Chapter 27: Epigenetics I

Edited by Trygve Tollefsbol

27.1 Introduction

Epigenetic inheritance describes the ability of different states, which may have different phenotypic consequences, to be inherited without any change in the sequence of DNA. This means that two individuals with the same DNA sequence at the locus that controls the effect may show different phenotypes. The basic cause of this phenomenon is the existence of a self-perpetuating structure in one of the individuals that does not depend on the DNA sequence. Several different types of structures have the ability to sustain epigenetic effects:

A covalent modification of DNA (methylation of a base)
A proteinaceous structure that assembles on DNA
A protein aggregate that controls the conformation of new subunits as they are synthesized

In each case the epigenetic state results from a difference in function that is determined by the structure.

In the case of DNA methylation, a gene methylated in its control region may fail to be transcribed, whereas an unmethylated version of the gene will be expressed (this idea is introduced in the Eukaryotic Transcription chapter). FIGURE 27.1 shows how this situation is inherited. One allele has a sequence that is methylated on both strands of DNA, whereas the other allele has an unmethylated sequence. Replication of the methylated allele creates hemimethylated daughters that are restored to the methylated state by a constitutively active DNA methyltransferase (DNMT). Replication does not affect the state of the unmethylated allele. If the state of methylation affects transcription, the two alleles differ in their state of gene expression, even though their sequences are identical.

FIGURE 27.1 Replication of a methylated site produces hemimethylated DNA, in which only the parental strand is methylated. A perpetuation methylase recognizes hemimethylated sites and adds a methyl group to the base on the daughter strand. This restores the original situation, in which the site is methylated on both strands. An unmethylated site remains unmethylated after replication.

Self-perpetuating structures that assemble on DNA usually have a repressive effect by forming heterochromatic regions that prevent the expression of genes within them. Their perpetuation depends on the ability of proteins in a heterochromatic region to remain bound to those regions after replication, and then to recruit more protein subunits to sustain the complex. If individual subunits are distributed at random to each daughter duplex at replication, the two daughters will continue to be marked by the protein, though its density will be reduced to half of the level before replication. FIGURE 27.2 shows that the existence of epigenetic effects forces us to the view that a protein responsible for such a situation must have some sort of self-templating or self-assembling capacity to restore the original complex.

FIGURE 27.2 Heterochromatin is created by proteins that associate with histones. Perpetuation through division requires that the proteins associate with each daughter duplex and then recruit new subunits to reassemble the repressive complexes.

It can be the state of protein modification, rather than the presence of the protein per se, that is responsible for an epigenetic effect. Usually the tails of histones H3 and H4 are not acetylated in constitutive heterochromatin. If heterochromatin becomes acetylated, though, silenced genes in the region may become active. The effect may be perpetuated through mitosis and meiosis, which suggests that an epigenetic effect has been created by changing the state of histone acetylation.

Independent protein aggregates that cause epigenetic effects (called prions) work by sequestering the protein in a form in which its normal function cannot be displayed. Once the protein aggregate has formed, it forces newly synthesized protein subunits to join it in the inactive conformation.

27.2 Heterochromatin Propagates from a Nucleation Event

An interphase nucleus contains both euchromatin and heterochromatin. The condensation state of heterochromatin is close to that of mitotic chromosomes. Heterochromatin is generally inert. It remains condensed in interphase, is transcriptionally repressed, replicates late in S phase, and may be localized to the nuclear periphery. Centromeric heterochromatin typically consists of repetitive satellite DNAs; however, the formation of heterochromatin is not rigorously defined by sequence. When a gene is transferred, either by a chromosomal translocation or by transfection and integration, into a position adjacent to heterochromatin, it may become inactive as the result of its new location, implying that it has become heterochromatic.

Such inactivation is the result of an epigenetic effect (see the section later in this chapter titled Epigenetic Effects Can Be Inherited). It may differ between individual cells in an organism, in which case it results in the phenomenon of position-effect variegation (PEV), in which genetically identical cells have different phenotypes. Genes affected by PEV have two states—active or silenced—depending on their position relative to the boundary of heterochromatin, which can lead to variegated phenotypes. This has been well characterized in Drosophila. FIGURE 27.3 shows an example of PEV in the fly eye. Some of the regions in the eye lack color, whereas others are red. This is because the white gene (required to develop red pigment) is inactivated by adjacent heterochromatin in some cells but remains active in others.

FIGURE 27.3 Position-effect variegation (PEV) in eye color results when the white gene is integrated near heterochromatin. Cells in which white is inactive give patches of white, whereas cells in which white is active give red patches. The severity of the effect is determined by the closeness of the integrated gene to heterochromatin.

Photo courtesy of Steven Henikoff, Fred Hutchinson Cancer Research Center.

The explanation for this effect is shown in FIGURE 27.4. Inactivation spreads from heterochromatin into the adjacent region for a variable distance. In some cells it goes far enough to inactivate a nearby gene, whereas in others it does not. This happens at a certain point in embryonic development, and after that point the state of the gene is stably inherited by all the progeny cells. Cells descended from an ancestor in which the gene was inactivated form patches corresponding to the phenotype of loss of function (in the case of white, the absence of color).

FIGURE 27.4 Extension of heterochromatin inactivates genes. The probability that a gene will be inactivated depends on its distance from the heterochromatin region.

The closer a gene lies to heterochromatin, the higher the probability that it will be inactivated. This is due to the fact that formation of heterochromatin is typically a two-stage process: A nucleation event occurs at a specific sequence or region (triggered by binding of a protein that recognizes the DNA sequence or other identifiers in the region), and then the inactive structure propagates along the chromatin fiber. The distance by which the inactive structure extends is not precisely determined and may be stochastic, being influenced by parameters such as the quantities of limiting protein components. Another factor that may affect the spreading process is the activation of promoters in the region; an active promoter may inhibit spreading. Genes near heterochromatin are more likely to be inactivated; however, insulators can protect a transcriptionally active region by preventing heterochromatin from spreading (see the Chromatin chapter).

The effect of telomeric silencing in yeast is analogous to PEV in Drosophila; genes translocated to a telomeric location show the same sort of variable loss of activity. This results from a spreading effect that propagates from the telomeres. In this case, the binding of the Rap1 protein to telomeric repeats triggers the nucleation event, which results in the recruitment of heterochromatin proteins, as described in the next section, Heterochromatin Depends on Interactions with Histones.

In addition to the telomeres, heterochromatin is nucleated at two other sites in yeast. Yeast mating type is determined by the activity of a single active locus (MAT), but the genome contains two other copies of the mating-type sequences (HML and HMR), which are maintained in an inactive form. The silent loci HML and HMR nucleate heterochromatin via binding of several proteins (rather than the single protein, Rap1, required at telomeres), which then leads to propagation of heterochromatin, similar to that at telomeres. Heterochromatin in yeast exhibits features typical of heterochromatin in other species, such as transcriptional inactivity and self-perpetuating protein structures superimposed on nucleosomes (which are generally deacetylated). The only notable difference between yeast heterochromatin and that of most other species is that histone methylation in yeast is not associated with silencing, whereas specific sites of histone methylation are a key feature of heterochromatin formation in most eukaryotes.

27.3 Heterochromatin Depends on Interactions with Histones

Inactivation of chromatin occurs via a combination of covalent modifications and the addition of proteins to the nucleosomal fiber. The inactivation may be due to a variety of effects, including condensation of chromatin to make it inaccessible to the apparatus needed for gene expression, addition of proteins that directly block access to regulatory sites, or proteins that directly inhibit transcription.

Two systems that have been characterized at the molecular level involve HP1 in mammals and the SIR complex in yeast. Although many of the proteins involved in each system are not evolutionarily related, the general reaction mechanism is similar: The points of contact in chromatin are the N-terminal tails of the histones.

Insight into the molecular mechanisms that regulate the formation of heterochromatin originated with mutants that affect PEV. Twenty-eight genes have been identified in Drosophila that affect PEV. They are named systematically as Su(var) for genes whose products act to suppress variegation and E(var) for genes whose products enhance variegation. These genes were named for the behavior of the mutant loci; thus, Su(var) mutations lie in genes whose products are needed for the formation of heterochromatin. They include enzymes that act on chromatin, such as histone deacetylases, and proteins that are localized to heterochromatin. In contrast, E(var) mutations lie in genes whose products are needed to activate gene expression. They include members of the SWI/SNF chromatin remodeling complex (see the Eukaryotic Transcription Regulation chapter).

HP1 (heterochromatin protein 1) is one of the most important Su(var) proteins. It was originally identified as a protein that is localized to heterochromatin by staining polytene chromosomes with an antibody directed against the protein. It was later shown to be the product of the gene Su(var)2–5. Its homolog in the yeast Schizosaccharomyces pombe is encoded by swi6. HP1 is now called HP1α because two related proteins, HP1β and HP1γ, have since been found.

HP1 contains a chromodomain near the N-terminus and another domain that is related to it (the chromo shadow domain) at the C-terminus. HP1 is able to interact with many chromosomal proteins through the chromo shadow domain while the HP1 chromodomain binds to histone H3 that is dimethylated or trimethylated at lysine 9 (H3K9me3). FIGURE 27.5 shows the structures of the chromodomain and chromo shadow domains of HP1, as well as a structure showing the interaction between the chromodomain and the methylated lysine. This interaction is a hallmark of inactive chromatin.

FIGURE 27.5 (a, b) HP1 contains a chromodomain and a chromo shadow domain. (c)Trimethylation of histone H3 K9 creates a binding site for HP1.

(a, b) Photo reproduced from G. Lomberk, L. Wallrath, and R. Urrutia, Genome Biol. 7 (2006): p. 228. Used with permission of Raul A. Urrutia and Gwen Lamberk, Mayo Clinic. (c) Structure from Protein Data Bank 1KNE. S. A. Jacobs and S. Khorasanizadeh, Science 275 (2002): 2080–2083.

Mutation of a deacetylase that acts on H3K14Ac prevents the methylation at K9, resulting in loss of the HP1 binding site. This suggests the model for initiating formation of heterochromatin shown in FIGURE 27.6. First the deacetylase acts to remove the modification at K14, and this allows the SUV39H1 methyltransferase (also known as KMT1A) to methylate H3K9 to create the methylated signal to which HP1 will bind. FIGURE 27.7 shows that the inactive region may then be extended by the ability of further HP1 molecules to interact with one another.

FIGURE 27.6 SUV39H1 is a histone methyltransferase that acts on K9 of histone H3. HP1 binds to the methylated histone.

FIGURE 27.7 Binding of HP1 to methylated histone H3 forms a trigger for silencing because additional molecules of HP1 aggregate along the methylated chromatin domain.

The state of histone methylation is important in the control of heterochromatin or euchromatin states. Methylation of H3K9 demarcates heterochromatin, whereas H3K4 methylation demarcates euchromatin. A trimethyl H3K4 demethylase found in S. pombe referred to as Lid2 interacts with the Clr4 H3K9 methyltransferase, resulting in H3K4 hypomethylation and heterochromatin formation. The link between H3K4 demethylation and H3K9 methylation suggests that the two reactions act in a coordinated manner to control the relative state of heterochromatin or euchromatin of a specific region.

Heterochromatin formation at telomeres and silent mating-type loci in yeast relies on an overlapping set of genes known as silent information regulators (SIR genes). Binding of SIR proteins can actually silence any promoter or coding region, but under normal conditions nucleation or the recruitment of SIR proteins to specific sequences allows for silencing to be targeted to specific regions of the genome—specifically the telomeres and HM loci. Mutations in SIR2, SIR3, or SIR4 cause HML and HMR to become activated and also relieve the inactivation of genes that have been integrated near telomeric heterochromatin. The products of these SIR genes therefore function to maintain the inactive state of both types of heterochromatin.

FIGURE 27.8 shows a model for the actions of these proteins. Only one of them—Rap1—is a sequence-specific DNA-binding protein. It binds to the C_1–3A repeats at the telomeres and also binds to the cis-acting silencer elements that are needed for repression of HML and HMR. The proteins Sir3 and Sir4 interact with Rap1 and also with one another (they may function as a heteromultimer). Sir3 and Sir4 interact with the N-terminal tails of the histones H3 and H4, with a preference for unacetylated tails. Another SIR protein, Sir2, is a deacetylase, and its activity is necessary to maintain binding of the Sir3/Sir4 complex to chromatin.

FIGURE 27.8 Formation of heterochromatin is initiated when Rap1 binds to DNA. Sir3/4 bind to Rap1 and also to histones H3/H4. Sir2 deacetylates histones. The SIR complex polymerizes along chromatin and may connect telomeres to the nuclear matrix.

Rap1 has the crucial role of identifying the DNA sequences at which heterochromatin forms. It recruits Sir4, which, in turn, recruits both its binding partner Sir3 and the HDAC Sir2. Sir3 and Sir4 then interact directly with histones H3 and H4. Once Sir3 and Sir4 have bound to histones H3 and H4, the complex (including Sir2) can polymerize further and spread along the chromatin fiber. This may inactivate the region, either because coating with the Sir3/Sir4 complex itself has an inhibitory effect, or because Sir2-dependent deacetylation represses transcription. It is not known what limits the spreading of the complex. The C-terminus of Sir3 has a similarity to nuclear lamin proteins (constituents of the nuclear matrix) and may be responsible for tethering heterochromatin to the nuclear periphery.

A similar series of events forms the silenced regions at HMR and HML. Three sequence-specific factors are involved in triggering formation of the complex: Rap1, Abf1 (a transcription factor), and the origin replication complex (ORC). In this case, Sir1 (which is not required for telomeric silencing) binds to a sequence-specific factor and recruits Sir2, -3, and -4 to form the repressive structure. As at the telomeres, Sir2-dependent deacetylation is necessary to maintain binding of the SIR complex to chromatin.

Formation of heterochromatin in the yeast S. pombe utilizes an RNAi-dependent pathway (see the Regulatory RNA chapter). This pathway is initiated by the production of siRNA molecules resulting from transcription of centromeric repeats. These siRNAs result in formation of the RNA-induced transcriptional silencing (RITS) complex. The siRNA components are responsible for localizing the complex at centromeres. The complex contains proteins that are homologs of those involved in heterochromatin formation in other organisms, including plants, Caenorhabditis elegans, and D. melanogaster. This complex includes Argonaute, which is involved in targeting RNA-induced silencing complex (RISC) remodeling complexes to chromatin. The siRNA complex promotes methylation of histone H3K9 by the Clr4 methyltransferase (also known as KMT1, a homolog of Drosophila Su[Var]3–9). H3K9 methylation recruits the S. pombe homolog of HP1, Swi6.

How does a silencing complex repress chromatin activity? It could condense chromatin so that regulator proteins cannot find their targets. The simplest case would be to suppose that the presence of a silencing complex is mutually incompatible with the presence of transcription factors and RNA polymerase. The cause could be that silencing complexes block remodeling (and thus indirectly prevent factors from binding) or that they directly obscure the binding sites on DNA for the transcription factors. The situation may not be that simple, though, because transcription factors and RNA polymerase can be found at promoters in silenced chromatin. This could mean that the silencing complex prevents the factors from working rather than from binding as such. In fact, competition may exist between gene activators and the repressing effects of chromatin so that activation of a promoter inhibits spread of the silencing complex.

Centromeric heterochromatin is particularly interesting, because it is not necessarily nucleated by simple sequences (as is the case for telomeres and the mating-type loci in yeast), but instead depends on more complex mechanisms, some of which are RNAi dependent. The specialized chromatin structure that forms at the centromere may be associated with the formation of heterochromatin in the region. The unique centromeric chromatin structure and the centromere-specific histone H3 variants are discussed in the Chromosomes and Chromatin chapters. In human cells, the centromere-specific protein CENP-B is required to initiate modifications of histone H3 (deacetylation of K9 and K14, followed by methylation of K9) that trigger an association with HP1 that leads to the formation of heterochromatin in the region. Moreover, heterochromatin and RNAi are required to establish the human CenH3 homolog, CENP-A, at centromeres. Heterochromatin is often present near CENP-A chromatin and the RNAi-directed heterochromatin flanking the central kinetochore domain is required for kinetochore assembly. Several factors, such as the Suv39 methyltransferase, HP1, and components of the RNAi pathway (see the Regulatory RNA chapter), are required to form the CENP-A chromatin.

Studies of the propagation of the pathogenic yeast Candida albicans have shown that naked centromeric DNA that can confer centromeric activity in vivo is not able to assemble functional centromeric chromatin de novo when reintroduced into cells. This suggests that C. albicans centromeres are dependent on their preexisting chromatin state and provides an example of epigenetic propagation of a centromere.

27.4 Polycomb and Trithorax Are Antagonistic Repressors and Activators

Regions of constitutive heterochromatin, such as at telomeres and centromeres, provide one example of the specific repression of chromatin. Another is provided by the genetics of homeotic genes (which affect the identity of body segments) in Drosophila, which has led to the identification of a protein complex that may maintain certain genes in a repressed state. Polycomb (Pc) mutants show transformations of cell type that are equivalent to gain-of-function mutations in the genes Antennapedia (Antp) or Ultrabithorax, because these genes are expressed in tissues in which they are usually repressed. This implicates Pc in negatively regulating transcription. Furthermore, Pc is the prototype for a class of about 15 loci called the Pc-group (Pc-G); mutations in these genes generally have the same result of derepressing homeotic genes, which suggests the possibility that the group of proteins has some common regulatory role.

The Pc proteins function in large complexes. PRC1 (Polycomb repressive complex 1) contains Pc itself, several other Pc-G proteins, and five general transcription factors. The Esc-E(z) complex contains Esc (extra sex combs), E(z) (enhancer of zeste), other Pc-G proteins, a histone-binding protein, and a histone deacetylase. Pc itself has a chromodomain that binds to methylated H3, and E(z) is a methyltransferase that trimethylates histone H3K27. These properties directly support the connection between chromatin remodeling and repression that was initially suggested by the properties of brahma, a fly counterpart to SWI2. The brahma gene encodes a component of the SWI/SNF remodeling complex (see the Eukaryotic Transcription Regulation chapter), and loss of brahma function suppresses mutations in Polycomb.

Consistent with the pleiotropy of Pc mutations, Pc is a nuclear protein that can be visualized at approximately 80 sites on polytene chromosomes. These sites include the Antp gene. Another member of the Pc-G, polyhomeotic, is visualized at a set of polytene chromosome bands that are identical to those bound by Pc. The two proteins coimmunoprecipitate in a complex of approximately 2.5 × 10⁶ Da that contains 10 to 15 polypeptides. The relationship between these proteins and the products of the 28 or so Pc-G genes remains to be established. One possibility is that some of these gene products form a general repressive complex, and then some of the other proteins associate with it to determine its specificity.

The Pc-G proteins are not conventional repressors. They are not responsible for determining the initial pattern of expression of the genes on which they act. In the absence of Pc-G proteins, these genes are initially repressed as usual, but later in development the repression is lost without Pc-G group functions. This suggests that the Pc-G proteins in some way recognize the state of repression when it is established, and they then act to perpetuate it through cell division of the daughter cells. FIGURE 27.9 shows a model in which Pc-G proteins bind in conjunction with a repressor, but the Pc-G proteins remain bound after the repressor is no longer available. This is necessary to maintain repression; otherwise, the gene becomes activated if Pc-G proteins are absent.

FIGURE 27.9 Pc-G proteins do not initiate repression, but they are responsible for maintaining it.

A Polycomb response element (PRE) is a region of DNA that is sufficient to enable the response to the Pc-G genes. It can be defined operationally by the property that it maintains repression in its vicinity throughout development. The assay for a PRE is to insert it close to a reporter gene that is controlled by an enhancer that is repressed in early development, and then to determine whether the reporter becomes expressed subsequently in the descendants. An effective PRE will prevent such re-expression.

The PRE is a complex sequence that measures about 10 kb. Several proteins with DNA-binding activity for sites within the PRE, including Pho, Pho1, and GAGA factor (GAF), have been identified, but there could be others. When a locus is repressed by Pc-G, however, the Pc-G proteins occupy a much larger length of DNA than the PRE itself. Pc is found locally over a few kilobases of DNA surrounding a PRE. This suggests that the PRE may provide a nucleation center from which a structural state depending on Pc-G proteins may propagate. This model is supported by the observation of effects related to PEV (see Figure 27.4); that is, a gene near a locus whose repression is maintained by Pc-G may become heritably inactivated in some cells but not others. In one typical situation, crosslinking experiments in vivo show that Pc protein is found over large regions of the bithorax complex locus that are inactive, but the protein is excluded from regions that contain active genes. The idea that this could be due to cooperative interactions within a multimeric complex is supported by the existence of mutations in Pc that change its nuclear distribution and abolish the ability of other Pc-G members to localize in the nucleus. The role of Pc-G proteins in maintaining, as opposed to establishing, repression must mean that the formation of the complex at the PRE also depends on the local state of gene expression.

The effects of Pc-G proteins are vast in that hundreds of potential Pc-G targets in plants, insects, and mammals have been identified. A working model for Pc-G binding at a PRE is suggested by the properties of the individual proteins. First, Pho and Pho1 bind to specific sequences within the PRE. Esc-E(z) is recruited to Pho/Pho1; it then uses its methyltransferase activity to methylate K27 of histone H3. This creates the binding site for the PRC1, because the chromodomain of Pc binds to the methylated lysine. The dRING component of PRC1 then monoubiquitinates histone H2A on K119, which is linked to chromatin compaction and RNA polymerase II pausing. In addition, long intergenic noncoding RNAs (lincRNAs) play an important role in assembly of Polycomb complexes. For example, the HOTAIR lincRNA acts as a scaffold for assembly of the PRC2 complex (see the Regulatory RNA chapter). The Polycomb complex induces a more compact structure in chromatin; each PRC1 complex causes about three nucleosomes to become less accessible.

In fact, the chromodomain was first identified as a region of homology between Pc and the protein HP1 found in heterochromatin. Binding of the chromodomain of Pc to K27 on H3 is analogous to HP1’s use of its chromodomain to bind to methylated K9. Variegation is caused by the spreading of inactivity from constitutive heterochromatin, and as a result it is likely that the chromodomain is used by Pc and HP1 in a similar way to induce the formation of heterochromatic or inactive structures. This model implies that similar mechanisms are used to repress individual loci or to create heterochromatin.

In contrast, Trithorax group (TrxG) proteins have the opposite effect of Pc-G proteins: They act to maintain genes in an active state. TrxG proteins are quite diverse; some comprise subunits of chromatin-remodeling enzymes such as SWI/SNF, whereas others also possess important histone-modification activities (such as histone demethylases), which could oppose the activities of Pc-G proteins. The actions of the two groups may share some similarities: Mutations in some loci prevent both Pc-G and TrxG from functioning, suggesting that they could rely on common components. The GAGA factor, which is encoded by the Trithorax-like gene, has binding sites in the PRE. In fact, the sites where Pc binds to DNA coincide with the sites where GAGA factor binds. What does this mean? GAGA is probably needed for activating factors, including TrxG members, to bind to DNA. Is it also needed for Pc-G proteins to bind and exercise repression? This is not yet clear, but such a model would demand that something other than GAGA determines which of the alternative types of complex subsequently assemble at the site.

The TrxG proteins act by making chromatin continuously accessible to transcription factors. Although Pc-G and TrxG proteins promote opposite outcomes, they bind to the same PREs, which can regulate homeotic gene promoters some distance away from the PRE through looping of DNA.

27.5 CpG Islands Are Subject to Methylation

Methylation of DNA occurs at specific sites. In bacteria, it is associated with identifying the bacterial restriction-methylation system used for phage defense and also with distinguishing replicated and nonreplicated DNA. In eukaryotes, its principal known function is connected with the control of transcription: Methylation of a control region is usually associated with gene inactivation. Methylation in eukaryotes principally occurs at CpG islands in the 5′ regions of some genes; these islands are defined by the presence of an increased density of the dinucleotide sequence CpG (see the Eukaryotic Transcription chapter).

From 2% to 7% of the cytosines of animal cell DNA are methylated (the value varies with the species). The methylation occurs at the fifth carbon position of cytosine, producing 5-methylcytosine (5mC). Most of the methyl groups are found in CG dinucleotides in CpG islands, where the C residues on both strands of this short palindromic sequence are methylated.

Such a site is described as fully methylated. Consider, though, the consequences of replicating this site. FIGURE 27.10 shows that each daughter duplex has one methylated strand and one unmethylated strand. Such a site is considered to be hemimethylated.

FIGURE 27.10 The state of methylated CpGs can be perpetuated by an enzyme (Dnmt1) that recognizes only hemimethylated sites as substrates.

The perpetuation of the methylated site now depends on what happens to hemimethylated DNA. If methylation of the unmethylated strand occurs, the site is restored to the fully methylated condition. If replication occurs first, though, the hemimethylated condition will be perpetuated on one daughter duplex, but the site will become unmethylated on the other daughter duplex. FIGURE 27.11 shows that the state of methylation of DNA is controlled by DNA methyltransferases (often shortened to methylases), or Dnmts, which add methyl groups to the 5 position of cytosine, and demethylases, which remove the methyl groups.

FIGURE 27.11 The state of methylation is controlled by three types of enzymes. Numerous de novo and perpetuation methylases are known, and methylation occurs in a single enzymatic step. Demethylation is more complex, and no single-step demethylases have been identified.

Two types of DNA methyltransferases have been identified. Their actions are distinguished by the state of the methylated DNA. To modify DNA at a new position requires the action of a de novo methyltransferase, which recognizes DNA by virtue of a specific sequence. It acts only on unmethylated DNA to add a methyl group to one strand. The mouse has two de novo methyltransferases (Dnmt3A and Dnmt3B); they have different target sites, and both are essential for development.

A maintenance methyltransferase acts constitutively only on hemimethylated sites to convert them to fully methylated sites. Its existence means that any methylated site is perpetuated after replication. The mouse has one maintenance methyltransferase (Dnmt1), and it is essential: Mouse embryos in which its gene has been disrupted do not survive past early embryogenesis.

Maintenance methylation is almost 100% efficient. The result is that if a de novo methylation occurs on one allele but not on the other the difference will be perpetuated through ensuing cell divisions, maintaining a difference between the alleles that does not depend on their sequences. The fact that maintenance methylation actually falls short of 100% efficiency may lead to a decrease in genomic methylation with progressive cell replication, as is often observed in aging cells. Moreover, this change in methylation status with aging, known as epigenetic drift, is thought to be a contributing factor to the increasing phenotypic variability that is observed with aging of monozygotic twins.

How does a maintenance methyltransferase such as Dnmt1 target methylated CpG sites to preserve DNA methylation patterns with each cell replication? One possibility is that Dnmt1 is brought to hemimethylated sites by factors that recognize methylated CpG sites. Consistent with this concept, a protein has been identified, UHRF1, that is important for the maintenance of methylation both locally and globally through its association with Dnmt1. This protein is able to recognize CpG dinucleotides and to preferentially bind to hemimethylated DNA. Most important, however, is that UHRF1 binds to Dnmt1 and appears to increase the efficacy of Dnmt1 for maintenance methylation at hemimethylated CpG dinucleotides. Thus, UHRF1 has dual functions in recognizing sites for maintenance methylation as well as in recruitment of the maintenance methyltransferase to these sites for methylation of the unmethylated CpG on the newly synthesized strand, thereby preserving methylation patterns with each cell replication.

Strikingly, UHRF1 also interacts with methylated histone H3, which connects the maintenance of DNA methylation with the stabilization of heterochromatin structure (see the Eukaryotic Transcription Regulation chapter). DNA methylation and heterochromatin are, in fact, mutually reinforcing in several ways, such as in the example depicted in FIGURE 27.12. Recall that HP1 is recruited to regions in which histone H3 has been methylated at lysine 9, a modification involved in heterochromatin formation. It turns out that HP1 can also interact with Dnmt1, which can promote DNA methylation in the vicinity of HP1 binding. Furthermore, Dnmt1 can directly interact with the methyltransferase responsible for H3K9 methylation, creating a positive feedback loop to ensure continued DNA and histone methylation. These interactions (and other similar networks of interactions) contribute to the stability of epigenetic states, allowing a heterochromatin region to be maintained through many cell divisions.

FIGURE 27.12 Mammalian HP1 is recruited to regions where lysine 9 of histone H3 (H3K9) has been methylated by a histone methyltransferase. HP1 then binds to Dnmt1 and potentiates its DNA methyltransferase activity (blue arrow), thereby enhancing cytosine methylation (meCG) on nearby DNA. Dnmt1 may, in turn, assist HP1 loading onto chromatin (red arrow). Furthermore, association of Dnmt1 with the histone methyltransferase could allow a positive feedback loop to stabilize inactive chromatin.

Methylation has various functional targets. Gene promoters are a common target. The promoter may be methylated when a gene is inactive and is always unmethylated when it is active. The absence of Dnmt1 in mice causes widespread demethylation at promoters; it is assumed that this is lethal because of the uncontrolled gene expression. Satellite DNA is another target. Mutations in Dnmt3B prevent methylation of satellite DNA, which causes centromere instability at the cellular level. Mutations in the corresponding human gene cause the disease ICF (immunodeficiency/centromere instability, facial anomalies). The importance of methylation is emphasized by another human disease, Rett syndrome, which is caused by mutation of the gene encoding the protein MeCP2 that binds methylated CpG sequences. People with Rett syndrome exhibit autism-like symptoms that appear to be the result of a failure of normal gene silencing in the brain.

How are demethylated regions established and maintained? If a DNA site has not been methylated, a protein that recognizes the unmethylated sequence could protect it against methylation. Once a site has been methylated, demethylated sites can be generated in several possible ways. Loss of methylation at a site can occur due to incomplete fidelity of Dnmt1 during maintenance methylation; this is a “passive” demethylation event. Another passive (i.e., nonenzymatic) mechanism is to block the maintenance methylase from acting on the site when it is replicated. After a second replication cycle, one of the daughter duplexes will be unmethylated. A third mechanism is to actively demethylate the site, either by removing the methyl group directly from cytosine or by excising the methylated cytosine or cytidine from DNA for replacement by a repair system.

Plants transmit genomic methylation patterns through each generation, though methylation is removed from repeated sequences to prevent interference with nearby gene expression. Plants therefore can easily remove DNA methylation. Plants use the DEMETER family of 5mC DNA glycosylases, followed by cleavage of the DNA backbone phosphodiester bond by apurinic/apyrimidinic (AP) endonuclease and insertion of the unmethylated dCMP base through the base excision repair (BER) pathway (see the Repair Systems chapter).

In mammals, however, the genomic methylation patterns are erased in primordial germ cells—the cells that ultimately give rise to the germline (discussed in the section on imprinting in the chapter titled Epigenetics II). Primordial germ cells have low levels of Dnmt1, thereby eliminating the need for demethylation on larger scales, as seen in plants. This reduced need for DNA demethylation in mammals relative to plants may explain the challenges in characterizing their mechanisms for DNA demethylation. DNMT3A and DNMT3B (de novo methyltransferases) may paradoxically participate in active DNA demethylation in mammals, though. DNMT3A and DNMT3B may possess deaminase activity and are involved in not only gene demethylation but also cyclical demethylation and remethylation within the cell cycle. These enzymes appear to mediate oxidative deamination at cytosine C4 in the absence of the methyl donor (S-adenosylmethionine) to convert 5-methylcytosine to thymine. The resulting guanine-thymine (G-T) mismatch is repaired by base excision, thereby returning the mismatch to a guanine-cytosine (G-C) pair and leading to demethylation of a previously methylated CpG site.

Recent work has identified a new family of proteins that may be involved not only in active demethylation but also potentially in producing novel epigenetic marks, such as 5-hydroxymethylcytosine (5hmC). The ten-eleven translocation 1-3, or Tet1-3, proteins are DNA hydroxylase enzymes that can convert 5mC to 5hmC and can further convert 5hmC to 5-formylcytosine (5fC) and then 5-carboxylcytosine (5caC) in successive reactions. These derivatives, especially 5hmC, can be detected in genomic DNA and have been proposed to represent stages of demethylation and to create functionally significant modifications themselves. Proteins that normally recognize 5mC, such as MeCP2, do not bind to 5hmC, suggesting that generation of 5hmC might serve to reverse methylation-dependent silencing. Similarly, Dnmt1 does not recognize 5hmC during DNA replication, thus the presence of 5hmC can lead to passive demethylation by preventing maintenance methylation. It has also been suggested that, as in plants, 5mC oxidation by TET proteins could also lead to glycosylase action and removal of the methylated site via BER. Alternatively, 5hmC could promote deamination by deaminases such as activation-induced (cytidine) deaminase (AID), which can act on 5mC to create a mismatched T-G base pair or on 5hmC to produce 5-hydroymethyluracil (5hmU), which a repair system can then correct to a standard (unmethylated) C-G pair.

TET proteins/5hmC have been shown to be critical in genome-wide demethylation during zygotic development, and TET proteins also play a role in preventing hematopoietic malignancies (the original identification and name of TET proteins came from the discovery that Tet1 is oncogenically fused to the histone methyltransferase MLL in a translocation in acute myeloid leukemia). Genome-wide analyses in embryonic stem cells have suggested that Tet1 and 5hmC may have important roles in transcriptional regulation. TET proteins (such as Tet1) contain CXXC motifs that bind to CpG islands and may result in maintaining the hypomethylated state of CpG islands at transcriptionally active (or potentially active) sites. Tet1 and 5hmC are enriched at promoters with so-called bivalent domains, which contain histone modifications associated with both active (H3K4me3) and repressive (H3K27me3) states; these types of promoters are usually present in developmentally regulated genes that are poised for expression in particular lineages. Other data suggest that Tet1/5hmC may be involved in both transcriptional activation and repression. Ongoing research is seeking factors that bind to 5hmC or other derivatives to mediate their activities as true epigenetic marks that define the local function of chromatin.

27.6 Epigenetic Effects Can Be Inherited

DNA may be modified by the covalent attachment of a moiety that is then perpetuated. Two alleles with the same sequence may have different states of methylation that confer different properties.
A self-perpetuating protein state may be established. This might involve assembly of a protein complex, modification of specific protein(s), or establishment of an alternative protein conformation.

Methylation establishes epigenetic inheritance so long as the maintenance methyltransferase acts constitutively to restore the methylated state after each cycle of replication, as shown in Figure 27.10. A state of methylation can be perpetuated through an indefinite series of somatic mitoses. This is probably the “default” situation. Methylation can also be perpetuated through meiosis. For example, in the fungus Ascobolus epigenetic effects can be transmitted through both mitosis and meiosis by maintaining the state of methylation. In mammalian cells, epigenetic marks are first erased in primordial germ cells and then reestablished in new patterns by resetting the state of methylation differently in male and female meioses during gametogenesis.

Situations in which epigenetic effects appear to be maintained by means of protein states are less well understood in molecular terms. PEV shows that constitutive heterochromatin may extend for a variable distance, and the structure is then perpetuated through somatic divisions. There is no methylation of DNA in Saccharomyces and a vanishingly small amount in Drosophila, and as a result the inheritance of epigenetic states of PEV or telomeric silencing in these organisms is likely to be due to the perpetuation of protein structures.

FIGURE 27.13 considers two extreme possibilities for the fate of a protein complex at replication:

A complex could perpetuate itself if it splits symmetrically, so that half complexes associate with each daughter duplex. If the half complexes have the capacity to nucleate formation of full complexes, the original state will be restored. This is basically analogous to the maintenance of methylation. The problem with this model is that there is no evident reason why protein complexes should behave in this way.
A complex could be maintained as a unit and segregate to one of the two daughter duplexes. The problem with this model is that it requires a new complex to be assembled de novo on the other daughter duplex, and it is not evident why this should happen.

FIGURE 27.13 What happens to protein complexes on chromatin during replication?

Consider now the need to perpetuate a heterochromatic structure consisting of protein complexes. As described earlier, random distribution of proteins to each daughter duplex at replication can result in restoration of the heterochromatic state if the protein has a self-assembling property that causes new subunits to associate with it (Figure 27.2).

In some cases, it may be the state of protein modification, rather than the presence of the protein per se, that is responsible for an epigenetic effect. A general correlation exists between the activity of chromatin and the state of acetylation of the histones, in particular the acetylation of the N-terminal tails of histones H3 and H4. Activation of transcription is associated with acetylation in the vicinity of the promoter, and repression of transcription is associated with deacetylation (see the Eukaryotic Transcription Regulation chapter). The most dramatic correlation is that the inactive X chromosome in mammalian female cells is underacetylated.

The inactivity of constitutive heterochromatin may require that the histones are not acetylated. If a histone acetyltransferase is tethered to a region of telomeric heterochromatin in yeast, silenced genes become active. When yeast is exposed to trichostatin (an inhibitor of deacetylation), centromeric heterochromatin becomes acetylated, and silenced genes in centromeric regions may become active. The effect may persist even after trichostatin has been removed. In fact, it may be perpetuated through mitosis and meiosis. This suggests that an epigenetic effect has been created by changing the state of histone acetylation.

How might the state of acetylation be perpetuated? Suppose that the H3₂–H4₂ tetramer is distributed at random to the two daughter duplexes. This creates the situation shown in FIGURE 27.14, in which each daughter duplex contains some histone octamers that are acetylated on the H3 and H4 tails, whereas others are unacetylated. To account for the epigenetic effect, we could suppose that the presence of some acetylated histone octamers provides a signal that causes the unacetylated octamers to be acetylated.

FIGURE 27.14 Acetylated histones are conserved and distributed at random to the daughter chromatin fibers at replication. Each daughter fiber has a mixture of old (acetylated) cores and new (unacetylated) histones.

It is not yet fully understood how epigenetic changes are inherited mitotically in somatic cells, but it is clear that this occurs. Surprisingly, several lines of evidence indicate that epigenetic effects may also be transmitted across generations in a process referred to as transgenerational epigenetics. Evidence that DNA methylation is a central coordinator that secures stable transgenerational inheritance in plants comes from studies of an Arabidopsis thaliana mutant deficient in maintaining DNA methylation. The loss of DNA methylation triggers genome-wide activation of alternative epigenetic mechanisms such as RNA-directed DNA methylation, DNA demethylase inhibition, and retargeting of histone H3K9 methylation. In the absence of maintenance methylation, new and aberrant patterns of epigenetic marks accumulate over several generations, leaving these plants dwarfed and sterile. As a result—at least in plants—the case is strong that intact maintenance methylation plays a major role in transgenerational epigenetics.

In mammals, support for transgenerational epigenetics is less strong, but several lines of evidence indicate that this process occurs in mammals as well. Metastable epialleles are dependent on the epigenetic state for their transcription. This state can vary not only between cells but also between tissues. Although the epigenetic state of the genome undergoes reprogramming in the parental genomes and during early embryogenesis, some loci may transmit the epigenetic state through the gametes to the next generation (transgenerational epigenetics). For example, in mice there is a dominant mutation of the agouti locus (a coat color gene) known as agouti viable yellow, which is caused by the insertion of a retrotransposon upstream of the agouti coding region. This allele shows variegation, resulting in coat colors ranging from solid yellow, to mottled, to completely agouti (dark). It has been observed that agouti females are more likely to produce agouti offspring and yellow females are more likely to produce yellow offspring—in other words, the variable level of expression of agouti in the mother appears to be transmitted to the offspring (while the color of the father is irrelevant). It turns out that DNA methylation of the inserted retrotransposon determines the coat color of the agouti mice, indicating transgenerational conservation of expression levels due to incomplete erasure of the epigenetic mark between generations.

Metastable alleles may also play a role in transgenerational epigenetic inheritance in humans, as suggested by the high degree of copy-number variation within monozygotic twins. Moreover, in some cases of Prader–Willi syndrome no mutation is apparent, but there is an epimutation involving aberrant DNA methylation. The cause for the epimutation may be due to an allele that has passed through the male germline without erasure of the silent epigenetic state established in the grandmother. Thus, the evidence for transgenerational epigenetic inheritance is emerging not only in plants and mammals but also as a potential cause for gene control or diseases due to aberrant epigenetic control of transcription in humans.

As an interesting and important extension of this concept, a number of human diseases may have an etiological basis in transgenerational epigenetic inheritance that may be preventable. For example, in utero exposure can occur from certain diets that have epigenetic-modifying potential through their bioactive compounds, such as maternal diets lacking methyl donors (e.g., folate or choline) that result in lifelong undermethylation of certain regions in the offspring. This could lead to reprogramming of primary epigenetic profiles such as DNA methylation and histone modifications in the fetal genome that could impact disease risk later in life.

27.7 Yeast Prions Show Unusual Inheritance

One of the clearest cases of the dependence of epigenetic inheritance on the condition of a protein is provided by the behavior of prions. They have been characterized in two circumstances: (1) by genetic effects in yeast and (2) as the causative agents of neurological diseases in mammals, including humans. A striking epigenetic effect is found in yeast, where two different states can be inherited that map to a single genetic locus, though the sequence of the gene is the same in both states. The two different states are [psi⁻] and [PSI⁺]. A switch in condition occurs at a low frequency as the result of a spontaneous transition between the states.

The [psi] genotype maps to the locus SUP35, which codes for a translation termination factor. FIGURE 27.15 shows the effects of the Sup35 protein in yeast. In wild-type cells, which are characterized as [psi⁻], the gene is active, and the Sup35 protein terminates protein synthesis. In cells of the mutant [PSI⁺] type, the oligomerized factor does not function, which causes a failure of proper termination of protein synthesis. (This was originally detected by the lethal effects of the enhanced efficiency of suppressors of ochre codons in [PSI⁺] strains.)

FIGURE 27.15 The state of the Sup35 protein determines whether termination of translation occurs.

[PSI⁺] strains have unusual genetic properties. When a [psi⁻] strain is crossed with a [PSI⁺] strain, all of the progeny are [PSI⁺]. This is a pattern of inheritance that would be expected of an extrachromosomal agent, but the [PSI⁺] trait cannot be mapped to any such nucleic acid. The [PSI⁺] trait is metastable, which means that, though it is inherited by most progeny, it is lost at a higher rate than is consistent with mutation. Similar behavior also is shown by the locus URE2, which encodes a protein required for nitrogen-mediated repression of certain catabolic enzymes. When a yeast strain is converted into an alternative state called [URE3], the Ure2 protein is no longer functional.

The [PSI⁺] state is determined by the conformation of the Sup35 protein. In a wild-type [psi^–] cell, the protein displays its normal function. In a [PSI⁺] cell, though, the protein is present in an alternative conformation in which its normal function has been lost. To explain the unilateral dominance of [PSI⁺] over [psi^–] in genetic crosses, we must suppose that the presence of protein in the [PSI⁺] state causes all the protein in the cell to enter this state. This requires an interaction between the [PSI⁺] protein and newly synthesized protein, which probably reflects the generation of an oligomeric state in which the [PSI⁺] protein has a nucleating role, as illustrated in FIGURE 27.16.

FIGURE 27.16 Newly synthesized Sup35 protein is converted into the [PSI⁺] state by the presence of preexisting [PSI⁺] protein.

A feature common to both the Sup35 and Ure2 proteins is that each consists of two domains that function independently. The C-terminal domain is sufficient for the activity of the protein. The N-terminal domain is sufficient for formation of the structures that make the protein inactive. Thus, yeast in which the N-terminal domain of Sup35 has been deleted cannot acquire the [PSI⁺] state, and the presence of a [PSI⁺] N-terminal domain is sufficient to maintain Sup35 protein in the [PSI⁺] condition. The critical feature of the N-terminal domain is that it is rich in glutamine and asparagine residues.

Loss of function in the [PSI⁺] state is due to the sequestration of the protein in an oligomeric complex. Sup35 protein in [PSI⁺] cells is clustered in discrete foci, whereas the protein in [psi^–] cells is diffused in the cytosol. Sup35 protein from [PSI⁺] cells forms amyloid fibers in vitro—these have a characteristic high content of β-sheet structures. These amyloid fibers consist of a parallel in-register β-sheet structure, which allows the prion amyloid to induce a “templating” action at the end of filaments. This templating action provides the faithful transmission of variant differences in these molecules and allows self-reproduction encoding heritable information reminiscent of the behavior of genes.

The involvement of protein conformation (rather than covalent modification) is suggested by the effects of conditions that affect protein structure. Denaturing treatments cause loss of the [PSI⁺] state. In particular, the chaperone Hsp104 is involved in inheritance of [PSI⁺]. Its effects are paradoxical. Deletion of HSP104 prevents maintenance of the [PSI⁺] state, and overexpression of Hsp104 also causes loss of the [PSI⁺] state through elimination of Sup35 proteins. The Ssa and Ssb components of the Hsp70 chaperone system affect Sup35 prion genesis directly through cooperation with Hsp104. Ssa and Ssb binding is facilitated by Hsp40 chaperones through interactions with Sup35 oligomers. At high concentrations, Hsp104 eliminates Sup35 prions while low levels of Hsp104 stimulate prion genesis and alleviate some Hsp70–Hsp40 pairs. Thus, the interplay among Hsp104, Hsp70, and Hsp40 regulates the formation, growth, and elimination of Sup35 prions.

Using the ability of Sup35 to form the inactive structure in vitro, it is possible to provide biochemical proof for the role of the protein. FIGURE 27.17 illustrates a striking experiment in which the protein was converted to the inactive form in vitro, put into liposomes (where in effect the protein is surrounded by an artificial membrane), and then introduced directly into cells by fusing the liposomes with [psi^–] yeast. The yeast cells were converted to [PSI⁺]! This experiment refutes all of the objections that were raised to the conclusion that the protein has the ability to confer the epigenetic state. Experiments in which cells are mated, or in which extracts are taken from one cell to treat another cell, always are susceptible to the possibility that a nucleic acid has been transferred. When the protein by itself does not convert target cells, but the protein converted to the inactive state can do so, the only difference is the treatment of the protein—which must therefore be responsible for the conversion.

FIGURE 27.17 Purified protein can convert the [psi^–] state of yeast to [PSI⁺].

The ability of yeast to form the [PSI⁺ ] prion state depends on the yeast’s genetic background. The yeast must be [PIN⁺] in order for the [PSI⁺] state to form. The [PIN⁺] condition itself is an epigenetic state. It can be created by the formation of prions from any one of several different proteins. These proteins share a key characteristic of Sup35, which is that they have Gln/Asn-rich domains. Overexpression of these domains in yeast stimulates formation of the [PSI⁺] state. This suggests that there is a common model for the formation of the prion state that involves aggregation of the Gln/Asn domains into self-propagating amyloid structure.

How does the presence of one Gln/Asn protein influence the formation of prions by another? We know that the formation of Sup35 prions is specific to Sup35 protein; that is, it does not occur by cross-aggregation with other proteins. This suggests that the yeast cell may contain soluble proteins that antagonize prion formation. These proteins are not specific for any one prion. As a result, the introduction of any Gln/Asn-domain protein that interacts with these proteins will reduce the concentration. This will allow other Gln/Asn proteins to aggregate more easily.

Prions have recently been linked to chromatin-remodeling factors. Swi1 is a subunit of the SWI/SNF chromatin-remodeling complex (see the Eukaryotic Transcription Regulation chapter), and this protein can become a prion. Swi1 aggregates in [SWI⁺] cells but not in nonprion cells, and is dominantly and cytoplasmically transmitted. This suggests that inheritance through proteins can impact chromatin remodeling and potentially affect gene regulation throughout the genome.

Summary

The formation of heterochromatin occurs by proteins that bind to specific chromosomal regions (such as telomeres) and that interact with histones. The formation of an inactive structure may propagate along the chromatin thread from an initiation center. Similar events occur in silencing of the inactive yeast mating-type loci. Repressive structures that are required to maintain the inactive states of particular genes are formed by Polycomb repressive complexes (PRCs). They share with heterochromatin the property of propagating from an initiation center.

Formation of heterochromatin may be initiated at certain sites and then propagated for a distance that is not precisely determined. When a heterochromatic state has been established, it is inherited through subsequent cell divisions. This gives rise to a pattern of epigenetic inheritance, in which two identical sequences of DNA may be associated with different protein structures and therefore have different abilities to be expressed. This explains the occurrence of position-effect variegation (PEV) in Drosophila.

Modification of histone tails is a trigger for chromatin reorganization. Acetylation is generally associated with gene activation. Histone acetyltransferases are found in activating complexes, whereas histone deacetylases are found in inactivating complexes. Histone methylation is associated with gene inactivation or activation, depending on the specific histone residues that are affected. Some histone modifications may be exclusive or synergistic with others.

Inactive chromatin at yeast telomeres and silent mating-type loci appears to have a common cause and involves the interaction of certain proteins with the N-terminal tails of histones H3 and H4. Formation of the inactive complex may be initiated by binding of one protein to a specific sequence of DNA; the other components may then polymerize in a cooperative manner along the chromosome.

Methylation of DNA is inherited epigenetically. Replication of DNA creates hemimethylated products, and a maintenance methylase restores the fully methylated state. Epigenetic effects can be inherited during mitosis in somatic cells or they may be transmitted through organisms from one generation to another. Demethylation occurs through glycosylase action and base excision repair (BER) in plants. In mammals TET proteins convert 5mC to 5hmC and other products, which can serve as glycosylase/BER targets or lead to passive demethylation. These products may also act as epigenetic marks.

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27.2 Heterochromatin Propagates from a Nucleation Event

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27.3 Heterochromatin Depends on Interactions with Histones

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27.4 Polycomb and Trithorax Are Antagonistic Repressors and Activators

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Fischle, W., Wang, Y., Jacobs, S. A., Kim, Y., Allis, C. D., and Khorasanizadeh, S. (2003). Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromo domains. Genes Dev. 17, 1870–1881.
Francis, N. J., Kingston, R. E., and Woodcock, C. L. (2004). Chromatin compaction by a Polycomb group protein complex. Science 306, 1574–1577.
Franke, A., DeCamillis, M., Zink, D., Cheng, N., Brock, H. W., and Paro, R. (1992). Polycomb and polyhomeotic are constituents of a multimeric protein complex in chromatin of D. melanogaster. EMBO J. 11, 2941–2950.
Geyer, P. K., and Corces, V. G. (1992). DNA position-specific repression of transcription by a Drosophila zinc finger protein. Genes Dev. 6, 1865–1873.
Orlando, V., and Paro, R. (1993). Mapping Polycomb-repressed domains in the bithorax complex using in vivo formaldehyde cross-linked chromatin. Cell 75, 1187–1198.
Strutt, H., Cavalli, G., and Paro, R. (1997). Colocalization of Polycomb protein and GAGA factor on regulatory elements responsible for the maintenance of homeotic gene expression. EMBO J. 16, 3621–3632.
Wang, L., Brown, J. L., Cao, R., Zhang, Y., Kassis, J. A., and Jones, R. S. (2004). Hierarchical recruitment of Polycomb group silencing complexes. Mol. Cell 14, 637–646.

27.5 CpG Islands Are Subject to Methylation

Reviews

Bird, A. (2002). DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21.
Franchini, D. M., Schmitz, K. M., and Petersen-Mahrt, S. K. (2012). 5-Methylcytosine DNA demethylation: more than losing a methyl group. Annu. Rev. Genet. 46, 419–441.
Matarese, F., Carillo-de Santa Pau, E., and Stunnenberg, H. G. (2011). 5-hydroxymethylcytosine: a new kid on the epigenetic block? Molec. Syst. Biol. 7, 562.
Schübeler, D. (2015). Function and information content of DNA methylation. Nature. 517, 321–326.
Williams, K., Christensen, J., and Helin, K. (2012). DNA methylation: TET proteins—guardians of CpG islands? EMBO Rep. 13, 28–35.
Wu, H., and Zhang, Y. (2012). Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation. Genes Dev. 25, 2436–2452.

Research

Amir, R. E., Van den Veyver, I. B., Wan, M., Tran, C. Q., Francke, U., and Zoghbi, H. Y. (1999). Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat. Genet. 23, 185–188.
Avvakumov, G. V., Walker, J. R., Xue, S., Li, Y., Duan, S., Bronner, C., Arrowsmith, C. H., and Dhe-Paganon, S. (2008). Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1. Nature 455, 822–825.
Kangaspeska, S., Stride, B., Métivier, R., Polycarpou-Schwarz, M., Ibberson, D., Carmouche, R. P., Benes, V., Gannon, F., and Reid, G. (2008). Transient cyclical methylation of promoter DNA. Nature 452, 112–115.
Ficz, G., Branco, M. R., Seisenberger, S., Santos, F., Krueger, F., Hore, T. A., Marques, C. J., Andrews, S., and Reik, W. (2011). Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473, 398–402.
Hahn, M. A., Szabó, P. E., and Pfeifer, G. P. (2014). 5-Hydroxymethylcytosine: a stable or transient DNA modification? Genomics 104, 314–323.
He, Y. F., Li, B. Z., Li, Z., Liu, P., Wang, Y., Tang, Q., Ding, J., Jia, Y., Chen, Z., Li, L., Sun, Y., Li, X., Dai, Q., Song, C. X., Zhang, K., He, C., and Xu, G. L. (2011). Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307.
Hill, P. W., Amouroux, R., and Hajkova, P. (2014). DNA demethylation, Tet proteins and 5-hydroxymethylcytosine in epigenetic reprogramming: an emerging complex story. Genomics 104, 324–333.
Ito, S., Shen, L., Dai, Q., Wu, S. C., Collins, L. B., Swenberg, J. A., He, C., and Zhang, Y. (2011). Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303.
Li, E., Bestor, T. H., and Jaenisch, R. (1992). Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926.
Métivier, R., Gallais, R., Tiffoche, C., Le Péron, C., Jurkowska, R. Z., Carmouche, R. P., Ibberson, D., Barath, P., Demay, F., Reid, G., Benes, V., Jeltsch, A., Gannon, F., and Salbert, G. (2008). Cyclical DNA methylation of a transcriptionally active promoter. Nature 452, 45–50.
Morgan, H. D., Dean, W., Coker, H. A., Reik, W., and Petersen-Mahrt, S. K. (2004). Activation-induced cytidine deaminase deaminates 5-methylcytosine in DNA and is expressed in pluripotent tissues: implications for epigenetic reprogramming. J. Biol. Chem. 279, 52353–52360.
Okano, M., Bell, D. W., Haber, D. A., and Li, E. (1999). DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–257.
Penterman, J., Uzawa, R., and Fischer, R. L. (2007). Genetic interactions between DNA demethylation and methylation in Arabidopsis. Plant Physiol. 145, 1549–1557.
Wu, H., D’Alessio, A. C., Ito, S., Wang, Z., Cui, K., Zhao, K., Sun, Y. E., and Zhang, Y. (2011). Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. Genes Dev. 25, 679–684.
Xu, G. L., Bestor, T. H., Bourc’his, D., Hsieh, C. L., Tommerup, N, Bugge, M., Hulten, M., Qu, X., Russo, J. J., and Viegas-Paquignot, E. (1999). Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature 402, 187–191.
Xu, Y., Wu, F., Tan, L., Kong, L., Xiong, L., Deng, J., Barbera, A. J., Zheng, L., Zhang, H., Huang, S., Min, J., Nicholson, T., Chen, T., Xu, G., Shi, Y., Zhang, K., and Shi, Y. G. (2011). Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells. Molec. Cell 42, 451–464.

27.6 Epigenetic Effects Can Be Inherited

Reviews

Heard, E., and Martienssen, R. A. (2014). Transgenerational epigenetic inheritance: myths and mechanisms. Cell 157, 95–109.
Jirtle, R. L., and Skinner, M. K. (2007). Environmental epigenomics and disease susceptibility. Nat. Rev. Genet. 8, 253–262.
Li, Y., Saldanha, S. N., and Tollefsbol, T. O. (2014). Impact of epigenetic dietary compounds on transgenerational prevention of human diseases. AAPS J. 16, 27–36.
Li, Y., and Tollefsbol, T. O. (2010). Impact on DNA methylation in cancer prevention and therapy by bioactive dietary components. Curr Med Chem. 17, 2141–2151.
Morgan, D. K., and Whitelaw, E. (2008). The case for transgenerational epigenetic inheritance in humans. Mamm. Genome 19, 394–397.

Research

Bruder, C. E., Piotrowski, A., Gijsbers, A. A., Andersson, R., Erickson, S., de Ståhl, T. D., Menzel, U., Sandgren, J., von Tell, D., Poplawski, A., Crowley, M., Crasto, C., Partridge, E. C., Tiwari, H., Allison, D. B., Komorowski, J., van Ommen, G. J., Boomsma, D. I., Pedersen, N. L., den Dunnen, J. T., Wirdefeldt, K., and Dumanski, J. P. (2008). Phenotypically concordant and discordant monozygotic twins display different DNA copy-number-variation profiles. Am. J. Hum. Genet. 82, 763–771.
Mathieu, O., Reinders, J., Caikovski, M., Smathajitt, C., and Paszkowski, J. (2007). Transgenerational stability of the Arabidopsis epigenome is coordinated by CG methylation. Cell 130, 851–862.

27.7 Yeast Prions Show Unusual Inheritance

Reviews

Byers, J. S., and Jarosz, D. F. (2014). Pernicious pathogens or expedient elements of inheritance: the significance of yeast prions. PLoS Pathog. 10, e1003992.
Garcia, D. M., and Jarosz, D. F. (2014). Rebels with a cause: molecular features and physiological consequences of yeast prions. FEMS Yeast Res. 14, 136–147.
Horwich, A. L., and Weissman, J. S. (1997). Deadly conformations: protein misfolding in prion disease. Cell 89, 499–510.
Lindquist, S. (1997). Mad cows meet psi-chotic yeast: the expansion of the prion hypothesis. Cell 89, 495–498.
Serio, T. R., and Lindquist, S. L. (1999). [PSI⁺]: an epigenetic modulator of translation termination efficiency. Annu. Rev. Cell Dev. Biol. 15, 661–703.
Wickner, R. B., Edskes, H. K., Roberts, B. T., Baxa, U., Pierce, M. M., Ross, E. D., and Brachmann, A. (2004). Prions: proteins as genes and infectious entities. Genes Dev. 18, 470–485.
Wickner, R. B., Shewmaker, F., Kryndushkin, D., and Edskes, H. K. (2008). Protein inheritance (prions) based on parallel in-register beta-sheet amyloid structures. Bioessays 30, 955–964.

Research

Derkatch, I. L., Bradley, M. E., Hong, J. Y., and Liebman, S. W. (2001). Prions affect the appearance of other prions: the story of [PIN(1)]. Cell 106, 171–182.
Derkatch, I. L., Bradley, M. E., Masse, S. V., Zadorsky, S. P., Polozkov, G. V., Inge-Vechtomov, S. G., and Liebman S. W. (2000). Dependence and independence of [PSI(1)] and [PIN(1)]: a two-prion system in yeast? EMBO J. 19, 1942–1952.
Du, Z., Park, K. W., Yu, H., Fan, Q., and Li, L. (2008). Newly identified prion linked to the chromatin-remodeling factor Swi1 in Saccharomyces cerevisiae. Nat. Genet. 40, 460–465.
Glover, J. R., et al. (1997). Self-seeded fibers formed by Sup35, the protein determinant of [PSI⁺], a heritable prion-like factor of S. cerevisiae. Cell 89, 811–819.
Osherovich, L. Z., and Weissman, J. S. (2001). Multiple gln/asn-rich prion domains confer susceptibility to induction of the yeast. Cell 106, 183–194.
Shorter, J., and Lindquist, S. (2008). Hsp104, Hsp70 and Hsp40 interplay regulates formation, growth and elimination of Sup35 prions. EMBO J. 27, 2712–2724.
Sparrer, H. E., Santoso, A., Szoka, F. C, and Weissman, J. S. (2000). Evidence for the prion hypothesis: induction of the yeast [PSI1] factor by in vitro-converted Sup35 protein. Science 289, 595–599.