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Chapter 28: Epigenetics II

Edited by Trygve Tollefsbol

CHAPTER OUTLINE

1. 28.1 Introduction

2. 28.2 X Chromosomes Undergo Global Changes

3. 28.3 Chromosome Condensation Is Caused by Condensins

4. 28.4 DNA Methylation Is Responsible for Imprinting

5. 28.5 Oppositely Imprinted Genes Can Be Controlled by a Single Center

6. 28.6 Prions Cause Diseases in Mammals

28.1 Introduction

KEY CONCEPT

• Many biological processes, including X chromosome inactivation and genomic imprinting, are mediated through epigenetic mechanisms such as DNA methylation.

The process of X chromosome inactivation in female (eutherian) mammals is a random process between the maternally and paternally derived X chromosomes. The X-inactivation center, or Xic, serves as the locus that ultimately determines X-inactivation. A key gene that is transcribed from the Xic is known as Xist (X inactive-specific transcript). Xist is a nontranslated RNA molecule that acts in cis to silence the X chromosome from which it is transcribed. The X-inactivation process is mediated by epigenetic processes, including DNA methylation, that maintain the inactive X in a silent state.

Genomic imprinting also relies on epigenetic processes, especially DNA methylation, for marking specific maternally or paternally derived genes. The expression of these genes during early development contributes to many biological phenotypes, including embryonic and postnatal growth. Moreover, aberrations of imprinting can lead to a number of imprinting diseases, such as Prader–Willi and Angelman syndromes.

Epigenetic processes may also directly impact proteins as well as nucleic acids, and an important example of this concept is prions. Prions are proteinaceous structures that can act as infectious agents. In fact, prions can cause human diseases such as Creutzfeldt-Jakob Disease (CJD), which is an example of the growing list of infectious diseases that are mediated through epigenetic modifications of proteins.

28.2 X Chromosomes Undergo Global Changes

KEY CONCEPTS

• One of the two X chromosomes is inactivated at random in each cell during embryogenesis of eutherian mammals.

• In exceptional cases where there are more than two X chromosomes, all but one are inactivated.

• The X-inactivation center (Xic) is a cis-acting region on the X chromosome that is necessary and sufficient to ensure that only one X chromosome remains active.

• Xic includes the Xist gene, which codes for an RNA that is found only on inactive X chromosomes.

• Xist recruits Polycomb complexes, which modify histones on the inactive X chromosome.

• Xist spreads along the X chromosome by binding to distal sites relative to the Xic.

• The mechanism that is responsible for preventing Xist RNA from accumulating on the active chromosome is unknown.

For species with chromosomal sex determination, the sex of the individual presents an interesting problem for gene regulation because of the variation in the number of X chromosomes. If X-linked genes were expressed equally in each sex, females would have twice as much of each product as males. The importance of avoiding this situation is shown by the existence of dosage compensation, which equalizes the level of expression of X-linked genes in the two sexes. Dosage compensation mechanisms used in different species are summarized in FIGURE 28.1:

• In mammals, one of the two female X chromosomes is inactivated during embryogenesis. The result is that females have only one active X chromosome, which is the same situation found in males. The active X chromosome of females and the single X chromosome of males are expressed at the same level. (Note that both X chromosomes are active during early embryogenesis in females, and the inactive X chromosome actually retains about 5% activity.)

• In Drosophila, the expression of the single male X chromosome is doubled relative to the expression of each female X chromosome.

• In Caenorhabditis elegans, the expression of each female (hermaphrodite) X chromosome is halved relative to the expression of the single male X chromosome.

The common feature in all these mechanisms of dosage compensation is that the entire chromosome is the target for regulation. A global change occurs that quantitatively affects almost all of the promoters on the chromosome. Inactivation of the X chromosome in mammalian females is well documented, with the entire chromosome becoming heterochromatic.

FIGURE 28.1 Different means of dosage compensation are used to equalize X chromosome expression in males and females.

The twin properties of heterochromatin are its condensed state and associated inactivity (introduced in the Chromosomes chapter). It can be divided into two types:

• Constitutive heterochromatin contains specific sequences that have no coding function. These include satellite DNAs, which are often found at the centromeres. These regions are invariably heterochromatic because of their intrinsic nature.

• Facultative heterochromatin takes the form of chromosome segments or entire chromosomes that are inactive in one cell lineage, though they can be expressed in other lineages. The best example is the mammalian X chromosome. The inactive X chromosome is perpetuated in a heterochromatic state, whereas the active X chromosome is euchromatic. Either X chromosome has an equal chance of being inactivated; thus, identical DNA sequences are involved in both states. Once the inactive state has been established, it is inherited by descendant cells. This is an example of epigenetic inheritance, because it does not depend on the DNA sequence.

The basic view of the situation of the female mammalian X chromosomes was formed by the single X hypothesis in 1961. Female mice that are heterozygous for X-linked coat color mutations have a variegated phenotype in which some areas of the coat are wild type but others are mutant. FIGURE 28.2 shows that this can be explained if one of the two X chromosomes is inactivated at random in each cell of a small precursor population. Cells in which the X chromosome carrying the wild-type gene is inactivated give rise to progeny that express only the mutant allele on the active chromosome. Cells derived from a precursor where the other chromosome was inactivated have an active wild-type gene. In the case of coat color, cells descended from a particular precursor stay together and thus form a patch of the same color, creating the pattern of visible variegation (calico cats are a familiar example of this phenomenon). In other cases, individual cells in a population will express one or the other of X-linked alleles; for example, in heterozygotes for the X-linked locus G6PD, any particular red blood cell will express only one of the two allelic forms. (Random inactivation of one X chromosome occurs in eutherian mammals. In marsupials, the choice is directed: It is always the X chromosome inherited from the father that is inactivated.)

FIGURE 28.2 X-linked variegation is caused by the random inactivation of one X chromosome in each precursor cell. Cells in which the wild-type allele (pink) is on the active chromosome have the wild-type phenotype; cells in which the mutant allele (green) is on the active chromosome have the mutant phenotype.

Inactivation of the X chromosome in females is governed by the n – 1 rule: Regardless of how many X chromosomes are present, all but one will be inactivated. Normal females of course have two X chromosomes, but in rare cases where nondisjunction has generated a genotype of three or more X chromosomes, only one X chromosome remains active. This suggests a general model in which a specific event is limited to one X chromosome that protects it from an inactivation mechanism that applies to all the others.

A single locus on the X chromosome is sufficient for inactivation. When a translocation occurs between the X chromosome and an autosome, this locus is present on only one of the reciprocal products, and only that product can be inactivated. By comparing different translocations, it is possible to map this locus, which is called the Xic (X-inactivation center). A cloned region of 450 kb contains all the properties of the Xic. When this sequence is inserted as a transgene onto an autosome, the autosome becomes subject to inactivation (at least in a cell culture system). Pairing of Xic loci on the two X chromosomes has been implicated in the mechanism for the random choice of X-inactivation. Moreover, differences in sister chromatid cohesion correlates with the outcome of the choice of the X chromosome to be inactivated, indicating that alternate states present before the inactivation process may direct the choice of which X chromosome will become inactivated.

Xic is a cis-acting locus that contains the information necessary to count X chromosomes and inactivate all copies but one. Inactivation spreads from Xic along the entire X chromosome. When Xic is present on an X chromosome–autosome translocation, inactivation spreads into the autosomal regions (although the effect is not always complete).

Xic is a complex genetic locus that expresses several long noncoding RNAs (ncRNAs). The most important of these is a gene called Xist (X inactive-specific transcript), which is stably expressed only on the inactive X chromosome. The behavior of this gene is effectively the opposite of all other loci on the chromosome, which are turned off. Deletion of Xist prevents an X chromosome from being inactivated. It does not, however, interfere with the counting mechanism (because other X chromosomes can be inactivated). Thus, we can distinguish two features of Xic: (1) an unidentified element(s) required for counting and (2) the Xist gene required for inactivation.

The n – 1 rule suggests that stabilization of Xist RNA is the “default” and that some blocking mechanism prevents stabilization at one X chromosome (which will be the active X chromosome). This means that even though Xic is necessary and sufficient for a chromosome to be inactivated, the products of other loci are necessary for the establishment of an active X chromosome.

The Xist transcript is regulated in a negative manner by Tsix, its antisense partner. Loss of Tsix expression on the future inactive X chromosome permits Xist to become upregulated and stabilized, and persistence of Tsix on the future active X chromosome prevents Xist upregulation. Tsix is, in turn, regulated by Xite, which has a Tsix-specific enhancer and is located 10 kb upstream of Tsix.

FIGURE 28.3 illustrates the role of Xist RNA in X-inactivation. Xist codes for an ncRNA that lacks open reading frames. The Xist RNA “coats” the X chromosome from which it is synthesized, which suggests that it has a structural role. Prior to X-inactivation, it is synthesized by both female X chromosomes. Following inactivation, the RNA is found only on the inactive X chromosome. The transcription rate remains the same before and after inactivation, so the transition depends on posttranscriptional events.

FIGURE 28.3 X-inactivation involves stabilization of Xist RNA, which coats the inactive chromosome. Tsix prevents Xist expression on the future active X chromosome.

Prior to X-inactivation, Xist RNA decays with a half-life of approximately 2 hours. X-inactivation is mediated by stabilizing the Xist RNA on the inactive X chromosome. The Xist RNA shows a punctate distribution along the X chromosome, which suggests that association with proteins to form particulate structures may be the means of stabilization. Xist spreads along the X chromosome beginning at the Xic and moves distally to silence regions of the X chromosome. It is not yet known what other factors may be involved in this reaction or how the Xist RNA is limited to spreading in cis along the chromosome.

Accumulation of Xist on the future inactive X chromosome results in exclusion of transcription machinery (such as RNA polymerase II) and leads to the recruitment of Polycomb repressor complexes (PRC1 and PRC2), which trigger a series of chromosome-wide histone modifications (H2AK119 ubiquitination, H3K27 methylation, H4K20 methylation, and H4 deacetylation). Late in the process, an inactive X-specific histone variant, macroH2A, is incorporated into the chromatin, and promoter DNA is methylated, resulting in gene silencing. These changes are shown in FIGURE 28.4. At this point, the heterochromatic state of the inactive X is stable, and Xist is not required to maintain the silent state of the chromosome.

FIGURE 28.4 Xist RNA produced from the Xic locus accumulates on the future inactive X chromosome (Xi). This excludes transcription machinery, such as RNA polymerase II (Pol II). Polycomb group complexes are recruited to the Xist-covered chromosome and establish chromosome-wide histone modifications. Histone macroH2A becomes enriched on the Xi, and promoters of genes on the Xi are methylated. In this phase X-inactivation is irreversible and Xist is not required for maintenance of the silent state.

Data from A. Wutz and J. Gribnau, Curr. Opin. Genet. Dev. 17 (2007): 387–393.

Despite these findings, none of the chromatin components or modifications found have been shown on their own to be essential for X chromosome silencing, indicating potential redundancy among them or the existence of pathways that have yet to be identified.

Global changes also occur in other types of dosage compensation. In Drosophila, a large ribonucleoprotein complex, MSL, is found only in males, where it localizes to the X chromosome. This complex contains two noncoding RNAs, which appear to be needed for localization to the male X chromosome (perhaps analogous to the localization of Xist to the inactive mammalian X chromosome), and a histone acetyltransferase that acetylates histone H4 on K16 throughout the male X chromosome. The net result of the action of this complex is the twofold increase in transcription of all genes on the male X chromosome. The next section presents a third mechanism for dosage compensation, a global reduction in X-linked gene expression in XX (hermaphrodite) nematodes.

28.3 Chromosome Condensation Is Caused by Condensins

KEY CONCEPTS

• SMC proteins are ATPases that include condensins and cohesins.

• A heterodimer of SMC proteins associates with other subunits.

• Condensins cause chromatin to be more tightly coiled by introducing positive supercoils into DNA.

• Condensins are responsible for condensing chromosomes at mitosis.

• Chromosome-specific condensins are responsible for condensing inactive X chromosomes in C. elegans.

The structures of entire chromosomes are influenced by interactions with proteins of the structural maintenance of chromosome (SMC) family. These are ATPases that fall into two functional groups: condensins and cohesins. Condensins are involved in the control of overall structure and are responsible for the condensation into compact chromosomes at mitosis. Cohesins play a role in the connections between sister chromatids that concatenate through a cohesin ring, which must be released at mitosis. Both consist of dimers formed by SMC proteins. Condensins form complexes that have a core of the heterodimer SMC2–SMC4 associated with other (non-SMC) proteins. Cohesins have a similar organization but consist of SMC1 and SMC3 and also interact with smaller non-SMC subunits, Scc1/Rad21 and Scc3/SA.

FIGURE 28.5 shows that an SMC protein has a coiled-coil structure in its center that is interrupted by a flexible hinge region. Both the amino and carboxyl termini have ATP- and DNA-binding motifs. The ATP-binding motif is also known as a Walker module. SMC monomers fold at the hinge region, forming an antiparallel interaction between the two halves of each coiled coil. This allows the amino and carboxyl termini to interact to form a “head” domain. Different models have been proposed for the actions of these proteins depending on whether they dimerize by intra- or intermolecular interactions.

(a)

(a)

FIGURE 28.5 (a) An SMC protein has a Walker module with an ATP-binding motif and DNA-binding site at each end, which are connected by coiled coils that are linked by a hinge region. (b) SMC monomers fold at the hinge regions and interact along the length of the coiled coils. The N- and C-termini interact to form a head domain.

Data from I. Onn, et al., Annu. Rev. Cell Dev. Biol. 24 (2008): 105–129.

Folded SMC proteins form dimers via several different interactions. The most stable association occurs between hydrophobic domains in the hinge regions. FIGURE 28.6 shows that these hinge–hinge interactions result in V-shaped structures. Electron microscopy shows that in solution cohesins tend to form Vs, with the arms separated by a large angle, whereas condensins form more linear structures, with only a small angle between the arms. In addition, the heads of the two monomers can interact, closing the V, and the coils of the individual monomers may also interact with each other. Various non-SMC proteins interact with SMC dimers and can influence the final structure of the dimer.

FIGURE 28.6 (a) The basic architecture of condensin and cohesin complexes. (b) Condensin and cohesin consist of V-shaped dimers of two SMC proteins interacting through their hinge domains. The two monomers in a condensin dimer tend to exhibit a very small separation between the two arms of the V; cohesins have a much larger angle of separation between the arms.

Data from T. Hirano, Nat. Rev. Mol. Cell Biol. 7 (2006): 311–322.

The function of cohesins is to hold sister chromatids together, but it is not yet clear how this is achieved. Several different models have been proposed for cohesin function. FIGURE 28.7 shows one model in which a cohesin could take the form of extended dimers, interacting hinge to hinge, that crosslink two DNA molecules. Head–head interactions would create tetrameric structures, adding to the stability of cohesion. An alternative “ring” model is shown in FIGURE 28.8. In this model, dimers interact at both their head and hinge regions to form a circular structure. Instead of binding directly to DNA, a structure of this type could hold DNA molecules together by encircling them.

FIGURE 28.7 One model for DNA linking by cohesins. Cohesins may form an extended structure in which each monomer binds DNA and connects via the hinge region, allowing two different DNA molecules to be linked. Head domain interactions can result in binding by two cohesin dimers.

Data from I. Onn, et al., Annu. Rev. Cell Dev. Biol. 24 (2008): 105–129.

FIGURE 28.8 Cohesins may dimerize by intramolecular connections and then form multimers that are connected at the heads and at the hinge. Such a structure could hold two molecules of DNA together by surrounding them.

Whereas cohesins act to hold separate sister chromatids together, condensins are responsible for chromatin condensation. FIGURE 28.9 shows that a condensin could take the form of a V-shaped dimer, interacting via the hinge domains, that pulls together distant sites on the same DNA molecule, causing it to condense. It is thought that dynamic head–head interactions could act to promote the ordered assembly of condensed loops, but the details of condensin action are still far from clear.

FIGURE 28.9 Condensins may form a compact structure by bending at the hinge, causing DNA to become compacted.

Visualization of mitotic chromosomes shows that condensins are located all along the length of the chromosome, as shown in FIGURE 28.10. (By contrast, cohesins are found at discrete locations in a focal nonrandom pattern with an average spacing of about 10 kb.) The condensin complex was named for its ability to cause chromatin to condense in vitro. It has an ability to introduce positive supercoils into DNA in an action that uses hydrolysis of ATP and depends on the presence of topoisomerase I. This ability is controlled by the phosphorylation of the non-SMC subunits, which occurs at mitosis. It is not yet known how this connects with other modifications of chromatin—for example, the phosphorylation of histones. The activation of the condensin complex specifically at mitosis makes it questionable whether it is also involved in the formation of interphase heterochromatin. Recent evidence indicates that chromosome condensation does not involve hierarchal folding of chromatin into scaffolds but rather that the condensation process is dynamic. This dynamic process involves interactions of condensin between segments of chromatin that can be quite some distance apart. Therefore, chromosome condensation may involve a scaffold-free organization that consists of nucleosome fibers folded in an irregular manner in a polymer structure.

FIGURE 28.10 Condensins are located along the entire length of a mitotic chromosome. DNA is red; condensins are yellow.

Photo courtesy of Ana Losada and Tatsuya Hirano.

As discussed in the previous section, dramatic chromosomal changes occur during X-inactivation in female mammals and in X chromosome upregulation in male flies. In the nematode C. elegans, a third approach is used: twofold reduction of X-chromosome transcription in XX hermaphrodites relative to XO males. A dosage compensation complex (DCC) is maternally provided to both XX and XO embryos, but it then associates with both X chromosomes only in XX animals, while remaining diffusely distributed in the nuclei of XO animals. The protein complex contains an SMC core and is similar to the condensin complexes that are associated with mitotic chromosomes in other species. This suggests that it has a structural role in causing the chromosome to take up a more condensed, inactive state. Recent studies have shown, though, that SMC-related proteins may also have roles in dosage compensation in mammals: The protein SmcHD1 (SMC-hinge domain 1) may actually contribute to the deposition of DNA methylation on the mammalian inactive X chromosome. SMCs could recruit DNA methyltransferase via a component of the SMC core that is involved in RNAi-directed DNA methylation, such as occurs in Arabidopsis via the DMS3 protein (another SMC-related protein).

Whatever the mechanism of transcriptional downregulation, multiple sites on the X chromosome appear to be needed for the DCC to be fully distributed along it, and short DNA sequence motifs have been identified that appear to be key for localization of DCC. The complex binds to these sites and then spreads along the chromosome to cover it more thoroughly.

Changes affecting all the genes on a chromosome, either negatively (mammals and C. elegans) or positively (Drosophila), are therefore a common feature of dosage compensation. The components of the dosage compensation apparatus may vary, however, as well as the means by which it is localized to the chromosome. Dosage compensation in mammals and Drosophila both entail chromosome-wide changes in histone acetylation and involve noncoding RNAs that play central roles in targeting X chromosomes for global change. In C. elegans, chromosome condensation by condensin homologs is used to accomplish dosage compensation. It remains to be seen whether there are also global changes in histone acetylation or other modifications in XX C. elegans that reflect the twofold reduction in transcription of the X chromosomes.

28.4 DNA Methylation Is Responsible for Imprinting

KEY CONCEPTS

• Paternal and maternal alleles may have different patterns of methylation at fertilization.

• Methylation is usually associated with inactivation of the gene.

• When genes are differentially imprinted, survival of the embryo may depend on whether a functional allele is provided by the parent with the unmethylated allele.

• Survival of heterozygotes for imprinted genes is different, depending on the direction of the cross.

• Imprinted genes occur in clusters and may depend on a local control site where de novo methylation occurs unless specifically prevented.

The pattern of methylation of germ cells is established in each sex during gametogenesis by a two-stage process: First, the existing pattern is erased by a genome-wide demethylation in primordial germ cells and then a pattern specific for each sex is imposed during meiosis.

All allelic differences are lost when primordial germ cells develop in the embryo; irrespective of sex, the previous patterns of methylation are erased, and a typical gene is then unmethylated. In males, the pattern develops in two stages. The methylation pattern that is characteristic of mature sperm is established in the spermatocyte, but further changes are made in this pattern after fertilization. In females, the maternal pattern is imposed during oogenesis, when oocytes mature through meiosis after birth.

As may be expected from the inactivity of genes in gametes, the typical state is to be methylated. Some cases of differences between the two sexes have been identified, though, for which a locus is unmethylated in one sex. A major question is how the specificity of methylation is determined in the male and female gametes.

Systematic changes occur in early embryogenesis. Some sites will continue to be methylated, whereas others will be specifically unmethylated in cells in which a gene is expressed. From the pattern of changes, it may be inferred that individual sequence-specific demethylation events occur during somatic development of the organism as particular genes are activated.

The specific pattern of DNA methylation in germ cells is responsible for the phenomenon of imprinting, which describes a difference in behavior between the alleles inherited from each parent. The expression of certain genes in mouse embryos (and other mammals) depends upon the sex of the parent from which they were inherited. For example, the allele encoding insulin-like growth factor II (IGF-II) that is inherited from the father is expressed, but the allele that is inherited from the mother is not expressed. The IGF-II gene of oocytes is methylated in its promoter, whereas the IGF-II gene of sperm is not, so that the two alleles behave differently in the zygote. This is the most common pattern, but the dependence on sex is reversed for some genes. In fact, the opposite pattern (expression of maternal copy) is shown for IGF-IIR, a gene encoding a receptor that causes the rapid turnover of IGF-II.

This sex-specific mode of inheritance requires that the pattern of methylation be established specifically during each gametogenesis. The fate of a hypothetical locus in a mouse is illustrated in FIGURE 28.11. In the early embryo, the paternal allele is unmethylated and expressed, and the maternal allele is methylated and silent. What happens when this mouse itself forms gametes? If it is a male, the allele contributed to the sperm must be nonmethylated, irrespective of whether it was originally methylated or not. Thus, when the maternal allele finds itself in a sperm, it must be demethylated. If the mouse is a female, the allele contributed to the egg must be methylated; if it was originally the paternal allele, methyl groups must be added.

FIGURE 28.11 The typical pattern for imprinting is that a methylated locus is inactive. If this is the maternal allele, only the paternal allele is active, and it may be essential for viability. The methylation pattern is reset when gametes are formed so that all sperm have the paternal type and all oocytes have the maternal type.

The consequence of imprinting is that an embryo is hemizygous for any imprinted gene. Thus, in the case of a heterozygous cross where the allele of one parent has an inactivating mutation, the embryo will survive if the wild-type allele comes from the parent in which this allele is active but will die if the wild-type allele is the imprinted (silenced) allele. This type of dependence on the directionality of the cross (in contrast with Mendelian genetics) is an example of epigenetic inheritance, where some factor other than the sequences of the genes themselves influences their effects. Although the paternal and maternal alleles can have identical sequences, they display different properties, depending on which parent provided them. These properties are inherited through meiosis and the subsequent somatic mitoses.

Although imprinted genes are estimated to comprise 1% to 2% of the mammalian transcriptome, these genes are sometimes clustered. More than half of the 25 or so known imprinted genes in mice are contained in six particular regions, each containing both maternally and paternally expressed genes. This suggests the possibility that imprinting mechanisms may function over long distances. Some insights into this possibility come from deletions in the human population that cause Prader–Willi and Angelman syndromes. Most cases of these neurodevelopmental disorders involving the proximal long arm of chromosome 15 are caused by the same 4-Mb deletion, but the syndromes are different, depending on which parent contributed the deletion. The reason is that the deleted region contains at least one gene that is paternally imprinted and at least one that is maternally imprinted. Thus, affected individuals receive one chromosome missing a given allele due to the deletion, and the corresponding (intact) allele from the other parent is imprinted and thus silent. This results in affected individuals being functionally null for these alleles.

In some rare cases, however, affected individuals present with much smaller deletions. Prader–Willi syndrome can be caused by a 20-kb deletion that silences distant genes on either side of the deletion. The basic effect of the deletion is to prevent a father from resetting the paternal mode to a chromosome inherited from his mother. The result is that these genes remain in maternal mode so that both the paternal and maternal alleles are silent in the offspring. The inverse effect is found in some small deletions that cause Angelman syndrome. These mutations have led to the identification of a Prader–Willi/Angelman syndrome “imprint center” (PW/AS IC) that acts at a distance to regulate imprinting in either sex across the entire region.

A microdeletion resulting in removal of a cluster of small nucleolar RNAs (snoRNAs) that is paternally derived may result in the key aspects of Prader–Willi syndrome. Mutations that separate the snoRNA HBII-85 cluster from its promoter cause Prader–Willi syndrome, although other genes in the region could also contribute to the syndrome.

Six imprinted regions are often associated with disease in humans, and the phenotypic diversity of these disorders is related to the multiple genes in the imprinted regions. These defects in imprinted genes may take the form of aberrant expression involving loss or overexpression of genes. For example, in Russell–Silver syndrome, an overexpression of maternal alleles and loss of paternal gene expression for chromosome 11p15.5 result in this syndrome that is characterized by an undergrowth disorder.

Imprinting may also regulate alternative polyadenylation. A number of mammalian genes utilize multiple polyadenylation (polyA) sites to confer diversity on gene transcription. The H13 murine gene undergoes alternative polyadenylation in an allele-specific manner, in that polyA sites are differentially methylated in the maternal and paternal genome of this imprinted gene. Elongation proceeds to downstream polyadenylation sites when the allele is methylated, indicating that epigenetic processes may influence alternative polyadenylation, contributing to the diversity of gene transcription in mammals.

28.5 Oppositely Imprinted Genes Can Be Controlled by a Single Center

KEY CONCEPTS

• Imprinted genes are controlled by methylation of cis-acting sites.

• Methylation may be responsible for either inactivating or activating a gene.

Imprinting is determined by the state of methylation of a cis-acting site near a target gene or genes. These regulatory sites are known as differentially methylated domains (DMDs) or imprinting control regions (ICRs). Deletion of these sites removes imprinting, and the target loci then behave the same in both maternal and paternal genomes.

The behavior of a region containing the genes Igf2 and H19 illustrates the ways in which methylation can control gene activity. FIGURE 28.12 shows that these two genes react oppositely to the state of methylation at the ICR located between them. The ICR is methylated on the paternal allele. H19 shows the typical response of inactivation. Note, however, that Igf2 is expressed. The reverse situation is found on a maternal allele, where the ICR is not methylated; H19 now becomes expressed, but Igf2 is inactivated.

FIGURE 28.12 The ICR is methylated on the paternal allele, where Igf2 is active and H19 is inactive. The ICR is unmethylated on the maternal allele, where Igf2 is inactive and H19 is active.

The control of Igf2 is exercised by an insulator contained within the ICR (see the Chromatin chapter for a discussion of insulators). FIGURE 28.13 shows that when the ICR is unmethylated it binds the protein CTCF. This creates a functional insulator that blocks an enhancer from activating the Igf2 promoter. This is an unusual effect in which methylation indirectly activates a gene by blocking an insulator.

FIGURE 28.13 The ICR contains an insulator that prevents an enhancer from activating Igf2. The insulator functions only when CTCF binds to unmethylated DNA.

The regulation of H19 shows the more usual direction of control in which methylation creates an inactive imprinted state. This could reflect a direct effect of methylation on promoter activity, though the effect could also be due to additional factors. CTCF regulates chromatin by repressing H3K27 trimethylation at the Igf2 locus independent of repression by DNA hypermethylation. As a result, the effects of CTCF on chromatin, as well as on DNA methylation, likely contribute to the imprinting of H19 and Igf2.

28.6 Prions Cause Diseases in Mammals

KEY CONCEPTS

• The protein responsible for scrapie exists in two forms: the wild-type noninfectious form PrP^C, which is susceptible to proteases, and the disease-causing PrP^Sc, which is resistant to proteases.

• The neurological disease can be transmitted to mice by injecting the purified PrP^Sc protein into mice.

• The recipient mouse must have a copy of the PrP gene coding for the mouse protein.

• The PrP^Sc protein can perpetuate itself by causing the newly synthesized PrP protein to take up the PrP^Sc form instead of the PrP^C form.

• Multiple strains of PrP^Sc may have different conformations of the protein.

Prion diseases have been found in humans, sheep, cows, and, more recently, in wild deer and elk. The basic phenotype is an ataxia—a neurodegenerative disorder that is manifested by an inability to remain upright. The name of the disease in sheep, scrapie, reflects the phenotype: The sheep rub against walls in order to stay upright. Scrapie can be perpetuated by inoculating sheep with tissue extracts from infected animals. In humans, the disease kuru was found in New Guinea, where it appeared to be perpetuated by cannibalism, in particular the eating of brains. Related diseases in Western populations with a pattern of genetic transmission include Gerstmann–Straussler syndrome and the related Creutzfeldt–Jakob disease (CJD), which occurs sporadically. A disease resembling CJD appears to have been transmitted by consumption of meat from cows suffering from “mad cow” disease.

When tissue from scrapie-infected sheep is inoculated into mice, the disease occurs in a period ranging from 75 to 150 days. The active component is a protease-resistant protein. The protein is encoded by a gene that is normally expressed in the brain. The form of the protein in a normal brain, called PrP^C, is sensitive to proteases. Its conversion to the resistant form, called PrP^Sc, is associated with occurrence of the disease. Neurotoxicity is mediated by PrP^L, which is catalyzed by PrP^Sc and occurs when the PrP^L concentration becomes too high. Rapid propagation results in severe neurotoxicity and eventual death. The infectious preparation has no detectable nucleic acid, is sensitive to UV irradiation at wavelengths that damage protein, and has a low infectivity (1 infectious unit/10⁵ PrP^Sc proteins). This corresponds to an epigenetic inheritance in which there is no change in genetic information (because normal and diseased cells have the same PrP gene sequence), but the PrP^Sc form of the protein is the infectious agent (whereas PrP^C is harmless). The PrP^Sc form has a high content of β-sheets, which form an amyloid fibrillous structure that is absent from the PrP^C form. The basis for the difference between the PrP^Sc and PrP^C forms appears to lie with a change in conformation rather than with any covalent alteration. Both proteins are glycosylated and linked to the membrane by a glycosylphosphatidylinositol (GPI) linkage.

The assay for infectivity in mice allows the dependence on protein sequence to be tested. FIGURE 28.14 illustrates the results of some critical experiments. In the normal situation, PrP^Sc protein extracted from an infected mouse will induce disease (and ultimately kill) when it is injected into a recipient mouse. If the PrP gene is deleted, a mouse becomes resistant to infection. This experiment demonstrates two things. First, the endogenous protein is necessary for an infection, presumably because it provides the raw material that is converted into the infectious agent. Second, the cause of disease is not the removal of the PrP^C form of the protein, because a mouse with no PrP^C survives normally: The disease is caused by a gain of function in PrP^Sc. If the PrP gene is altered to prevent the GPI linkage from occurring, mice infected with PrP^Sc do not develop disease, which suggests that the gain of function involves an altered signaling function for which the GPI linkage is required.

FIGURE 28.14 A PrP^Sc protein can only infect an animal that has the same type of endogenous PrP^C protein.

The existence of species barriers allows hybrid proteins to be constructed to delineate the features required for infectivity. The original preparations of scrapie were perpetuated in several types of animal, but these cannot always be transferred readily. For example, mice are resistant to infection from prions of hamsters. This means that hamster PrP^Sc cannot convert mouse PrP^C to PrP^Sc. The situation changes, though, if the mouse PrP gene is replaced by a hamster PrP gene. (This can be done by introducing the hamster PrP gene into the PrP knockout mouse.) A mouse with a hamster PrP gene is sensitive to infection by hamster PrP^Sc. This suggests that the conversion of cellular PrP^C protein into the Sc state requires that the PrP^Sc and PrP^C proteins have matched sequences.

Different “strains” of PrP^Sc have been distinguished by characteristic incubation periods upon inoculation into mice. This implies that the protein is not restricted solely to alternative states of PrP^C and PrP^Sc but rather that there may be multiple Sc states. These differences must depend on some self-propagating property of the protein other than its sequence. If conformation is the feature that distinguishes PrP^Sc from PrP^C, then there must be multiple conformations, each of which has a self-templating property when it converts PrP^C.

The probability of conversion from PrP^C to PrP^Sc is affected by the sequence of PrP. Gerstmann–Straussler syndrome in humans is caused by a single amino acid change in PrP. This is inherited as a dominant trait. If the same change is made in the mouse PrP gene, mice develop the disease. This suggests that the mutant protein has an increased probability of spontaneous conversion into the Sc state. Similarly, the sequence of the PrP gene determines the susceptibility of sheep to develop the disease spontaneously; the combination of amino acids at three positions (codons 136, 154, and 171) determines susceptibility.

The prion offers an extreme case of epigenetic inheritance, in which the infectious agent is a protein that can adopt multiple conformations, each of which has a self-templating property. This property is likely to involve the state of aggregation of the protein.

Summary

Inactivation of one X chromosome in female (eutherian) mammals occurs at random. The Xic locus is necessary and sufficient to count the number of X chromosomes. The n – 1 rule ensures that all but one X chromosome are inactivated. Xic contains the gene Xist, which codes for an RNA that is expressed only on the inactive X chromosome. Stabilization of Xist RNA is the mechanism by which the inactive X chromosome is distinguished; it is then inactivated by the activities of Polycomb complexes, heterochromatin formation, and DNA methylation. The antisense RNA Tsix negatively regulates Xist on the future active X chromosome.

Condensins and cohesins control chromosome condensation and sister chromatid cohesion, respectively. Both are formed by SMC protein dimers. A specialized condensin complex mediates dosage compensation in C. elegans, reducing the level of expression of X chromosomes by half in XX hermaphrodites.

Methylation of DNA is inherited epigenetically. Epigenetic effects can be inherited during mitosis in somatic cells, or they may be transmitted through organisms from one generation to another. Some methylation events depend on parental origin. Sperm and eggs contain specific and different patterns of methylation, with the result that paternal and maternal alleles are differently expressed in the embryo. This is responsible for imprinting, in which the unmethylated allele inherited from one parent is essential because it is the only active allele; the allele inherited from the other parent is silent. Patterns of methylation are reset during gamete formation in every generation after erasure in primordial germ cells, the cells that ultimately give rise to the germline.

Prions are proteinaceous infectious agents that are responsible for the disease of scrapie in sheep and for related diseases in humans. The infectious agent is a variant of a normal cellular protein. The PrP^Sc form has an altered conformation that is self-templating: The normal PrP^C form does not usually take up this conformation but does so in the presence of PrP^Sc.

References

28.1 Introduction

Review

1. Tollefsbol, T., ed. (2012). Epigenetics in Human Disease. New York: Academic Press.

28.2 X Chromosomes Undergo Global Changes

Reviews

1. Briggs, S. F., and Reijo Pera, R. A. (2014). X chromosome inactivation: recent advances and a look forward. Curr. Opin. Genet. Dev. 28, 78–82.

2. Maclary, E., Hinten, M., Harris, C., and Kalantry, S. (2013). Long noncoding RNAs in the X-inactivation center. Chromosome Res. 21, 601–614.

3. Plath, K., Mlynarczyk-Evans, S., Nusinow, D. A., and Panning, B. (2002). Xist RNA and the mechanism of X chromosome inactivation. Annu. Rev. Genet. 36, 233–278.

4. Wutz, A. (2007). Xist function: bridging chromatin and stem cells. Trends Genet. 23, 457–464.

5. Wutz, A., and Gribnau, J. (2007). X inactivation Xplained. Curr. Opin. Genet. Dev. 17, 387–393.

Research

1. Changolkar, L. N., Costanzi, C., Leu, N. A., Chen, D., McLaughlin, K. J., and Pehrson, J. R. (2007). Developmental changes in histone macroH2A1-mediated gene regulation. Mol. Cell Biol. 27, 2758–2764.

2. Engreitz, J. M., Pandya-Jones, A., McDonel, P., Shishkin, A., Sirokman, K., Surka, C., Kadri, S., Xing, J., Goren, A., Lander, E. S., Plath, K., and Guttman, M. (2013). The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome. Science 341, 1237973.

3. Erwin, J. A., and Lee, J. T. (2008). New twists in X-chromosome inactivation. Curr. Opin. Cell Biol. 20, 349–355.

4. Lee, J. T., Strauss, W. M., Dausman, J. A., and Jaenisch, R. (1996). A 450 kb transgene displays properties of the mammalian X-inactivation center. Cell 86, 83–94.

5. Lyon, M. F. (1961). Gene action in the X chromosome of the mouse. Nature 190, 372–373.

6. Mlynarczyk-Evans, S., Royce-Tolland, M., Alexander, M. K., Andersen, A. A., Kalantry, S., Gribnau, J., and Panning, B. (2006). X chromosomes alternate between two states prior to random X-inactivation. PLoS Biol. 4, e159.

7. Penny, G. D., Kay, G. F., Sheardown, S. A., Rastan, S., and Brockdorff, N. (1996). Requirement for Xist in X chromosome inactivation. Nature 379, 131–137.

28.3 Chromosome Condensation Is Caused by Condensins

Reviews

1. Hirano, T. (2000). Chromosome cohesion, condensation, and separation. Annu. Rev. Biochem. 69, 115–144.

2. Hirano, T. (2006). At the heart of the chromosome: SMC proteins in action. Nat. Rev. Mol. Cell Biol. 7, 311–322.

3. Jessberger, R. (2002). The many functions of SMC proteins in chromosome dynamics. Nat. Rev. Mol. Cell Biol. 3, 767–778.

4. Lau, A. C., and Csankovszki, G. (2015). Condensin-mediated chromosome organization and gene regulation. Front Genet. 5, 473.

5. Meyer, B. J. (2005). X-chromosome dosage compensation. WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.8.1, http://www.wormbook.org.

6. Nasmyth, K. (2002). Segregating sister genomes: the molecular biology of chromosome separation. Science 277, 559–565.

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8. Peric-Hupkes, D., and van Steensel, B. (2008). Linking cohesin to gene regulation. Cell 132, 925–928.

9. Thadani, R., and Uhlmann, F. (2015). Chromosome condensation: weaving an untangled web. Curr. Biol. 25, R663–R666.

10. Thadani, R., Uhlmann, F., and Heeger, S. (2012). Condensin, chromatin crossbarring and chromosome condensation. Curr. Biol. 22, R1012–R1021.

Research

1. Blewitt, M. E., Gendrel, A. V., Pang, Z., Sparrow, D. B., Whitelaw, N., Craig, J. M., Apedaile, A., Hilton, D. J., Dunwoodie, S. L., Brockdorff, N, Kay, G. F., and Whitelaw E. (2008). SmcHD1, containing a structural-maintenance-of-chromosomes hinge domain, has a critical role in X inactivation. Nat. Genet. 40, 663–669.

2. Csankovszki, G., McDonel, P., and Meyer, B. J. (2004). Recruitment and spreading of the C. elegans dosage compensation complex along X chromosomes. Science 283, 1182–1185.

3. Ercan, S., Giresi, P. G., Whittle, C. M., Zhang, X., Green, R. D., and Lieb, J. D. (2007). X chromosome repression by localization of the C. elegans dosage compensation machinery to sites of transcription initiation. Nature Gen. 39, 403–408.

4. Haering, C. H., Farcas, A. M., Arumugam, P., Metson, J., and Nasmyth, K. (2008). The cohesin ring concatenates sister DNA molecules. Nature 454, 277–281.

5. Kanno, T., Bucher, E., Daxinger, L., Huettel, B., Böhmdorfer, G., Gregor, W., Kreil, D. P., Matzke, M., and Matzke, A. J. (2008). A structural-maintenance-of-chromosomes hinge domain-containing protein is required for RNA-directed DNA methylation. Nat. Genet. 40, 670–675.

6. Kimura, K, Rybenkov, V. V., Crisona, N. J., Hirano, T., and Cozzarelli, N. R. (1999). 13S condensin actively reconfigures DNA by introducing global positive writhe: implications for chromosome condensation. Cell 98, 239–248.

7. Liang, Z., Zickler, D., Prentiss, M., Chang, F. S., Witz, G., Maeshima, K., and Kleckner, N. (2015). Chromosomes progress to metaphase in multiple discrete steps via global compaction/expansion cycles. Cell 161, 1124–1137.

8. Nishino, Y., Eltsov, M., Joti, Y., Ito, K., Takata, H., Takahashi, Y., Hihara, S., Frangakis, A. S., Imamoto, N., Ishikawa, T., and Maeshima, K. (2012). Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibres without a 28-nm chromatin structure. EMBO J. 31, 1644–1653.

9. Shintomi, K., Takahashi, T. S., and Hirano, T. (2015). Reconstitution of mitotic chromatids with a minimum set of purified factors. Nat. Cell Biol. 17, 1014–1023.

28.4 DNA Methylation Is Responsible for Imprinting

Reviews

1. Horsthemke, B., and Wagstaff, J. (2008). Mechanisms of imprinting of the Prader-Willi/Angelman region. Am. J. Med. Genet. A. 146A, 2041–2052.

2. Kalish, J. M., Jiang, C., and Bartolomei, M. S. (2014). Epigenetics and imprinting in human disease. Int. J. Dev. Biol. 58, 271–278.

3. McStay, B. (2006). Nucleolar dominance: a model for rRNA gene silencing. Genes Dev. 20, 1207–1214.

4. Wood, A. J., and Oakey, R. J. (2006). Genomic imprinting in mammals: emerging themes and established theories. PLoS Genet. Nov 24; 2:e147.

Research

1. Chaillet, J. R., Vogt, T. F., Beier, D. R., and Leder, P. (1991). Parental-specific methylation of an imprinted transgene is established during gametogenesis and progressively changes during embryogenesis. Cell 66, 77–83.

2. Jacob, K. J., Robinson, W. P., and Lefebvre, L. (2013). Beckwith-Wiedemann and Silver-Russell syndromes: opposite developmental imbalances in imprinted regulators of placental function and embryonic growth. Clin. Genet. 84, 326–334.

3. Lawrence, R. J., Earley, K., Pontes, O., Silva, M., Chen, Z. J., Neves, N., Viegas, W., and Pikaard, C. S. (2004). A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance. Mol. Cell 13, 599–609.

4. Lecumberri, B., Fernández-Rebollo, E., Sentchordi, L., Saavedra, P., Bernal-Chico, A., Pallardo, L. F., Bustos, J. M., Castaño, L., de Santiago, M., Hiort, O., Pérez de Nanclares, G., and Bastepe, M. (2010). Coexistence of two different pseudohypoparathyroidism subtypes (Ia and Ib) in the same kindred with independent Gs{alpha} coding mutations and GNAS imprinting defects. J. Med. Genet. 47, 276–280.

5. Sahoo, T., del Gaudio, D., German, J. R., Shinawi, M., Peters, S. U., Person, R. E., Garnica, A., Cheung, S. W., and Beaudet, A. L. (2008). Prader-Willi phenotype caused by paternal deficiency for the HBII-85 C/D box small nucleolar RNA cluster. Nat. Genet. 40, 719–721.

6. Wood, A. J., Schulz, R., Woodfine, K., Koltowska, K., Beechey, C. V., Peters, J., Bourc’his, D., and Oakey, R. J. (2008). Regulation of alternative polyadenylation by genomic imprinting. Genes Dev. 22, 1141–1146.

28.5 Oppositely Imprinted Genes Can Be Controlled by a Single Center

Review

1. Edwards, C. A., and Ferguson-Smith, A. C. (2007). Mechanisms regulating imprinted genes in clusters. Curr. Opin. Cell Biol. 19, 281–289.

2. Plasschaert, R. N. and Bartolomei, M. S. (2014). Genomic imprinting in development, growth, behavior and stem cells. Development 141, 1805–1813.

Research

1. Bell, A. C, and Felsenfeld, G. (2000). Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405, 482–485.

2. Han, L., Lee, D. H., and Szabó, P. E. (2008). CTCF is the master organizer of domain-wide allele-specific chromatin at the H19/Igf2 imprinted region. Mol. Cell. Biol. 28, 1124–1135.

3. Hark, A. T., Schoenherr, C. J., Katz, D. J., Ingram, R. S., Levorse, J. M., and Tilghman, S. M. (2000). CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405, 486–489.

28.6 Prions Cause Diseases in Mammals

Reviews

1. Chien, P., Weissman, J. S., and DePace, A. H. (2004). Emerging principles of conformation-based prion inheritance. Annu. Rev. Biochem. 73, 617–656.

2. Collinge, J., and Clarke, A. R. (2007). A general model of prion strains and their pathogenicity. Science 318, 928–936.

3. Harris, D. A., and True, H. L. (2006). New insights into prion structure and toxicity. Neuron 50, 353–357.

4. Jeong, B. H., and Kim, Y. S. (2014). Genetic studies in human prion diseases. J. Korean Med. Sci. 27, 623–632.

5. Prusiner, S. B., and Scott, M. R. (1997). Genetics of prions. Annu. Rev. Genet. 31, 139–175.

6. Renner, M., and Melki, R. (2014). Protein aggregation and prionopathies. Pathol Biol (Paris). 62, 162–168.

Research

1. Basler, K., Oesch, B., Scott, M., Westaway, D., Walchli, M., Groth, D. F., McKinley, M. P., Prusiner, S. B., and Weissmann, C. (1986). Scrapie and cellular PrP isoforms are encoded by the same chromosomal gene. Cell 46, 417–428.

2. Bueler, H., Aguzzi, A., Sailer, A., Greiner, R. A., Autenried, P., Aguet, M., and Weissmann C. (1993). Mice devoid of PrP are resistant to scrapie. Cell 73, 1339–1347.

3. Hsiao, K., Baker, H. F., Crow, T. J., Poulter, M., Owen, F., Terwilliger, J. D., Westaway, D., Ott, J., and Prusiner, S. B. (1989). Linkage of a prion protein missense variant to Gerstmann-Straussler syndrome. Nature 338, 342–345.

4. McKinley, M. P., Bolton, D. C., and Prusiner, S. B. (1983). A protease-resistant protein is a structural component of the scrapie prion. Cell 35, 57–62.

5. Oesch, B., Westaway, D., Wälchli, M., McKinley, M. P., Kent, S. B., Aebersold, R., Barry, R. A., Tempst, P., Teplow, D. B., Hood, L. E., et al. (1985). A cellular gene encodes scrapie PrP27-28 protein. Cell 40, 735–746.

6. Scott, M., Groth, D., Foster, D., Torchia, M., Yang, S. L., DeArmond, S. J., and Prusiner, SB. (1993). Propagation of prions with artificial properties in transgenic mice expressing chimeric PrP genes. Cell 73, 979–988.