- Heterochromatin and Histone Interactions
- Telomeric silencing
- Telomeric silencing
- Polycomb and Trithorax
- CpG Islands
- Genomic Imprinting
- Epigenetic Effects and Inheritance
- The epigenetic inheritance of X chromosomes
- The epigenetic inheritance of X chromosomes
- Prion diseases
- Prion diseases
Epigenetics is the study of heritable changes in gene expression or cellular phenotype that is caused by mechanisms that do not involve changes in DNA sequence. In practice, epigenetics is the study of gene expression or phenotype changes. The Greek word ‘epi’ means over, above or outer and the word genetics means study of genes. The term ‘epigenome’ refers to the overall epigenetic state of a cell genome. ‘Epigenetic inheritance’ describes the ability of different states of the DNA, which may have different phenotypic consequences to be inherited without any change in the sequence of the DNA. This means that two individuals even if they have same DNA sequence may show different phenotypes. Some of the epigenetic effects include:
- A covalent modification of DNA (methylation of DNA bases).
- A proteinaceous structure that assembles on the DNA.
- A protein aggregate that controls the conformation of new subunit as they are synthesized.
These changes may remain through cell division for the remainder of the cell’s life and may also last for multiple generations. However, there is no change in the underlying DNA sequence of the organism instead, non-genetic factors cause the organism’s genes to behave (or ‘express themselves’) differently.
In case of DNA methylations, a DNA sequence methylated in its control region may fail to be transcribed, while the unmethylated sequence will be expressed. Certain proteins that assemble on the DNA usually have a repressive effect by forming heterochromatin regions that prevent the expression of genes within them. Usually the tails of histone H3 and H4 are not acetylated in constitutive heterochromatin. If the centromeric heterochromatin is acetylated, silenced genes may become active. The effect may be continued through mitosis and meiosis. The molecular basis of epigenetics is a complex phenomenon and involves changes in the switching ‘on’ and ‘off’ of certain genes.
Epigenetics is widely used in diagnostics and research. Epigenetics finds applications in a wide range of molecular biologic techniques including: methylation-specific PCR (MSP), popular chromatin immunoprecipitation, bisulphite sequencing, FISH, methylation-sensitive restriction enzymes and DNA adenine methyltransferase identification. The use of bioinformatic methods is playing an increasing role in computational epigenetics.
The important role of epigenetic defects analysis in cancer opens up new and exciting opportunities for improved diagnosis and therapy.
Euchromatin and heterochromatin can be localized during the interphase in the nucleus. Heterochromatin is highly inert and condensed, transcriptionally repressed, replicates during the late S-phase and is found in the periphery of the nucleus. Centromeric heterochromatin predominantly consists of satellite DNAs. When a gene is transferred to a position adjacent to the heterochromatin, it may also become inactive, i.e., it too becomes heterochromatic. Such inactivation is the result of an ‘epigenetic effect’. It differs between individual cells in an animal. This results in the phenomenon of ‘position effect variegation’, where genetically identical cells have different phenotypes; for example, the position effect variegation in the fly eye. A few regions in the eye are colourless, whereas a few others are red in colour. This is because the white gene required for developing the red pigment was inactivated by heterochromatin in some cells, while remained active in others.
Inactivation spreads from heterochromatin into adjacent region for varying distance, which may extend from a nearby gene to distant gene. This inactivation occurs during the embryonic state and thereafter is stably inherited by all progeny cells.
The formation of heterochromatin is a two-stage process that includes:
- A nucleation event occurs by the binding of a protein that recognizes the heterochromatic sequence.
- Propagation of the nucleated structure throughout the chromatin fibre.
However, such spreading of heterochromatic regions is prevented by the presence of an activated promoter of the nearby gene and by the presence of insulators.
Genes translocated to a telomeric location shows variable loss of activity. This results from the spreading effect that starts from the telomeres. The binding of a protein called Rapl to the telomeric sequence triggers the nucleation event, which recruits the heterochromatin proteins thereby inactivating the gene sequences.
The inactivation of chromatin occurs by the addition of proteins to the nucleosomal fibre that results in chromatin condensation thus making it inaccessible to the transcription machinery. The proteins added to the heterochromatic DNA may also directly block the regulatory sites or sometimes even can directly inhibit transcription. The molecular mechanisms needed for the formation of heterochromatin has been identified in Drosophila mutants. They include the following genes.
- Su(var)—The gene products of this gene suppress the position effect variegation.
- E(var)—The gene products of this gene enhance the position effect variegation.
The genes were so named for the behaviour of the mutant loci. The Su(var) mutations lie in the genes whose products are needed for the formation of heterochromatin. They include enzymes that act on chromatin-like histone deacetylase. The E(var) mutations lie in the genes whose products are required for activating gene expression. They include the members of SWI/SNF complex. HP1 (heterochromatic protein 1) is one of the most important Su(var) proteins.
The state of histone methylation is an important factor that determines whether a DNA sequence exists as heterochromatin or euchromatin state. The methylation of histone H3 lysine 9 favours heterochromatin formation, while histone H3 lysine methylation is required for euchromatin formation (Figure 10.1). A trimethyl H3K4 demethylase known as Lid2 in Schizo-saccharomyces pombe interacts with 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 co-ordinated manner to control the hetero-chromatic and euchromatic state of a specific region.
Figure 10.1 DNA methylation and histone modifications
Heterochromatin formation at telomeres depends on a set of genes known as ‘silent information regulators’ (SIR genes). The mutations of SIR genes relive the inactivation of the genes that are integrated near telomeric heterochromatin. Rap1 has the crucial role in the formation of heterochromatin. It recruits Sir4, which in turn recruits Sir3 and HDAC. Sir2, Sir3 and Sir4 then interact directly with the histones H3 and H4. This complex can polymerize further and gradually spreads along the chromatin. This process inactivates the DNA region because coating with Sir3/Sir4 complex has an inhibitory effect or because Sir2-dependent deacetylation that represses the transcription.
Silencing complexes repress chromatin activity because they condense chromatin, so that regulatory proteins cannot find their target sequences. Further, the presence of these proteins hinders the binding of transcription factors and RNA polymerase. They also block chromatin remodelling. The gene activation and the repressing effects of chromatin compete with each other and thus the activation of a promoter of an adjacent gene inhibits the spread of silencing complex.
In human cells, the centromeric specific protein CENP-B is required to initiate histone modification (H3 deacetylation of K9 and K14, followed by methylation of K9) that triggers the binding of HP1 and ultimately leads to the formation of heterochromatin in that region.
Polycomb group (PcG) proteins perpetuate a state of repression of genes through cell division. The polycomb responsive element (PRE) is a DNA sequence that is required for the action of PcG. The PRE provides a nucleation centre from which PcG proteins propagate an inactive structure. The trithorax group (trxG) proteins antagonize the action of PcG. The PcG and trxG can bind to the same PRE sequence with opposing effects.
The PcG proteins have some common regulatory roles. They function in large complexes; for example, the polycomb repressive complex (PRC1) contains Pc, several PcG proteins and five general transcription factors. They affect chromatin remodelling and induce repression. Once repression is established, the PcG proteins recognize it and perpetuate it through the cell division of the daughter cells.
The PRE is a complex structure of about 10 Kb. Several proteins have binding sites within the PRE sequence; for example, PcG, GAGA factor, etc. Once PcG binds the PRE, which provides a state of nucleation centre, a structural state depending on PcG proteins propagate.
The trxG proteins have the opposite effect to the PcG proteins. They maintain genes in an active state. They are quite diverse. Some comprise subunits of chromatin remodelling enzymes such as SWI/SNF, whereas others have histone-modifying activities. trxG also binds to the same PRE sequence to which PcG binds. The trxG proteins act by making chromatin continuously accessible to transcription factors. Both PcG and trxG can regulate homeotic gene promoters some distance away from PRE through DNA looping.
DNA methylation occurs at specific sites. In bacteria, the DNA methylation is used for identifying bacterial restriction-methylation system that is involved in phage defence and is also used for distinguishing replicated and non-replicated DNA. In eukaryotes, the DNA methylation is connected with the control of transcription. The methylation of a control region is usually accompanied by gene inactivation. Methylation in eukaryotes usually occurs at the CpG islands in the 5′ regions of some genes. These are the CG dinucleotide-rich regions of the gene. About 2–7 per cent of animal cell DNA is methylated. Methylation occurs at the 5th position of cytosine producing 5-methylcytosine. Most 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 considered fully methylated. Upon replicating this site, each daughter duplex will have one methylated strand and one unmethylated strand. Such site is called hemimethylated. If methylation of the unmethylated strand occurs, then the hemimethylated site also becomes fully methylated.
If replication occurs first, then the hemimethylated condition will be perpetuated on one daughter duplex and the other duplex will become unmethylated.
The state of methylation is controlled by DNA methyltransferases or methylases or DNMTs, which adds methyl groups to the 5th position of cytosine (Figure 10.2). There are two types of methyltransferases, namely:
- De novo methyltransferase: It modifies DNA at a new position and acts only on unmethylated DNA. There are two de novo methyltransferases, namely DNMT3A and DNMT3B in mouse; these have different target sites.
- Maintenance methyltransferase: It acts constitutively only on hemimethylated DNA and converts them to fully methylated sites.
A protein called UHRF1 is important for the maintenance of methylation and associates with DNMT1. This protein is able to recognize CpG dinucleotides and preferentially binds to hemimethylated DNA. UHRF1 increases the efficacy of DNMT1 for maintenance methylation at hemimethylated sites. UHRF1 also interacts with methylated histone H3. This shows that the maintenance of DNA methylation is connected with the stabilization of heterochromatin structure.
Promoters are the most common sites of methylation. They are methylated when gene is inactive and unmethylated when gene is active. DNA methylation plays a role in controlling gene expression. The transcriptional silencing of selected genes caused by DNA methylation plays a crucial role in the development and progression of human cancers.
Figure 10.2 SUVAR39H is a methyltransferase that specifically methylates the lysine 9 of histone H3. Such a methylation creates a binding site for the heterochromatin protein HP1 that recruits a DNA methyltransferase, capable to methylate the CpG in DNA (Me = methyl; methyl H3-K9 = methyl on lysine 9 of histone H3; HP1 = heterochromatin protein 1; DNMT = DNA methyltransferase)
Satellite DNAs are also methylated. The mutations of DNMT3B prevent the methylation of satellite DNA and this causes centromere instability; for example, a disease called ‘ICF—immunodeficiency centromere instability facial anomalies’ is caused by such mutations.
‘Rett syndrome’ is another disease that emphasizes the importance of DNA methylations. It is caused by the mutations of the gene for the protein MeCP2 that binds methylated CpG sequences. The disease is characterized by autism-like symptoms that are the result of a failure of normal gene silencing in the brain.
There are several ways to generate a demethylated site. These include:
- Loss of methylation at a site due to the incomplete fidelity of DNMT1 during maintenance methylation.
- Blocking the maintenance methylase from acting on the site when it is replicated. After the second round of replication, the daughter DNA will be unmethylated.
- Actively demethylating the site, i.e., removing methyl groups from methylated cytosine from the DNA and the excised region is then filled by repair system. Then, enzyme cytidine deaminase may be involved, which introduces a mismatch that is then corrected.
Active demethylation can occur to the paternal genome soon after fertilization. DNMT3A and DNMT3B participate in active DNA demethylation. They may possess deaminase activity and are involved in gene demethylation. The enzyme mediates the oxidative deamination of cytosine and converts it to 5-methylcytosine (thymine), the resulting guanine-thymine G-T mismatch is repaired by base excision repair thus leading to the demethylation of previously methylated CpG site.
The pattern of methylation of germ cells is established in each sex during gametogenesis. This takes place in two steps.
- First, the existing pattern of methylation is erased in primordial germ cells.
- The pattern that is specific for each sex is imposed during meiosis.
In males, the methylation pattern develops in two stages: first, the methylation pattern that is characteristic of mature sperm is established in the spermatocyte. Further changes in the pattern are made during fertilization. In females, the maternal pattern is imposed during oogenesis.
Systematic changes occur during early embryogenesis. Some sites will continue to be methylated, while some sites will be specifically unmethylated in cells in which a gene is expressed. As specific genes are activated, individual sequence-specific demethylation occurs during somatic cell development.
The specific pattern of methyl groups in germ cells is responsible for the phenomenon of imprinting. The difference in the behaviour between the alleles inherited from each parent is because of imprinting. For example, the allele coding for insulin-like growth factor II (IGF-II) that is inherited from the father is expressed, but the allele inherited from mother is not expressed. This is because the IGF-II gene of oocytes is methylated in its promoter whereas the IGF-II gene of sperm is not methylated so. Thus, the two alleles behave differently in the zygote. This sex-dependent pattern is reversed for some gene.
The consequence of imprinting is that an embryo is hemizygous for any imprinted gene. Thus, in the case of heterozygous cross over 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 allele. This type of dependence on the directionality of the cross is an example of epigenetic inheritance. In this type of inheritance, the factors other than the sequence of genes themselves influence their effects. Although the paternal and maternal alleles have identical sequences, they have different properties, depending on which parent provided them. These properties are inherited through meiosis and subsequent mitosis. About 1–2 per cent of the mammalian transcriptosome comprise of imprinted genes and these are sometimes clustered.
Prader-Willi syndrome is caused by the deletion of a 20-kb sequence that silences distant genes on either side of the deletion. This prevents the Male from resetting the paternal mode to a chromosome inherited from the mother. Consequently, both genes remain in maternal mode, so that the paternal and maternal alleles are silent in the offspring.
Epigenetic inheritance describes the ability of different states, which may have different phenotypic consequences to be inherited without any change in the sequence of the DNA (Figure 10.3). Epigenetic mechanisms can be divided into two classes.
- Covalent modification of the DNA by the attachment of a moiety and this is perpetuated. Two alleles with the same sequence may have different states of methylation and hence have different properties.
- A self-perpetuating protein state may be established involving the assembly of protein complex, modification of specific proteins and establishment of an alternative conformation.
Methylation establishes the epigenetic inheritance as long as the maintenance methyltransferase acts constitutively to restore the methylated state after each cycle of replication. In mammalian cells, epigenetic effects are first erased in primordial germ cells and then created latter by resetting the state of methylation differently in male and female meioses during gametogenesis.
The Epigenetic Inheritance of X Chromosomes
Variation in the number of X chromosomes in mammals poses a problem for gene regulation. If X-linked genes are expressed equally in each sex, females will have twice as much of the X genetic content as males. However, this does not happen and is prevented by dosage compensation, which equalizes the level of expression of X-linked genes in both male and female sexes. In mammals, one of the X chromosomes is inactivated completely. This inactivation of X chromosome is mediated by a gene called ‘Xic—X chromosome inactivation centre’. Inactivation spreads from Xic along the entire X chromosome. As the result, females have only one active X chromosome just like the males. This active X chromosomes of females and the single X chromosome of males are expressed equally. The inactive X chromosome is perpetuated in a heterochromatic state whereas the active X chromosome is euchromatic. Once the inactive state is established, it is inherited by descendant cells. This is an example of epigenetic inheritance as it is not dependant on the DNA sequence.
Independent protein aggregates that cause epigenetic effects are called ‘prions’. Prions are an unusual form of epigenetics. Their stable inheritance and complex phenotypes come about through protein folding rather than nucleic acid-associated changes. They are linked to chromosomal remodelling factors. Swil, a subunit of SWI/SNF chromatin remodelling complex and this protein can become a prion. Swil is cytoplasmically transmitted. This suggests that inheritance through proteins can influence chromatin remodelling and thus affect gene regulation throughout the genome.
Prions perpetuate by protein folding. A unique feature of prion-forming proteins is their ability to exist in different stable conformational states. Apart from a ‘native’ non-prion conformation, they occasionally fold into a prion conformation. This then replicates itself by templating the conformational conversion of other molecules of the same protein. These changes in conformation greatly alter the functions of the proteins involved, resulting in phenotypes specific to each determinant protein.
De novo prion formation appears to proceed through a high-energy oligomeric nucleus that is stabilized by interacting with, and converting, other prion proteins to the same conformation. The elongating prion polymer is then cut into smaller and actively growing pieces by the action of protein remodelling factors such as the disaggregase Hsp104. The resulting fragments are disseminated to daughter cells, ensuring the stable inheritance of the self-perpetuating prion template through each cell division. Prions are stable even during mating and meiosis, allowing their transmission through the germ line. Prion states are, however, reversible. Random fluctuations in prion dissemination to daughter cells, as well as changes in the activities of remodelling proteins and other factors, can generate daughter cells with the original non-prion state.
Many prion phenotypes result from qualitative changes in protein function. As the structure of the protein plays a role in determining its function, the refolding of a polypeptide into its prion form can alter the non-prion function and can even create gains of function.
The prion diseases are a large group of related neurodegenerative conditions, which affect both animals and humans. Prion diseases impair brain function, causing memory changes, personality changes, a decline in intellectual function (dementia) and problems with movement that worsen over time. The signs and symptoms of these conditions typically begin in adulthood and these disorders lead to death within a few months to several years. Familial prion diseases of humans include classic Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS) and fatal insomnia (FI) (Figure 10.4).
Figure 10.4 Prion diseases of the brain
One type of prion disease in humans, variant Creutzfeldt-Jakob disease (vCJD), is acquired by eating beef products obtained from cattle with prion disease. In cows, this form of the disease is known as bovine spongiform encephalopathy (BSE) or, more commonly, ‘mad cow’ disease. Another example of an acquired human prion disease is kuru, which was identified in the South Fore tribe in Papua New Guinea. The disorder was transmitted when tribe members ate the tissue of affected people during cannibalistic funeral rituals. Familial forms of prion disease are caused by inherited mutations in the PRNP gene. This gene encodes a protein called prion protein (PrP). Normally, this protein is likely to be involved in transporting copper into cells. It may also play a role in brain cells protection and communication. In familial cases of prion disease, mutations in the PRNP gene cause cells to produce an abnormal form of the prion protein known as PrPSc.
- Epigenetics is the study of heritable changes in gene expression or cellular phenotype that is caused by the mechanisms that do not involve changes in DNA sequence.
- Epigenome refers to the overall epigenetic state of a cell genome.
- Epigenetic inheritance describes the ability of different states of the DNA, which may have different phenotypic consequences to be inherited without any change in the sequence of the DNA. This means that two individuals even if they have same DNA sequence may show different phenotypes.
- Position effect variegation refers to the concept where genetically identical cells have different phenotype.
- PcG proteins perpetuate a state of repression of genes through cell division. The PRE is a DNA sequence that is required for the action of PcG.
- The trxG proteins have the opposite effect to the PcG proteins. They maintain genes in an active state.
- Independent protein aggregates that cause epigenetic effects are called prions. Prions are an unusual form of epigenetics. Their stable inheritance and complex phenotypes come about through protein folding rather than nucleic acid-associated changes.
Describe epigenetic inheritance.
Explain the phenomenon of position effect varigation.
Discuss about the mechanism and significance of telomeric silencing.
Describe SIR genes.
What are CpG islands? Explain its connection with DNA methylation.
Briefly explain Genomic imprinting.
What are prions? Discuss about the diseases associated with it.
DNA methyl transferases adds methyl groups to the 5th position of———.
Which one of the following is not a disease connected with prions?
- Fatal insomnia
- Rett Syndrome
- Creutzfeldt-Jakob disease
- Gerstmann-Sträussler-Scheinker syndrome
———is the study of heritable changes in gene expression or cellular phenotype that is caused by mechanisms that does not involve changes in DNA sequence.
- Molecular Genetics
———refers to the concept where genetically identical cells have different phenotype.
- position effect variegation
- Epigenetic inheritance
- Chromosome inactivation center
- Genomic imprinting
What is the usual site where Methylation in eukaryotes usually occurs?
- Polycomb group proteins (Pc-G)
- CpG islands in the 3′ regions of some genes
- CpG islands in the 5′ regions of some genes
- None of the above
Heterochromatin formation at telomeres depends on set of genes known as———.
- IGF - II genes
- CpG sequences
- X chromosome inactivation center
- silent information regulators (SIR genes)
———modifies DNA at a new position and acts only on unmethylated DNA.
- denovo methyl transferase
- maintanence methyl transferase
Blasco, MaríA. 2007. ‘The Epigenetic Regulation of Mammalian Telomeres’, Nature Reviews Genetics, 8(4): 299–309.
Feinberg, Andrew P. 2008. ‘Epigenetics at the Epicenter of Modern Medicine, The Journal of American Medical Association, JAMA, 299(11): 1345–1350.
Halfmann, Randal and Lindquist, Susan. 2010. ‘Epigenetics in the Extreme: Prions and the Inheritance of Environmentally Acquired Traits’, Science, 330(6004): 629–632.
Krebs, Jocelyn E., Lewin, Benjamin, Goldstein, Elliott S., and Kilpatrick, Stephen T. 2009. Lewin’s GENES X. Jones and Bartlett.