. However, approximately 7% of women have highly skewed patterns of X-inactivation, that is, greater than 90:10 (blue bar) in favor of the inactivation of a particular X chromosome. (b) Moving average of change in X-inactivation score in individual females (over nearly two decades; see Sandovici et al ., 2004) as a function of age. Females who were greater than 60 years of age when the first sample was taken show significantly more variation over time than younger females. (c) Heritable effects on X-chromosome inactivation variation. Distribution of X-inactivation ratios in heterozygous Xcea /Xcec mouse females – each circle represents an individual female mouse (de la Casa-Esperon et al., 2002). The X-controlling element locus affects the probability that an X chromosome will become inactive, so that X chromosomes carrying the Xcea allele have a higher probability of being inactivated than X chromosomes carrying the Xcec allele. The observed mean X-inactivation ratio of this population of females is 25% of cells with an active Xcea -carrying X chromosome inactivated in some human samples, but escape inactivation in others (Carrel and Willard, 2005). In addition, the level of expression of such “escapees” also differs between samples (Carrel and Willard, 2005). Therefore, even genetically identical women (monozygotic twins) can differ in their mosaic pattern of X-inactivation, the number of genes that escape X-inactivation, and the levels of expression of some X-linked genes. In addition, some genes that have been inactivated may become reactivated as a function of age (Wareham et al ., 1987) or other environmental factors, although not all X-linked genes appear equally susceptible to reactivation (Migeon et al ., 1988; Pagani et al ., 1990). A similar variability in a long-term inactivation phenomenon has been observed for another class of monoallelically expressed genes located in the autosomes, the imprinted genes. Imprinted genes are expected to be expressed exclusively, or 4 Epigenetics nearly exclusively, from the paternal or the maternal copy (Article 37, Evolution of genomic imprinting in mammals, Volume 1). Several studies have shown that some imprinted genes (e.g., IGF2 , HTR2A genes) are expressed from both alleles in a small fraction of normal individuals (Bunzel et al ., 1998; Sakatani et al ., 2001), while others (IGF2R) exhibit the reciprocal characteristic of being imprinted in only a small fraction of individuals (Xu et al ., 1993). Expression levels between alleles have been found to be variable for several imprinted genes in human tissues (Dao et al ., 1998; McMinn et al ., 2006), and have also been observed at nonimprinted autosomal genes. In fact, large-scale transcription profiling studies in humans have shown differential expression of alleles at a large proportion of loci (up to 54%, depending on the cutoff level of differential expression selected (Lo et al ., 2003)) and, interestingly, the degree of difference in expression between particular alleles varies between individuals (Lin et al ., 2005; Lo et al ., 2003; Pant et al ., 2006; Pastinen et al ., 2004). Moreover, skewing of allelic expression is not necessarily in the same direction: in some individuals who are heterozygous for the same alleles, the allele that is preferentially expressed differs (Lo et al ., 2003; Pastinen et al ., 2004). This observation suggests that trans-modifiers and epigenetic variation are involved in the control of allelic differences in expression, in addition to polymorphisms in cis-regulatory sequences. Such extensive variation in allelic expression must have a large impact in generating phenotypic diversity. 2.2. Variability in the biochemical “marks” associated with epigenetic variation The types of epigenetic marks that result in allelic variation in gene expression can be of diverse nature. The best known and most extensively investigated are covalent modifications of DNA and core histones. DNA methylation at CpG sites shows a degree of variability between different individuals at multiple loci. This is the case for imprinted genes like IGF2/H19 and IGF2 R, for which interindividual variation in methylation patterns has been observed in the differentially methylated regions associated with their expression (Sandovici et al ., 2003). Interestingly, alterations in normal methylation patterns of these regions have been associated with loss of imprinting (LOI), a common observation in several types of cancer (Cui et al ., 1998; Nakagawa et al ., 2001). Another interesting example of interindividual variation at an imprinted gene is PEG1 : this gene codes two isoform, one imprinted (isoform 1) and one expressed biallelically in multiple tissues (isoform 2). However, in a large subset of human placentae, isoform 2 allelic expression differences are observed, as well as interindividual variation in methylation of an associated CpG island (McMinn et al ., 2006). Interindividual variability in methylation patterns has been also described outside of imprinted genes or even protein coding regions: this is the case of methylation differences between humans that is observed in specific Alu repeated sequences (Sandovici et al ., 2005). These observations reflect the fact that DNA methylation may have roles in addition to transcriptional control (de la Casa-Esperon and Sapienza, 2003; Pardo-Manuel de Villena et al ., 2000; Sandovici et al ., 2005). Specialist Review Studies in other organisms also support the idea that variation in DNA methylation could be a widespread phenomenon. For instance, variation in cytosine methylation has been described in rRNA genes of natural accessions of the flowering plant Arabidopsis thaliana (Riddle and Richards, 2002), as well as in retrotransposons (Rangwala et al ., 2006). Also, differentially methylated P1 pigment gene alleles have been observed in maize (Das and Messing, 1994). Importantly, studies in Arabidopsis have also shown that both natural and induced methylation changes can be transmitted to the offspring and result in developmental abnormalities in some instances (Kakutani et al ., 1999; Rangwala et al ., 2006; Riddle and Richards, 2005). 3. Epigenetic variation: what are the causes? Epigenetic variation is the result of three types of processes: stochastic, environmental, and heritable. Variation in X-inactivation illustrates all three of these processes: during embryogenesis, one of the two X chromosomes is inactivated in each cell and clonally transmitted through successive mitotic divisions. Because this choice has a stochastic component (although some deterministic models are also capable of explaining the observations (Williams and Wu, 2004)), the X-inactivation patterns of a population of females approximates a normal distribution. The average female has about half of her cells with the maternal X chromosome inactive and half with the paternal X chromosome inactive. However, a small proportion of females show skewed patterns with a particular X chromosome being inactive in most cells (Figure 1a). Therefore, females are a mosaic for the expression of X-linked genes, and not even genetically identical females need show the same mosaic pattern. The so-called skewing of X-inactivation is not always the rare consequence of the stochastic nature of the choice process. In some instances, skewing is the result of selection against X chromosomes carrying deleterious mutations, and the cell type-specificity of this skewing, as in X-linked agammaglobulinemia (skewing for inactivation of the mutant XLA/BTK allele in B-lymphocytes but not in T-lymphocytes), highlights the role of functional cellular selection (Fearon et al ., 1987; reviewed in Belmont, 1996). In addition, skewing appears more common in older women, which suggests the contribution of environmental factors throughout their lifespan (Busque et al ., 1996; Gale et al ., 1997; Sharp et al ., 2000). In this regard, X-inactivation seems to remain quite stable over many years during earlier ages (Sandovici et al ., 2004) (Figure 1b). Many older females, however, exhibit substantial changes over the timescales at which younger females do not exhibit changes (Sandovici et al ., 2004). In this regard, we have speculated (Sandovici et al ., 2004) that acquired skewing of X-inactivation in older females may result from discontinuous or catastrophic processes that result in decreased numbers of stem cells or an age-related tendency toward bone marrow clonality or myelodysplasia. Additionally, preference for the inactivation of a particular X chromosome can have a completely different origin compared to the selection for particular clonal cell populations or against disadvantageous mutations. Several studies in human and mice have shown that preference for X-inactivation can be heritable and genetically 5 6 Epigenetics controlled (Cattanach and Isaacson, 1967; Naumova et al ., 1996, 1998; Plenge et al ., 1997) In the mouse, the X-controlling element (Xce) is well known for its participation in the X-inactivation choice, so chromosomes carrying different alleles of Xce have different probabilities of being inactivated (Cattanach and Isaacson, 1967) (Figure 1c). Additional autosomal loci also participate in the genetic control of the choice of the X chromosome to be inactivated in mice (Chadwick and Willard, 2005; Percec et al ., 2002, 2003). Moreover, parent-of-origin effects have also been observed in both mice (Takagi and Sasaki, 1975) and humans (Chadwick and Willard, 2005). Stochastic, environmental, and genetic factors result in variability in X-chromosome inactivation and, consequently, generate a gamut of phenotypes for each of the X-linked genes, with multiple implications. The relative abundance of transcripts of each allele of any gene subject to X-inactivation reflects the fraction of cells with each of the two chromosomes active, as well as any allelic differences in expression that are intrinsic to specific alleles. Variations in such relative expression result in the spectrum of phenotypes observed in the population. For instance, a correlation between X-inactivation patterns and meiotic recombination levels (genomewide) has been described in female mice (de la Casa-Esperon et al ., 2002). The biological importance of this trait (recombination levels) in the human population cannot be overestimated as it is a major determinant of female fecundity and reproductive lifespan. If recombination levels are controlled by gene/s in the X chromosome, then levels of recombination can change accordingly with the relative expression of different alleles of such gene/s. Because this is only one of the numerous genes in the X chromosome, the phenotypic diversity generated by similar phenomena related to X-inactivation processes is expected to be large in female mammals. Similarly, epigenetic variability between individuals at multiple autosomal loci can be the result of multiple processes. Since erasure and establishment of epigenetic marks is a dynamic process that occurs during the lifespan of organisms, especially during gametogenesis and embryogenesis (reviewed in Latham, 1999; Mann and Bartolomei, 2002; Article 33, Epigenetic reprogramming in germ cells and preimplantation embryos, Volume 1), there is ample room for stochastic factors to contribute to the diversity of patterns observed. Environmental effects have also been described. Nutritional factors can induce epigenetic modifications such as changes in the expression of imprinted genes; moreover, maternal diet can affect the methylation status of transposable elements and the expression of nearby genes in mice (reviewed in Waterland and Jirtle, 2004). Examples of environmental effects have also been reported in rats, in which variations in maternal care behavior result in epigenetic changes in the offspring at the level of histone acetylation and DNA methylation of the consensus sequence for the NGFI-A transcription factor of the glucocorticoid receptor gene. Consequently, expression of this gene in the hippocampus can be modified by maternal care, which might be the basis for the changes in stress response observed in this gene in the offspring (reviewed in Fish et al ., 2004). Environmental effects could be also the basis for the changes observed in epigenetic marks over time. DNA methylation patterns change with aging in a complex fashion, although overall hypomethylation has been observed in most vertebrate tissues (Mays-Hoopes et al ., 1986; Richardson, 2003). For Specialist Review instance, changes in the methylation profile of the c-myc proto-oncogene have been described during the aging process of mice. Because this is a gene involved in many tumor processes, similar temporal alterations of epigenetic marks might be part of the basis of the increasing incidence of cancer with age (Ono et al ., 1986, 1989). Finally, epigenetic diversity can be the result of heritable variants that affect the formation or stability of epigenetic marks. It has been observed that allelic differences in the expression of several genes are transmitted in families, although the patterns of transmission are variable (Pastinen et al ., 2004; Yan et al ., 2002). In some instances, the transmission of allelic imbalance is compatible with Mendelian inheritance, and even associated with transmission of particular polymorphisms (haplotypes), suggesting the participation of cis-acting elements in the regulation of allelic expression (Yan et al ., 2002), whether they are of genetic or epigenetic origin. In fact, studies showing transmission of de novo induced methylation changes indicate that chromatin modifications, per se, are heritable (Kakutani et al ., 1999; Stokes et al ., 2002). Moreover, abnormal methylation patterns at the differentially methylated regions of the IGF2/H19 and IGF2R imprinted genes have been found to cluster in families (Sandovici et al ., 2003). Also, methylation levels at particular Alu repeated sequences show interindividual differences when the insertions were paternally versus maternally transmitted (Sandovici et al ., 2005). In the case of imprinting defects, epimutations in an imprinting control region of human chromosome 15 have been associated with a substantial percentage of cases of the neurodevelopmental disorders Angelman and Prader–Willi syndromes (see Article 29, Imprinting in Prader–Willi and Angelman syndromes, Volume 1). Recent studies have shown that both cis- and trans-acting factors seem to increase the risk of conceiving a child with Angelman syndrome (AS) (Zogel et al ., 2006). Trans-acting genetic elements have also been involved in changes in the imprinting status of the Dlk1 gene in mouse brain (Croteau et al ., 2005). In this case, reactivation of the normally silent maternal allele correlates with the methylation status of a differentially methylated region. Therefore, epigenetic information constitutes a code superimposed on the genetic information, thereby increasing phenotypic diversity. Much future research will no doubt focus on determining whether epigenetic variation makes a significant contribution to common “complex genetic disorders”, such as diabetes, hypertension, schizophrenia, Alzheimer’s disease and the like, in humans. 4. Epigenetic variation: what are the consequences? Phenotypic diversity is the direct consequence of much epigenetic variation. As we mentioned before, epigenetic modifications can result in allelic expression imbalance within (differential expression levels) or between cells (monoallelic and mosaic expression). This, in turn, can result in phenotypic differences between cells, tissues, and/or individuals. The most obvious example is that of monozygotic twins: although genetically identical, numerous phenotypic differences appear during their life span. The same is true at the epigenetic level: recent studies have shown that differences in DNA methylation and histone acetylation between twins are present 7 8 Epigenetics throughout the genome (Fraga et al ., 2005). Therefore, epigenetic differences could be the basis of many phenotypic discordances observed between twins, including their susceptibility to complex diseases (Wong et al ., 2005). 4.1. Epigenetic variation and disease Epigenetic variation is particularly important for genes involved in diseases. For instance, the fragile-X syndrome of mental retardation is associated with an expansion in the number of CGG repeats in the promoter and 5 untranslated region of the FMR1 gene on chromosome X. This expansion results in hypermethylation of the region and silencing of the FMR1 gene (Hansen et al ., 1992). Short expansions (premutations) do not have apparent phenotypic effects, while long expansions are observed in affected individuals. Notably, the severity of the disease ranges from severe mental retardation to only mild learning disabilities. It is possible that the observed gamut of symptoms depends, at least in part, on epigenetic differences, because variability in methylation in this region has been observed between and within individuals (Genc et al ., 2000; Stoger et al ., 1997) and changes in the CGG repeat length might also result in additional chromatin and transcriptional modifications. Another interesting example of mosaicism has been observed in a small group of AS patients, in whom an imprinting defect silences the maternal copy of the UBE3A gene. However, some of these patients show mosaic maternal expression and methylation of this gene, which, again, suggests the possibility of an epigenetic effect on the observed variability in the severity of clinical symptoms (Nazlican et al ., 2004). Cancer has also been associated with epigenetic alterations, such as losses and gains of methylation and LOI (Feinberg et al ., 1988, 2002; Cui et al ., 2003; Jones and Baylin, 2002; Nakagawa et al ., 2001). Interestingly, some of these alterations are also observed in normal tissues of the same individuals, as highlighted by the gain of DNA methylation in the imprinting control region upstream of H19 in human Wilms tumors and in the non-neoplastic kidney parenchyma adjacent to these tumors (Cui et al ., 1998, 2003; Moulton et al ., 1994). Hence, epigenetic variation between individuals is probably involved in susceptibility to develop cancer as well as other genetic diseases. Moreover, since heritable epigenetic variation has been observed in many instances, it can actually play an important role in quantitative trait variation, and selection acting on such epialleles might result in rapid phenotypic changes, making it a formidable force in evolution (Rutherford and Henikoff, 2003; Sollars et al ., 2003). 4.2. Epigenetic variation and development Epigenetic variation also has important consequences in development and differentiation. A potentially important example of epigenetic changes as a result of environmental effects is the effects of culture conditions on the expression of imprinted genes in mouse embryos. It has been shown that some culture media Specialist Review perturbs gene expression and results in aberrant methylation and expression of imprinted genes (Doherty et al ., 2000; Mann et al ., 2004; Rinaudo and Schultz, 2004). Although some of these abnormalities can be restored in the embryo proper (Mann et al ., 2004), many persist in the extraembryonic tissues and can potentially affect the development of the embryo. In fact, several epidemiological studies suggest that assisted reproductive technologies (ART) might result in an increased frequency of diseases caused by imprinting defects, such as AS and BeckwithWiedemann syndrome (BWS) (Article 30, Beckwith–Wiedemann syndrome, Volume 1). Despite the many reassuring reports on the safety of ART, there have been a small number of recent reports suggesting that ART children may be at increased risk for rare congenital malformation syndromes that are related to defects in genome imprinting (Cox et al ., 2002; DeBaun et al ., 2003; Halliday et al ., 2004; Horsthemke et al ., 2003; Niemitz et al ., 2004; Olivennes et al ., 2001; Orstavik et al ., 2003). At least three children conceived by intracytoplasmic sperm injection (ICSI) have been diagnosed with AS (Horsthemke et al ., 2003; Orstavik et al ., 2003) and at least 28 ART children (both in vitro fertilization (IVF) and ICSI cases) have been diagnosed with BWS (Boerrigter et al ., 2002; Bonduelle et al ., 2002; DeBaun et al ., 2003; Gicquel et al ., 2003; Halliday et al ., 2004; Koudstaal et al ., 2000; Maher et al ., 2003; Olivennes et al ., 2001; Sutcliffe et al ., 1995). Because both AS and BWS are rare disorders (each affects approximately 1 in 15 000 children (Nicholls et al ., 1998)), the appearance of even small numbers of cases is unexpected except among a large sample of births. Therefore, the current data strongly suggests that there is an association between increased risk for AS and BWS and ART. With respect to BWS, the number of affected individuals observed is estimated to be up to nine times the expected incidence (Halliday et al ., 2004). The epidemiological assessment that ART may lead to an increase in the frequency of defective genome imprints is also supported by biochemical characterization of alleles at the relevant disease loci. All three cases of AS show allelic DNA methylation patterns characteristic of a sporadic imprinting defect at the AS locus (i.e., complete or mosaic absence of methylation on both maternal and paternal alleles (Horsthemke et al ., 2003; Orstavik et al ., 2003)). None of the patients has a cytogenetically visible alteration of chromosome 15 (which occurs in 70% of all AS cases (Nicholls et al ., 1998)) and none has a detectable microdeletion at the imprinting center, suggesting that all three cases are due to sporadic, primary, epigenetic defects rather than genetic changes. Given that such imprinting defects account for less than 5% of all AS cases (Buiting et al ., 2001, 2003; Nicholls et al ., 1998), there is at least a suspicion that all three cases occurring in patients following ICSI are of this type. The case for the presence of primary epigenetic defects in the majority of the BWS patients found among ART children is also supported by molecular analyses of alleles at the BWS locus on chromosome 11. Nineteen of the 24 patients have been analyzed for “loss of imprinting” (“LOI”; defined, in this context, as transcription of both maternal and paternal alleles; or the specific changes in DNA methylation that track with this phenomenon and provide a more robust marker in clinical samples) at one or more imprinted genes within the BWS locus and 13 of the 19 cases showed LOI at either KCNQ10 T1 (DeBaun et al ., 2003; Gicquel 9 10 Epigenetics et al ., 2003; Maher et al ., 2003) or H19/IGF2 (DeBaun et al ., 2003). With the addition of the BWS patients described by Halliday et al ., 2004, 16 out of a total of 22 cases examined showed LOI. Although imprinting defects are more common in BWS than in AS, LOI still appears to be overrepresented among BWS cases in ART children and ART is, in turn, overrepresented among BWS cases. 4.3. Epigenetic variation diversity During the last several years, there has been a dramatic increase in the number of studies attempting to elucidate the patterns and interrelationship between DNA methylation, histone modifications, noncoding RNAs, binding of nonhistone chromatin proteins, nuclear positioning and interactions, and so on, which are part of the “epigenetic code” (Article 27, The histone code and epigenetic inheritance, Volume 1). Alterations of the chromatin configuration can affect interactions between DNA regions, between chromosomes, and with other molecules. Most of the studies in epigenetic variation have been focused on the different mechanisms and effects on gene expression and its phenotypic consequences, including allelic differences and disease, enhancers and insulators, trans-sensing and paramutation, long-range interactions and nuclear colocation, and so on. However, epigenetic changes have also been found to affect many other chromosomal functions (see the following text). A classical example is the centromere, in which multiple chromatin modifications and proteins play a major role in binding to the poles of the spindle and promoting chromosome segregation. Interestingly, epigenetic changes can generate new domains with similar properties (neocentromeres) that affect the segregation of chromosomes during mitosis and meiosis (PardoManuel de Villena and Sapienza, 2001; Rhoades and Dempsey, 1966; Warburton, 2004). Consequently, changes in the segregation of chromosomes or chromatids can favor the transmission of particular alleles to the next generations, with important consequences in evolution and disease (Pardo-Manuel de Villena and Sapienza, 2001). Another example of a biochemical process for which there is a strong epigenetic effect is asynchronous DNA replication. Asynchronous replication is characteristic of regions containing monoallelically expressed genes (Mostoslavsky et al ., 2001; Simon et al ., 1999) and, therefore, epigenetic differences seem to be the basis for the differential replication between homologs at such regions. Consequently, these chromosomal regions are interesting examples of how epigenetic modifications of chromosomal regions have not one but multiple effects (on replication and expression). In addition, a recent survey of asynchronously replicated regions have found that they are located in close proximity to areas of tandem gene duplication (Gimelbrant and Chess, 2006) – although whether such epigenetic marks play a role in chromosome stability in regions of duplications remains to be determined. Meiotic pairing and recombination constitute another example of a cellular process in which epigenetic marking appears to play an important role. Functional and epigenetic differences between paternal and maternal chromosomes are a common observation in sexually reproducing organisms (reviewed in de la CasaEsperon and Sapienza, 2003; Pardo-Manuel de Villena et al ., 2000). However, only Specialist Review a few of such differences have been associated with imprinted gene expression. Consequently, it has been postulated that parent-of-origin epigenetic differences share a common origin and function in all sexually reproducing organisms: to allow the recognition (and distinction) between homologous chromosomes during the processes of recombination and repair (de la Casa-Esperon and Sapienza, 2003; Pardo-Manuel de Villena et al ., 2000). Indeed, a recent study has shown that DNA methylation has a role in early meiotic stages: mice deficient in the DNA methyltransferase 3-like (Dnmt3L) gene are sterile and display abnormal chromosome synapsis during meiosis (Bourc’his and Bestor, 2004). Curiously, normal expression of Dnmt3L occurs not in the meiotic cells, but in their precursors. Hence, the epigenetic signals must be inherited through multiple cell divisions. Such epigenetic signals are observed as DNA methylation of retrotransposons, which appear demethylated in Dnmt3L knockout male germ cells. While methylation participates in the normal silencing of mobile elements, retrotransposons are transcribed in the mutant mice. Therefore, Dnmt3L mutant mice represent an example of how epigenetic changes can not only affect transcription but can also reshape the genome by affecting synapsis and allowing the mobilization of retrotransposons into new locations, with multiple consequences. Consequently, studies of epigenetic variation cannot be restricted to effects on gene expression, because it can also modulate many other chromosome functions (de la CasaEsperon and Sapienza, 2003; Pardo-Manuel de Villena et al ., 2000; Sandovici et al ., 2005). 5. Conclusions When discussing epigenetic variation, it is important to remember that we know little about either the underlying mechanisms or the consequences. To mention a few recent examples, studies on the viable yellow allele of the mouse agouti locus (Avy ) have shown that the expression of the agouti gene is correlated with the methylation status of upstream sequences (Article 36, Variable expressivity and epigenetics, Volume 1). Interestingly, epigenetic inheritance at this locus is not due to such methylation marks, because they are erased during embryonic development (Blewitt et al ., 2006). Therefore, other epigenetic marks are responsible for the transmission of this epiallele to the offspring. In this review, although we have mostly mentioned examples of variability in methylation (because it has been the most frequently studied epigenetic mark in mammals and is the first subject of the Human Epigenome Project (Eckhardt et al ., 2004)), we hope that current and future studies will bring to light epigenetic variation at many other levels. For instance, studies of the effects of histone tail modifications at multiple amino acid residues are an expanding field, because the spectrum of modifications and residues affected continues to grow (Article 27, The histone code and epigenetic inheritance, Volume 1). In addition, new epigenetic marks and inheritance modes are likely to be discovered. For instance, the role of small RNAs on epigenetic changes has become prominent since the discovery of RNA interference in Caenorhabditis elegans (Fire et al ., 1998). Recent studies have revealed striking new roles for RNA in non-Mendelian epigenetic inheritance, similar to paramutation in plants. 11 12 Epigenetics The homozygous wild-type progeny of mice that are heterozygous for a mutation in the Kit gene (Rassoulzadegan et al ., 2006) are found to exhibit the white spotting phenotype that is characteristic of mice that carry a Kit mutation. Elaboration of this phenotype is related to the zygotic inheritance of abnormally processed RNAs of the normal allele. A realistic description of the scale of epigenetic variation is hampered by the diversity of causes and consequences and because the mechanism by which many epigenetic marks are heritable remains obscure. An increasing number of studies are aiming to integrate profiles from different epigenetic marks and gene expression patterns of particular chromosomal regions, in order to better understand the possibilities of variations on the epigenetic code. The complexity and diversity of the epigenetic marks and their implications poses a tremendous challenge, but understanding the nature of the immense phenotypic diversity that surrounds us makes it worth the effort. Further Readings Kochanek S, Renz D and Doerfler W (1994) Variability in allelic DNA methylation in spermatozoa. Human Genetics, 94, 203–206. Maher ER (2005) Imprinting and assisted reproductive technology. 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