Although the majority of CpGs are methylated in the genome, CpG dinucleotides within CGI promoters are typically unmethylated during development and in normal (non-neoplastic/non-senescent) tissue types. The CGIs are genomic DNA regions with high frequency of CpG dinucleotides. Typically, a CGI is a region with at least 200 bp with a greater than 50% GC and an observed/expected CG ratio greater than 60% [17]. Comprehensive analysis of CGIs in human chromosomes 21 and 22 by Takai and Jones [18] revealed that regions of DNA of greater than 500 bp with a G+C equal to or greater than 55%, and observed CG/expected CG of 0.65 were more likely to be associated with the 5′ regions of genes. With this definition most of the Alu-repetitive elements were excluded. These islands overlap with promoter regions of 50–60% of human genes [19]. However, a subset of promoter CGIs are methylated in a tissue-specific manner during development, showing an exception to the general rule that CGI methylation in normal tissue is limited to X-inactivation and imprinted genes [8, 20]. This observation was supported further by recent findings of genome-wide profiling of DNA methylation demonstrating that non-X-linked promoter CGIs are methylated in normal tissues and escape methylation in germ line cells [21]. Another study has estimated 6–8% of CGIs to be methylated in the genomic DNA of human brain, blood, muscle and spleen [22]. Interestingly, in the same study, CGIs displayed tissue-specific methylation of genes essential for development, suggesting a programmed mechanism of DNA methylation. Another means of DNA methylation propagation is via methylation spreading that begins with genome-wide demethylation that starts shortly after fertilization. Remethylation of most of the genome occurs after the blastocyst stage [23] and continues at a slower pace during the rest of the developmental period. Even though the phenomenon of spreading has not been fully understood, it was proposed as a self-perpetuating interaction between chromatin-modifying proteins and DNA methylation [24]. Indeed, many of the chromatin modification enzymes responsible for gene silencing are found associated with each other in mammalian cells. Some of the examples of DNMT1-associated proteins are HDAC1 [25], histone methyltransferase G9a [26], ATP-dependent chromatin modeling enzyme SNF2H [27], and Polycomb protein EZH2 [28]. Therefore, the above hypothesis that initial DNA or histone methylation will attract repressive complexes, and create a transcriptionally unfavorable chromatin conformation is very plausible. This alteration in chromatin structure, in turn, influences the nearby chromatin and makes it more prone to methylation spreading. This phenomenon is well documented in Arabidopsis, where tandem repeats upstream of endogene SDC element recruit non-CG DNA methylation directed by histone methylation and siRNA, and display spreading of siRNAs and methylation beyond the repetitive DNA [29]. Existing pieces of evidence in mammalian cells show that there are certain cis-acting elements that are dispersed throughout the genome and they can either act as a methylation signal element or methylation boundary during methylation spreading. For example, in the mouse Aprt gene, two upstream B1 repetitive DNA elements were identified to provide de novo methylation signal for spreading [30]. These elements reside in the large stretches of DNA dubbed as methylation centers. Other retrotransposon elements such as B2, Alu (human equivalent of B1), and LINE- 1 (long interspersed nuclear element-1) are also considered to possess de novo signaling activity for methylation spreading [24]. In contrast, the Sp1 binding sites within the Aprt promoter provide the counteracting force against spreading. Indeed, site-directed mutation of one or more Sp1 sites eliminates the binding of transcription factors and allows methylation to spread at the Aprt promoter [31]. However, (ATAA)_n repeat sequences in the human GSTP1 gene, Sp1 and CTCF elements in the BRCA1 gene, act as boundary elements for prevention of methylation spreading onto CGIs [32, 33]. Recent experimental work on genome-wide DNA methylation analysis discovered an overrepresentation of putative zinc finger binding sites at the boundaries of methylation-resistant CGIs. This observation suggested that these sites may reinforce transcription factor binding and thereby block methylation spreading and promote transcription [34]. Dynamic equilibrium between methylation spreading and its suspension is likely to be responsible for establishing and maintaining stable DNA methylation patterns in human somatic cells. Furthermore, a combined study of bioinformatic approaches and methylation data from chromosome 21 has demonstrated that DNA sequence, repeat frequencies, and predicted DNA structures correlated with methylation status of CGIs [35].

Recently, a central role of acetyltransferase HBO1 (histone acetyltransferase binding to origin recognition complex) was demonstrated during DNA replication [52]. In the above study, HBO1 was found in a complex with ING family of tumor suppressors that are associated with cell proliferation. Low levels of HBO1 and ING5 correlate with reduction in DNA synthesis and affect progression into S phase [52]. These results suggest that the role of H4 acetylation by HBO1 is an important event for DNA synthesis. Furthermore, HBO1, ING4, ING5, and p53 have been shown to associate in shared protein complexes in cells. The tumor suppressor p53 physically interacts with HBO1 and negatively regulates its HAT activity in vitro and in cells, and thus connects p53-responsive stress signaling and HBO1-dependent chromatin modification pathways [53]. Another histone modification, phosphorylation may play a role in condensation/decondensation of chromatin during replication in mammalian cells. For example, phosphorylation of H3S10 may function to displace the HP1 complex from H3K9 methylated chromatin to facilitate cellular events for decondensation [54].

While correct establishment and maintenance of DNA methylation patterns are critical for normal development, DNA demethylation is also equally important for precise execution of developmental programs as evidenced by epigenetic reprogramming events in early embryos and primordial germ cells (PGCs). It is unknown whether DNA demethylation requires demethylase activities or can occur passively through DNA replication in the absence of DNMT1. Although no DNA demethylase activity has been convincingly identified, several mechanisms have been proposed to account for the loss of DNA methylation [64]. For example, DNA deaminases of the Aid/Apobec family have been shown to catalyze deamination of 5-methylcytosine resulting in T:G mismatch, which may lead to DNA demethylation if the mismatch is repaired [65]. Interestingly, a recent study has proposed thatDNMTs themselves have dual roles in CpG methylation and active demethylation of 5-methyl CpGs through deamination during periodic methylation/demethylation cycles of the pS2 gene promoter upon activation by estrogens [66]. Although precise roles of DNMT3A/B in this process are unclear, the study has demonstrated that DNMT3A/B can deaminate both cytosine and 5-methylcytosine in vitro, and that concordant recruitment of DNMT3A/B, DNA glycosylase, and other base excision repair proteins occurs during pS2 promoter demethylation.

Covalent modifications of histones add multiple layers of complexity to chromatin, ranging from small chemical changes such as acetylation and methylation to large peptide addition such as ubiquitylation and sumoylation. Over the past 10 years, several families of histone-modifying enzymes have been identified, as summarized in Table ​Table1.1. Recent reviews have extensively covered topics of histone modifications, their mechanism of action, and the biological functions derived from individual or combined modifications [42, 67]. Of particular interest, new nomenclature for some families of chromatinmodifying enzymes has recently been proposed since the current nomenclature of the enzymes is rather inconsistent and often creates additional complexity [68]. Most histone modifications are dynamically regulated as evidenced by identification of many enzymes that can remove the modification. One wellstudied example is histone demethylation that is carried out by two classes of enzymes, amine oxidases such as LSD1 and hydroxylases of JmjC family [69]. In contrast, arginine demethylation activity has not been identified yet, although the deimination process converting an arginine to citrulline has been proposed as an alternative mechanism to antagonize arginine methylation [70].

As supported by the number and type of histone-modifying enzymes (Table ​(Table1),1), lysine has emerged as a crucial amino acid residue for histone modifications over the past decade. Interestingly, lysine modifications of non-histone proteins are also mediated by some of the known histone-modifying enzymes, and can be reversed by antagonizing activities just as observed for histone modification. For example, lysine methylation has recently been identified as a novel modification of the p53 tumor suppressor in addition to previously known modifications such as acetylation and ubiquitylation [71]. Histone-modifying enzymes involved in methylation/demethylation of p53 include SYMD2, SET9, and LSD1.

Chromatin remodeling complexes are energy-driven, multi-protein machinery that allows access to specific DNA regions or histones by altering nucleosomal positions, histone-DNA interactions, and histone octamer positions (Fig. ​(Fig.1A).1A). These chromatin remodellers have a catalytic ATPase to induce changes in local chromatin structure covering one or two nucleosomes. The ATPases in chromatin remodeling complexes are grouped into three subfamilies: the SWI/SNF ATPases, the imitation switch (ISWI) ATPases, and the chromodomain and helicase-like domain (CHD) ATPases. Several recent reviews have summarized the current understanding of diversity and specialization of chromatin remodeling complexes and modulation of remodeller activity by nucleosome modifications [72, 73].

In many cases, chromatin modifications serve as recognition sites for the recruitment of effector modules that read and implement modification-encoded biological messages [42, 74]. Several distinct binding modules have been identified in various nuclear proteins, coupling a particular histone modification with cognate effector proteins (Table ​(Table2).2). Thus, the composition of modifications on a given histone can either recruit or occlude a set of proteins. Effector proteins may alter chromatin structure by binding two or more nucleosomes as found with HP1 and Polycomb group proteins [74]. Effector proteins can also act as an adaptor to attract additional chromatin-modifying enzymes or remodeling complexes to augment the chromatin alteration initiated by the modification. Such an example can be found in HP1 binding to trimethylated H3K9 [75] and DNMT1 [76]. These initial interactions can recruit SUV39H1 and/or DNMT1 and promote further H3K9 methylation, HP1 binding, and DNA methylation, which may in turn result in further transcriptional gene silencing or chromatin repression [77] (Fig. ​(Fig.1B).1B). Similarly, the PHD domain of BPTF, a component of the NURF chromatin remodeling complex, recognizes trimethylated H3K4 and brings the remodeller with it [78]. Some other effector proteins have enzymatic activities themselves, as exemplified by CHD1 remodeling ATPase, which binds to trimethylated H3K4 and introduces active structure remodeling [42]. Similar effector proteins have been identified for DNA methylation. A series of methyl CpG-binding proteins, such as MBDs and MeCP2, has demonstrated the ability to interpret DNA methylation marks in different biological contexts (reviewed in [56]). Specifically, it has been demonstrated that interpretation of DNA methylation marks by MBDs and MeCP2 has additional assurance via recruitment of HDACs for gene silencing [25, 79, 80]. Recently, another methyl CpG-binding protein UHRF1 has been shown to recruit DNMT1 itself onto chromatin to facilitate the faithful inheritance of genomic methylation patterns [81, 82].

PcG proteins form two distinct multi-protein complexes, Polycomb repressive complex 1 (PRC1) and PRC2, although mammals have two additional PRC2-related complexes. The components of each complex in different organisms and their recruitment/mechanism of action have been reviewed comprehensively [88, 89]. The catalytically active component of PRC2, EZH2, catalyzes trimethylation of histone H3K27, and this enzymatic activity is required for PRC2-mediated silencing. The H3K27 methylation mark deposited by PRC2 recruits PRC1 via its chromodomain-containing components, which is believed to facilitate condensation of chromatin structure. Other properties of PRC1 also contribute to transcriptional silencing. PRC1-mediated ubiquitylation of histone H2A is critical for Hox gene silencing by an unknown mechanism [90]. In mammalian cells this robust PcGmediated repression appears to be stabilized by DNA methylation since EZH2 can directly recruit DNA methyltransferases to target genes [28]. Furthermore, H3K27 methylation by PcG predisposes the marked genes to de novo methylation leading to aberrant silencing in cancer cells [91]. Although it remains unknown in mammalian cells, there may be additional mechanisms other than histone/DNA modifications in PcG-mediated repression, since studies in Drosophila have implicated other silencing mechanisms such as direct interactions with the transcriptional machinery and transcription of non-coding RNA (ncRNA) (reviewed in [9]). In fact, PcG complexes have been shown to participate in gene silencing during X-chromosome inactivation and genomic imprinting where ncRNAs play a critical role in silencing mechanisms, which is reviewed later in this contribution. As the mechanistic opposite of PcG, trxG proteins also form several multimeric complexes. The trxG-associated MLL1 has been shown to catalyze histone H3K4 trimethylation that is recognized by BPTF, a subunit of NURF chromatin remodeling complex. This targeting of the remodeling complex to histones methylated by trxG is thought to facilitate active chromatin formation by repositioning nucleosomes on the promoter [78].

In addition to activation of genomic programs leading to specific cell types, another equally important epigenetic event during development is that a cell must silence alternative gene expression programs specific to other cell types to secure its fate. The best example of this lineage restriction is found in neurogenesis, during which neural cell fates are acquired in the developing nervous system, and neuron-specific genes are repressed in non-neuronal cells outside the nervous system. This suggests that neuronal chromatin is epigenetically programmed in different cellular contexts. REST (repressor element 1-silencing transcription factor), a repressor of neuronal genes containing a conserved RE1 provides a link between epigenetic mechanisms and neurogenesis by establishing silent chromatin states in cooperation with other corepressors and chromatin modifiers (reviewed in [92]). The corepressor CoREST confers more specialized repression mechanisms, such that the RESTCoREST complex recruits various chromatin modifiers for long-term silencing of neuronal genes in terminally differentiated non-neuronal cells. Chromatin-modifying enzymes and other epigenetic silencing factors involved in this process have been extensively reviewed [92].

In contrast to stable and inheritable silencing of neuronal chromatin in terminally differentiated nonneuronal cells, the situation in ES cells and neuronal progenitors impose another aspect of epigenetic concern on gene expression since these cells should be able to relieve the silent chromatin state upon differentiation to allow a lineage-specific gene expression. A comparative analysis of neuronal gene chromatin in terminally differentiated fibroblasts and pluripotent ES cells has revealed that stem cells and progenitors possess a poised chromatin status for subsequent neuronal differentiation with distinct differences in epigenetic marks and transcriptional features [93]. This study suggests that the core REST complex establishes characteristic chromatin states by recruiting different chromatin modifiers in non-neuronal and ES cells, emphasizing the role of REST and its corepressors in building plasticity of neuronal chromatin.

As mentioned above, each imprinted gene cluster contains at least one ncRNA gene that plays a crucial role in silencing of the multiple protein-coding genes in the cluster by cis-acting mechanisms. Current understanding of imprinted gene expression/silencing has been gained from the studies on six well-characterized clusters, of which four (Igf2r/Air, IC2/Kcnq1, Gnas, and PWS/AS) are maternally imprinted, and two (Igf2/H19 and Dlk1) are paternally imprinted by DNA methylation acquired in the male gamete (as reviewed in [111]). Despite the differences in gene organization and ICR functions in different clusters, a few common features of imprinted gene expression/silencing can be derived. The unmethylated ICRs are implicated in all six clusters as positive regulators of ncRNA expression. In maternally imprinted clusters (Igf2r/Air and IC2/Kcnq1), the unmethylated ICR works as a promoter for a paternally expressed ncRNA that is an antisense orientation to at least one of the genes in the cluster. While deletion of the methylated maternal ICR has no effect on maternally inherited alleles, deletion of the unmethylated paternal ICR reverses the parental-specific expression pattern such that ncRNA expression is lost and biallelic gene expression is obtained by abrogation of paternal silencing. Truncation of ncRNAs at these loci also has similar effects on paternal gene expression, relieving silencing of the paternally inherited alleles (reviewed in [122]). ICRs in paternally imprinted clusters appear to utilize different mechanisms to control the imprinted gene expression. For example, the H19 ncRNA at the Igf2/H19 locus is expressed from the unmethylated maternal chromosome but the ICR does not act as a promoter. Rather, it serves as a boundary element for CTCF (CCCTCbinding factor) that is a chromatin insulator protein [83]. The CTCF protein binds the unmethylated maternal ICR blocking the interaction of downstream enhancers with Igf2 and Ins promoters , while it does not affect the interaction between the enhancers and H19 ncRNA promoter. On the paternal chromosome, the DNA methylation imprint prevents CTCF binding, thus allowing the enhancers to drive the expression of Igf2 and Ins genes [123]. Interestingly, the CTCF protein has been shown to have an additional function at the Igf2/H19 locus, protecting the maternal allele from methylation post-fertilization [124].

Retrotransposons transpose with the help of reverse transcriptase and they can be divided into two subfamilies depending on the presence or absence of direct repeats at the end of the element called LTR. LINE elements do not contain LTRs and account for 17% of the total human genome. A small percentage of these autonomous non-LTR retrotransposons in the human genome remain active [127]. Intracisternal Aparticles (IAPs), MaLR and Etn elements are active LTR retrotransposons present in the mouse genome [128]. In contrast, SINE elements are non-autonomous, non-LTR retrotransposons. The Alu repeats are most common SINE families in human genome and account for 10% of the whole genome mass [129]. B1 and B2 are major SINE elements in mouse genome [130]. DNA transposons do not require reverse transcriptase for integration event into the genome. Instead, a self-encoded protein called transposase can recognize terminal inverted repeats (TIR) of the DNA transposons for genome integration. To date, no evidence has been available for the presence of active DNA transposon, although many copies of inactive fossil DNA transposons are present [125].

There are many ways that transposable elements can interfere with the structure and regulation of gene expression in the genome. They include insertion, deletion or an inversion of genomic sequences. Recombination between non-allelic repeats can lead to rearrangements/translocations, and strong constitutive promoters of retrotransposons can express chimeric mRNA [128, 131]. Transposable elements also can serve as promoters, enhancers, silencers, and alternative splicing site and thereby modulate the expression of related genes [132]. In contrast to the huge number and different modes of gene disruption associated with these transposons, the damage that transposons cause to their host is generally minor. For instance, only 1 in 600 germ line mutations in human can be attributed to transposon insertions [133]. In fact, the damage caused by transposons is largely limited by active repression of these endogenous parasitic elements. Most transposon copies reside in heterochromatin, which by definition contains regions of silent DNA so that they possess little harm to the host genome.

Mammalian (and other vertebrates) genome structure is protected against these parasitic transposable elements. DNA cytosine methylation and modification of histone tails (methylation at H3K9 and deacetylation) are associated with the host-defense system [134, 135]. Drosophila suffers from abundant transposon-mediated mutations and lacks DNA methylation, which adds supportive evidence to the above scenario [136]. In mouse, the transcription of IAP is normally repressed but is greatly induced in embryos lacking DNMT1, demonstrating that methylation is responsible for the repressed state of these elements [137]. Human endogenous retroviruses (HERVs) resemble simple retroviruses in structure. The demethylation of HERVs has been examined in a limited number of cancers (germ cell tumors and cancers of the ovary, testicles and bladder). In these cases, HERV hypomethylation increases with malignancy [138]. In vitro transcription assays using site-specific mutagenesis and methylation demonstrate that methylation of critical CpG dinucleotides within the LINE promoter is enough to ensure repression of transcription. In a number of cancers, hypomethylation of LINE elements is evident, compared to their normal counterparts or unaffected adjacent tissues [139, 140]. LINE hypomethylation can occur early in cancer initiation, notably in colon and prostate cancers. In most other cancers studied (leukemias, urothelial, ovarian and breast cancers), LINE demethylation increases with the degree of malignancy. Therefore, depending on the cancer type, LINE hypomethylation may be useful as an early detector of cancer or a prognostic indicator [141].

Modification of histones also plays a role in suppressing TE transcription. Chromatins associated with TEs are enriched for methylation of histone H3K9, which is a signal for transcriptional suppression. Mutation in Suv39, a H3K9 methyltransferase, leads to reactivation of TE transcription in mouse ES cells [135]. In A. thaliana, DDM1 is required for TE silencing. Mutation of ddm1 results in a loss of DNA and H3K9 methylation, also leading to active TE [142]. Lsh1, mouse homolog of ddm1 is required for TE suppression, and elevated TE transcripts were observed in mutant Lsh1^−/− embryos [143].