With the recent completion of the human genome, approximately 23,800 genes have been identified. The use of functional genomic approaches has provided much insight into how genes shape a cell or an organism. However, our DNA sequence is not the sole determinant of a phenotype, since each cell of an organism contains the same genetic information. In fact, it is the differential regulation of genes in time (i.e., during development) and space (i.e., tissue) that determines cell fate and eventual phenotype. During every cell cycle, the entire genome needs to be accurately replicated during the S-phase. Similarly, the gene-expression profile needs to be replicated as well; after all, a dividing hepatocyte should give rise to a fully differentiated new hepatocyte even though it shares the same genome with a neuron. The study of “heritable changes in gene function that cannot be explained by changes in the DNA sequence” is referred to as epigenetics [1]. These epigenetic modifications can be stably maintained through mitosis; for example, cells with an epigenetically silenced transgene can be cultured for more than a year without losing the epigenetic information [2]. Additionally, epigenetic information can, in some cases, be transferred through meiosis and therefore affect the next generation [3]. Even though epigenetic information can be inherited through many cell divisions and even generations, it is also reversible. In fact, unlike DNA that, with exceptions, only changes due to mutagenesis or replication errors, epigenetic information is also a product of environmental factors. The reversibility of the system is illustrated by the epigenetic reprogramming of oocytes after fertilization, when widespread demethylation of the genome is observed. After implantation, de novo methylation restores DNA methylation levels in the embryo [4]. Besides physiological reprogramming during development, somatic cells can be reprogrammed into pluripotent stem cells through nuclear transfer or the expression of specific pluripotency-associated transcription factors [4, 5]. In addition, also cancers seem to exploit the reversible nature of the epigenome by silencing tumor suppressors or activating oncogenes [6, 7].

Much research on epigenetic inheritance has focused on DNA methylation, histone variants, and covalent modifications of histone tails [8]. However, throughout the cell cycle, these epigenetic marks face two major caveats; they need to be accurately replicated along with the DNA and, in addition, they need to survive mitosis. During DNA replication this means that any covalent modifications to the DNA need to be copied to the daughter strands. In addition, the passing replication machinery is likely to disrupt many DNA–protein interactions and higher-order chromatin organization and, hence, these need to be either maintained through the S-phase or reorganize after replication. During mitosis, chromatin can be condensed by more than a factor 10,000 [9], again disrupting higher-order chromatin structures and protein–DNA interactions. In this review we describe the molecular mechanisms behind epigenetic inheritance through the cell cycle in mammalian cells. We will focus on DNA methylation, histone modifications and how these are established and maintained by heterochromatin protein 1 (HP1), as well as the PcG/TrxG system. In addition, we consider the influence of small non-coding RNAs (ncRNAs) and nuclear organization of the genome on epigenetic inheritance.

DNA methylation has been demonstrated to play an important role in epigenetic inheritance.

For example, at the murine Agouti and Axin loci (determining coat color and tail phenotype, respectively [10]) where nearby retrotransposons or intracisternal A-particles (IAP) can affect gene expression. DNA methylation of these IAPs can silence IAP promoter activity and thereby restore wild-type expression. This can result in genetically identical mice that display different coat colors or tail phenotypes. Interestingly, the methylation status of these IAPs can be inherited across generations [11, 12]. Other examples that illustrate the role of DNA methylation in epigenetic inheritance are X-chromosome inactivation and parental imprinting (epigenetic silencing of one allele) which are impaired when DNA methylation machinery is disrupted [13, 14]. In addition, many cancers show local hypermethylation at genomic locations corresponding to tumor suppressors, such as cell cycle inhibitors and genes that mediate the DNA damage response [6].

In mammalian cells, DNA methylation occurs on cytosine residues (m5C) and this occurs almost exclusively at cytosine-guanidine dinucleotides (CpG) [15, 16]. These nucleotides are underrepresented in the genome, and approximately 70–80% of all CpGs are methylated [15, 17, 18]. Although most CpG dinucleotides are methylated, the genome also contains CpG islands: short CpG-rich genomic regions which are generally unmethylated (discussed below). Functionally, DNA methylation is associated with a repressed chromatin state and is required for both ‘genome housekeeping’ as well as for gene expression regulation and maintenance of cell identity [19, 20]. The former is illustrated by the widespread methylation of retrotransposons [19]. Abolishing methylation at these sites can result in reactivation of the transposon promoter activity [21]. In addition, methylation might be involved in maintaining chromosomal stability, as noted by chromosomal rearrangements and abnormalities as a result of chemical inhibition of methylation and genetic studies [15, 19]. Global hypomethylation as observed in certain cancers might therefore be a cause of chromosomal instability [22]. Of particular interest when examining the role of DNA methylation in gene regulation are CpG islands [15]. These CpG islands are enriched at genes, since approximately 60% of all genes in the human genome contain a CpG island upstream [15]. Although methylation of CpG islands is infrequent, it does result in a stable repression of genes [23]. The latter is also illustrated by experiments that showed that increased CpG density at a promoter induces more robust silencing of episomes [24]. In addition, although genes with low CpG content at their promoters are readily reactivated by the viral SV40 enhancer, transcriptional silencing of genes with an upstream CpG-dense region is not reversed by the SV40 enhancer [25].

The maintenance of DNA methylation throughout the cell cycle is mainly mediated by three DNA methyltransferases, namely DNMT1, DNMT3a, and DNMT3b [16]. DNMT1 is primarily involved in the propagation of DNA methylation during the cell cycle due to its affinity towards hemimethylated DNA [26]. During the S-phase, the two parental DNA strands are separated for DNA replication, resulting in two hemimethylated strands, and two newly synthesized strands. At this time, DNMT1 associates with proliferating cell nuclear antigen (PCNA), an important member of the replication machinery [27], and NP95, a protein that specifically binds hemimethylated DNA, and subsequently restores methylation on the daughter strands [28]. This process has been shown to be required for both in vitro as in vivo maintenance of DNA methylation [28, 29]. However, even though the accuracy of DNA methylation replication is reported to be approximately 95–99% [30], the maintenance activity of DNMT1 is not sufficient to explain all epigenetic inheritance considering DNA methylation. In addition, both de novo methyltransferases DNMT3a and -b seem to be required for complete epigenetic inheritance. For one, in murine ES cells abrogation of both DNMT3a and -b increases the amount of hemimethylated DNA at repeats by 30% [31]. Furthermore, ES cells deficient for both de novo methylases display reduced levels of methylation over time [32]. Likely, the epigenetic inheritance of DNA methylation requires the maintenance transferase DNMT1 as well as the de novo methyltransferases DNMT3a and -b for perhaps proofreading and methylation of specific DNA sequences [32, 33].

Throughout the cell cycle, the methyltransferases display differential temporal expression and localization. As expected, DNMT1 expression levels peak during the S-phase. During this phase, DNMT1 is responsible for the faithful replication of methylated DNA sequences and, hence, it colocalizes to DNA replication foci [28]. However, the de novo methyltransferases (DNMT3a and -b) show a more heterogeneous profile. DNMT3a seems to be primarily associated with heterochromatin, or inactive chromatin [34]. DNMT3b, however, displays diffuse nuclear localization although DNMT3a and -b colocalize at sites of pericentromeric heterochromatin [34]. The expression levels of the de novo DNMTs also differ during the cell cycle. DNMT3b mRNA levels, like DNMT1, are reduced during the G1/G0 phase of the cell cycle, while they peak during the S-phase [35]. DNMT3a, however, is expressed at approximately constant levels throughout the cell cycle [35]. One possibility is that DNMT3b, in addition to DNMT1, is primarily responsible for DNA methylation maintenance. Abrogation of DNMT3b results in decreased genome-wide methylation levels, while DNMT3a disruption results in a less severe phenotype [36, 37]. The expression peak of DNMT1 and DNMT3b during the S-phase might support the maintenance function DNMT1 and -3b. The abundant DNMT3a expression during the cell cycle indicates a more flexible mechanism for this particular methyltransferase, such as proofreading or DNA methylation of specific loci in response to certain extracellular or intracellular stimuli.

Currently, DNA methylation is the most established system implicated in epigenetic inheritance. The recognition of hemimethylated DNA by DNMT1 provides an efficient method for restoring DNA methylation during the S-phase, supplemented by the de novo methyltransferases DNMT3a and -3b. However, although DNA methylation is generally associated with robust silencing, it is not irreversible. This is illustrated by the demethylation of the paternal and maternal genomes in the zygote after fertilization [38]. In addition, DNA demethylation is required for the reactivation of multiple pluripotency-associated genes during reprogramming of differentiated cells into iPS cells [39]. Nevertheless, the molecular mechanism of DNA demethylation in mammals is disputed. One possibility for cells to demethylate their genome is by passive demethylation. By seizing maintenance methylation activity during DNA replication (i.e., preventing DNMT1 nuclear localization), the genomes of daughter cells become increasingly demethylated [40]. However, this process might not fully explain all observed demethylation events in the mammalian genome. For example, the paternal nucleus in the zygote undergoes rapid genome-wide demethylation after fertilization prior to DNA replication [41]. Furthermore, non-dividing peripheral monocytes undergo DNA demethylation upon differentiation [42], indicating active demethylation, as passive demethylation requires replication.

One candidate for mediating DNA demethylation is activation-induced deaminase (AID). In primordial germ cells, cells that normally undergo genome-wide demethylation, a knockout of this protein results in a significant increase of methylated DNA [43]. Furthermore, the demethylation of pluripotency-associated genes depends on AID [44]. In addition to AID, recent studies also identified several members of the elongator complex that are required for the previously discussed paternal genome demethylation in zygotes [45]. However, although both passive and active DNA demethylation are likely to occur during development, it remains to be determined whether it occurs outside specific developmental events and artificially induced conditions.

All in all, DNA methylation plays an important role in gene expression in mammalian cells. Methylation of CpG islands in promoter regions is often associated with gene silencing and aberrant DNA methylation has been shown to occur in many cancers, leading to silencing of some tumor suppressor genes [15]. Reversal of DNA methylation therefore has emerged as a potential strategy for treatment of cancer [46, 47].

Although DNA methylation is an established mechanism for stable epigenetic inheritance, organisms that hardly methylate their DNA, such as yeast and Drosophila, can still transmit epigenetic information through the cell cycle, likely by altering chromatin structure [48, 49]. The major determinants of chromatin structure are the histone proteins. DNA coils around histones (complexes referred to as nucleosomes), which eventually contributes to efficient packaging of approximately 2 m of DNA in a single nucleus. Each nucleosome contains an octamere of four core histone proteins, namely H3, H4, H2A, and H2B [50]. However, besides the structural role, histone proteins are crucial in the regulation of gene expression. By altering the local chromatin state, the accessibility of particular DNA sequences to, for example, transcription machinery, can be regulated resulting in activation or repression of genes [51]. In addition, effector proteins can be recruited to mediate transcriptional silencing or activation [52]. Since the local chromatin state contributes to maintaining transcriptional activation or repression, it is not surprising that it needs to be transmittable through the cell cycle, e.g., in order to maintain expression of tissue specific genes. However, this system also needs to allow for dynamic changes, such as required for stress responses or DNA repair. In general, chromatin functionality can be regulated either by the placement of specific histone variants throughout the genome (e.g., the placement of H3.3 at actively transcribed loci, discussed below), or by the covalent modification of histone N-terminal tails [53]. For the case of epigenetic inheritance, a number of histone modifications and histone variants have been identified that might be maintained through the cell cycle or play a role in epigenetic inheritance. Note that the concept of histone modifications as the direct carriers of epigenetic information is disputed as reviewed in [54–56].

The Polycomb and Trithorax (PcG/TrxG) family of proteins was first identified in Drosophila where it was shown to be essential for the stable inheritance of a repressed or active gene state, respectively [99]. During Drosophila development, specific homeotic genes (Hox) are activated in a patterned fashion throughout the embryo [99, 100], and although the PcG/TrxG proteins are not required for the initiation of Hox gene transcription, they are essential for the maintenance of these genetic programs through cell divisions [99]. More recent genome-wide analyses have identified hundreds genes besides Hox genes that can be silenced or activated by the PcG/TrxG proteins, which exert their effects at Polycomb regulatory elements (PRE) throughout the genome [101, 102]. In mammalian cells, PREs have not been identified on a large scale [103], although Woo et al. [104] recently proposed the existence of a mammalian PRE. However, PcG/TrxG proteins are known to play crucial roles in mammalian development, cell fate determination, cancer [7, 105], and even immunity [106]. On a molecular level, the PcG and TrxG proteins function in distinct multiprotein complexes and act as chromatin modifiers by both inducing the covalent modification of histones as well as through other mechanisms. Before examining the potential mechanisms by which PcG and TrxG complexes can confer epigenetic inheritance, first a more in-depth look at their specific methods of silencing is provided.

In addition to the mechanisms of epigenetic inheritance mentioned above, RNA might also mediate the stable transmission of epigenetic states, in particular small interfering RNAs (siRNAs) and long non-coding RNAs (ncRNA). An established example of this is Xist, a long ncRNA that mediates the inactivation of one of two X-chromosomes in females. Xist only associates with the inactive X-chromosome and subsequently recruits epigenetic silencing machinery, such as DNA methylases and PcG complexes [144]. Interestingly, the translocation of the Xist gene to autosomes results in subsequent silencing of the specific autosome [145]. Besides X-inactivation, there is also ample evidence for the involvement of ncRNAs in the previously discussed epigenetic inheritance mechanisms, especially heterochromatin formation by HP1, and the PcG/TrxG system. Since much of the knowledge on the role of ncRNAs in epigenetic inheritance is based on model organisms such as yeast (S. pombe), plants, and Drosophila, the exact involvement and specific mechanisms of mammalian ncRNA-mediated silencing is still uncertain.

RNA-mediated epigenetic inheritance is implicated in the formation and maintenance of heterochromatin [70, 146–148]. In S. pombe, the disruption of the RNAi machinery leads to heterochromatin defects, including H3K9 methylation defects [149]. One potential molecular model for RNAi-mediated heterochromatin silencing [70, 148, 150] involves the transcription of (peri) centromeric repeats [151, 152]. These transcripts are required for RNAi production; the resulting small interfering RNAs (siRNAs) together with Ago1, Tas3 and Chp1 form the RITS (RNA-induced transcriptional silencing) complex, which in turn recruits other heterochromatin-modifying factors, such as Swi6 (yeast homologue of HP1) and Clr4 (yeast homologue of Suv39h1/2). The recruitment of RITS, which in itself requires H3K9Me3 [150], results in subsequent heterochromatin formation and H3K9 methylation. Interestingly, the transcription of these repeats occurs mostly in the S-phase [153, 154], and therefore provides an interesting candidate for epigenetic inheritance of heterochromatin during DNA replication; transcription of repeats during the S-phase results in a positive feedback loop resulting in heterochromatin formation and maintenance [70, 148]. However, whether similar mechanisms occur in mammalian cells is somewhat disputed. There is evidence for the involvement of RNAi in mammalian heterochromatin formation [155–157]. For example, the formation of higher-order heterochromatin structures and HP1 localization to these requires an RNA component [157]. However, one study reports that Dicer-knockout ES cells display little pericentric heterochromatin defects [158].

Besides heterochromatin formation, siRNAs have also been implicated in mediating DNA methylation. Using short-hairpin RNAs (shRNA) against RASSF1A, Castanotto et al. [159] showed increased promoter methylation in HeLa cells. In plants, RNAi-mediated DNA methylation is readily observed, although in mammals this might be limited to the rare non-CpG DNA methylation as observed mostly early in development [160]. Currently, more research in mammalian cell systems and organisms are required for an established role of RNAi in DNA methylation.

RNAs are also likely involved in mediating some epigenetic functions of the PcG/TrxG system, although this seems mostly based on long ncRNAs rather than RNAi [161]. In Drosophila, many PREs are transcribed and produce ncRNAs, and although mammalian PREs are currently disputed, there is also evidence for the production of ncRNAs near mammalian PcG/TrxG targets [134, 161]. Furthermore, PcG/TrxG proteins have been shown to bind RNA, although whether this is sequence-dependent is disputed [161]. Interestingly, in Drosophila the mere transcription of PREs might promote transcriptional activity of PcG-targeted genes and thereby counteract PcG silencing [134]. For example, using a transgene containing a PRE followed by a reporter gene, Schmitt et al. [162] showed that mere transcription through the PRE is sufficient to activate the reporter gene. In addition, the transcribed ncRNA of a PRE results in TrxG recruitment, resulting in gene activation [163]. These findings support the previously discussed model of ‘standard silencing’, which states that active transcriptional sites should be marked by e.g., H3.3 replacement or RNA components, in order to transmit a heritable active chromatin state. In mammalian cells, the transcriptional activation of HoxA genes has also been linked to transcription of intergenic ncRNAs at the HoxA cluster [164]. However, several findings seem to contradict the standard silencing theory. Importantly, several mammalian ncRNAs have been detected that upon transcription induce PcG-mediated silencing, rather than to alleviate it [161]. For example, the Kcnq1ot1 ncRNA [165] is required for the silencing of the Kcnq1 locus. This ncRNA associates with PcG proteins in a tissue specific manner. In addition, the abrogation of the HOTAIR ncRNA results in upregulation of the HoxD cluster [166]. Interestingly, in mammalian cells most non-transcribed genes still produce short ncRNA transcripts [167]. Many of these ncRNAs can bind PRC2 [168, 169] and ncRNA production at activating genes declines. One model, as proposed by Guenther and Young [170], is that short transcribed ncRNAs recruit PRC2 and results in repression. However, transcription initiation as promoted by transcription factors recruits TrxG proteins and results in activation.

Although the exact mechanism and causality of the interaction between ncRNAs and the PcG/TrxG remains elusive, the involvement of an RNA component is highly likely. Similarly, an RNA component is likely to function in mammalian heterochromatin formation. However, how these ncRNAs might be regulated throughout the cell cycle is currently unclear.

The current view of the nucleus is a dynamic one; rather than being just a place of storage of genetic information, the nucleus contains specific functional domains, such as the nucleolus, the inactive heterochromatin, and the active euchromatin [171–173]. These latter two are even replicated in a spatio-temporal distinct manner during the S-phase. In recent years, much research has focused on long-range DNA interactions. These interactions can occur in cis, so in neighboring sequences on the same chromosome, as well as in trans, interactions with distant regions on the same chromosome or even interactions with sequences at different chromosomes. An example of each is the regulation of β-globin genes through looping to a cis-regulatory ‘master enhancer’ that also controls other globin genes, and the brief interaction of the X-inactivation centers (Xic) of two X-chromosomes in female cells just before the random X-inactivation of one of the copies [174]. These higher-order chromatin rearrangements can also be induced. For example HoxB genes can loop out of their ‘chromosome territory’ upon retinoic acid-induced expression, and steroid-induced activation of nuclear receptors results in the formation of interchromosomal contacts required for enhanced transcription of the induced genes [175, 176]. Interestingly, nuclear organization and long-range DNA structures might potentially function in epigenetic inheritance. For example, there is some evidence for mitotic transmissibility of chromosome organization in the nucleus, at least through several cell divisions [177, 178]. These findings, however, are somewhat disputed. Other studies confirm that chromosome positions in the nucleus are fixed throughout the cell cycle, although they change after mitosis. Subsequently, in G1 the chromosomes are unpacked into a different nuclear organization [179–181]. Regardless of the heritability of chromosome organization in the nucleus, considerable evidence has accumulated for long-range DNA interactions in heterochromatin formation and the PcG/TrxG system. However, are these DNA structures themselves involved in epigenetic inheritance? How could they survive DNA replication and mitosis? These questions will be addressed below.

Evidence for long-range DNA contacts during heterochromatin formation comes from studies in Drosophila, where the insertion of a heterochromatic sequence in the brown gene (bw), results in dominant repression and localization of both the mutant allele, as well as the wild type allele to heterochromatic regions, a process termed position-effect variegation (PEV) [182]. In mammalian cells there is also evidence for a role of DNA organization in heterochromatin formation. It was reported that localization of the β-globin locus away from heterochromatin influences its histone modification (acetylation) and transcriptional state [183]. Although localization of genes to heterochromatin at the nuclear periphery correlates with their transcriptional inactivity [184], the maintenance of these structures throughout the cell cycle is unclear. In Drosophila, it was reported that at least the interaction of the dominant repressive bw mutant with heterochromatin is lost during DNA replication and is re-established in G1 [185].

DNA organization is also likely involved in the PcG/TrxG system [173, 186]. For one, PcG proteins are known to localize to PcG bodies [187, 188] that might cluster Polycomb targets. In Drosophila, the introduction of extra copies of the PRE Fab-7 induces clustering with each other and the endogenous Fab-7 locus [189] in a PcG-dependent manner. Interestingly, the loss of the endogenous locus results in the loss of PcG silencing at the site of the inserted artificial Fab-7 fragment. This loss of silencing can be stably passed onto the next generation, even after restoring the endogenous Fab-7 locus. This indicates a potential role for nuclear organization in epigenetic inheritance of PcG silencing [186, 189]. Although it was previously discussed that a direct role for RNAi in PcG silencing is currently disputed [161], the nuclear localization of the Fab-7 fragments depends on the RNAi machinery [190, 191].

Insulator proteins, and the insulator sequences that they bind, may play a role in mediating epigenetic inheritance. Although several functions have been published, the most established function of insulator proteins is to insulate promoters from enhancer elements and thereby affecting transcription. In Drosophila the expression of a dominant-negative form of the insulator protein BEAF (boundary element-associated factors) results in enhanced PEV through the disruption of chromatin organization [192]. In mammalian cells, CTCF, an insulator protein, is an interesting candidate for mediating epigenetic inheritance by altering nuclear organization [193–195]. CTCF binds to insulator sequences throughout the mammalian genome where it performs its function by looping DNA and hence altering nuclear organization of genes [193, 195]. One example of CTCF function is the expression of the paternally imprinted Igf2 gene [193, 194] on the murine chromosome 7. Genomic imprinting of genes is the monoallelic expression of certain genes transferred either paternally or maternally [196]. Under normal circumstances, the maternal imprinting control region (ICR) is unmethylated leading to CTCF binding, subsequent looping, and silencing of the maternal Igf2 gene. However, the paternal ICR is methylated which prevents CTCF binding and therefore facilitates transcription. In addition to disrupting Igf2, the abrogation of CTCF function at the ICR can also disrupt the transcription at specific genes located at different chromosomes [197].

Interestingly, there are several indications that CTCF might be stably transmitted through replication and mitosis. For one, some insulator sequences and CTCF-binding sites have been detected within introns of genes. In experiments in Drosophila, insertion of an insulator binding sequence in an intron retains its function [198]. Also in mammalian cells, CTCF-binding sites have been detected in introns [199]. Although this does not directly implicate maintenance through DNA replication, at least local double-strand disruption by transcription machinery does not hamper insulator function. In addition, several studies have shown continued CTCF binding at mitotic chromosomes, and even continued Igf2–ICR interaction through mitosis [200]. Note that CTCF has also been shown to interact with PRC2 and is a known regulator of heterochromatin spreading, which also might indicate a role for CTCF in epigenetic inheritance through these systems.

As discussed, cells depend on the inheritance of epigenetic information throughout the cell cycle to maintain their state of differentiation. So far, DNA methylation is the most established epigenetic mark that can stably confer heritable phenotypes. However, more and more evidence is accumulating for the epigenetic inheritance of chromatin states and even higher-order chromatin structures. The H3K9Me3-HP1 system and the PcG/TrxG systems are the obvious candidates for the transfer of chromatin states through cell divisions. Interestingly, although HP1-mediated heterochromatin and PcG-silenced areas generally do not colocalize, DNA methylation seems to be involved in both systems.

Heterochromatin is, in general, fairly gene-poor, repeat-rich, and relatively CpG-poor, although most CpG dinucleotides that are present are methylated [70, 201]. The interaction between HP1, H3K9 KMTs and the DNA methylating enzymes have been shown in multiple studies [202, 203]. However, the causality of the interaction is still disputed and evidence exists for both the recruitment of the HP1-KMT machinery to methylated DNA, as well as the recruitment of DNMTs to sites of H3K9 methylation [204]. This could indicate a self-reinforcing mechanism of both DNA methylation and H3K9 methylation as proposed by Fuks [204].

Interestingly, although the methylation of H3K9 does occur at promoters with relatively low CpG content, it does not occur at promoters containing CpG islands [88, 201, 205]. As previously discussed, CpG islands are generally unmethylated CpG-dense areas found upstream of ~60% of human genes and the methylation of such islands results in robust silencing. The PcG/TrxG system and hence H3K27Me3 and H3K4Me3, however, does associate with CpG-islands. In fact, in ES cells more than 97% of PRC2 target sites contain CpG-islands or CpG-rich areas [205]. One option is that DNA methylation might reinforce PcG-mediated silencing [89, 137, 206]. In agreement with this, there is evidence that PcG target genes become methylated during development [207]. Both PRC2 as PRC1 might be responsible for this methylation by direct recruitment of DNMTs [208, 209]. Intriguingly, H3K4Me3 does not colocalize with DNA methylation [201]. In fact, DNA methylation can not occur at sequences that contain methylated H3K4, and conversely, DNA demethylation is correlated with H3K4 methylation [42]. Since the TrxG proteins depend on H3K4 trimethylation in order to maintain an open chromatin conformation, it is possible that DNA methylation at CpG islands can only occur at monovalent PcG sites that do not contain TrxG.

All in all, it is tempting to suggest a model as depicted in Fig. 3, where the DNMTs can interact with both PcG/TrxG and HP1-KMT system to alter the local chromatin state.Fig. 3

Interactions of the PcG/TrxG system and the HP1-KMT system with DNA methyltransferases. Both the repressive PcG proteins as well as HP1 are known to interact with DNMTs. For the former, both PRC1 as PRC2 directly interact with DNMTs, and methylation of PcG sites might reflect a state of stable transcriptional silencing. HP1 also interacts with DNMTs although the causality of the interaction is not yet resolved; there is evidence for both the recruitment of DNMTs by H3K9Me and HP1, as well as recruitment of HP1-KMT to methylated DNA. The TrxG proteins, however, are likely to oppose DNMT action, as H3K4Me3 and DNA methylation are mutually exclusive. The fact that both repressive chromatin-modifying systems (PcG and HP1) interact with DNMTs, while the activating TrxG antagonizes DNMTs could support the notion that DNA methylation might be, in certain genomic regions, an additional ‘layer’ of silencing

All in all, we have reviewed the major epigenetic systems that are implicated in the stable maintenance of cell fate. In order to confer a similar phenotype after cell division, the epigenetic systems need to be faithfully replicated at their genomic locations during the S-phase and survive mitosis, or at least be re-recruited after the M-phase. From the reviewed literature two general mechanisms become apparent for the transmission of epigenetic information, namely self-recruitment and maintained binding. In the case of DNA methylation, hemimethylated DNA recruits DNMT1 in order to copy the majority of methylated CpGs. For the propagation of methylated H3K27 and H3K9 through the S-phase, cells could rely on PRC2 and HP1, respectively. The former binds H3K27Me3 directly and possesses intrinsic KMT activity for that specific residue, while the latter binds H3K9Me3 and can associate with known H3K9 KMTs as well as histone chaperones, In addition to self-recruitment, there is also evidence for the continued binding of certain epigenetic modifiers through phases of the cell cycle. PRC1, for example, can remain bound to DNA during replication, and PRC2 was shown can be detected on chromosomes during mitosis. Even CTCF can remain bound at imprinted loci in order to maintain DNA organization during mitosis at specific loci.

Nonetheless, many questions remain on the exact mechanisms of epigenetic inheritance. For one, although the previously discussed positive-feedback mechanisms suggest transmission of histone methylation, it remains a question whether the histone modifications themselves are the primary determinants of epigenetic inheritance or whether other mechanisms (e.g., ncRNA transcription or genome organization) underlie this inheritance. Furthermore, although much research has focused on epigenetic repression, the stability and heritability of the activating TrxG and the histone variant H3.3 requires additional research in mammalian cells, as well as how these interact. Overall, an in-depth understanding of epigenetic inheritance can also aid in our understanding of human disease, as already DNA methylation and the PcG/TrxG proteins have been implicated in the pathogenesis of numerous diseases, such as cancers and infectious diseases.