Epigenetics: an overview
Epigenetics: an overview
Cristina Hernández Rollán
Líf- og umhverfisvísindadeild
Háskóli Íslands
2014
Epigenetics: an overview
Cristina Hernández Rollán
12 eininga ritgerð sem er hluti af
LÍF219F Námskeið til meistaraprófs í líffræði
Leiðbeinandi
Zophonías O. Jónsson (Ph.D)
Líf- og umhverfisvísindadeild
Háskóli Íslands
2014
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Epigenetics: an overview
12 eininga ritgerð sem er hluti af LÍF219F Námskeið til meistaraprófs í líffræði
Höfundarréttur © 2014 Cristina Hernández Rollán
Öll réttindi áskilin
Líf- og umhverfisvísindadeild
Háskóli Íslands
Askja, Sturlugatu 7
101, Reykjavik
Iceland
Sími: 525 4000
Skráningarupplýsingar:
Cristina Hernández Rollán, 2014, Epigenetics: an overview, LÍF219F Námskeið til meistaraprófs í líffræði, Líf- og umhverfisvísindadeild, Háskóli Íslands.
Reykjavík, Janúar 2014
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TABLE OF CONTENTS
SOME OTHER SYNDROMES CAUSED BY EPIGENETIC MISFUNCTION .................................. 28
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LIST OF FIGURES
Figure 1: Waddingston’s Epigenetics Landscape………………………………………………………………… ……………………5
Figure 15: Schematic description of the increase methylation experience by infected lymphocytes T by the
HIV virus……………………………………………………………………………………………………………………………………………….. 35
LIST OF TABLES
Table 1 . Human DNA methyltransferases …………...………...........……………………………………..……………………....9
Table 2 . Histone modifications associated with transcription ………………………...……………………………………..12
Table 3 . Bacterial species, bacterial factors and effects ……………………………………………………………………..….32
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1.INTRODUCTION
The publication of the human genome in Science and Nature on February 16 th
, 2001 in many ways brought with it more questions than answers. We are now aware of how the human genome is organized, but we have yet to understand how all the genomic components interrelate to drive gene expression, inheritance, regulation, etc. DNA sequences alone give us very incomplete knowledge about functions of the genome or how it directs cell development.
Cells respond in different ways to external stimuli, they gain and retain responses to these changes as they develop and are able to transfer this information as changes that occur within the chromatin to the daughter cells (Ashe et al., 2012). Responses that are potentially inheritable without altering of the DNA sequence are called epigenetic mechanisms. Epigenetic modifications can be further classified into three major groups:
DNA methylation and hydroxymethylation, posttranslational modifications of histone tails and regulation of gene expression by non-coding RNAs (ncRNAs) (Gonzalo 2010;
Minocherhomji et al., 2012).
Many external factors such as temperature, viral infections, bacteria and diet for example, can affect the chromatin organization and structure, driving the cells to respond to them by epigenetic changes (Abraham et al., 2012; Bierne et al., 2012) and impacting the phenotype without disruption of the genotype.
The term epigenetic was first introduced by Conrad Waddington in the 1940s.
Although he did not believe that some external factors could be inherited without changes in the DNA sequences, he was aware of the interactions of the environment with inheritance. He coined the term “epigenetic landscape”.
Figure 1: Waddingston’s Epigenetics Landscape. The figure represents the complex process of cellular development where the ball represents the cell and the slopes (lines) represent metaphorically the possible pathways to follow. Every possibility will give different outcomes. Figure adapted from Waddington, 1957 adapted from Goldberg et al., 2007.
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The discovery of the epigenome and how the epigenetic changes can be phenotypically inherited over generations have given rise to a new area of study in biology. It is now clear that epigenetic changes influence cell fate and development, and studying them will help understanding more complex areas of biology such as embryogenesis and development. In addition, the study of epigenetics can also contribute understanding of many diseases such as some types of cancer, immune disorders, even bacterial and viral infections that have eluded old fashioned approaches of understanding genetics and inheritance (Rodríguez et al., 2003; Gal-Yam et al., 2007;
Paschos and Allday, 2010; Bierne et al., 2012).
The father of epigenetics Conrad Waddington proposed the name epigenetics based on the Aristotelian theory of epigenesis in his book “On the generation of animals”. He believed that environmental changes can gradually influence hereditability in an animal.
Waddington choose the term “epi” that means over, upon or above and “genetics” to infer that genes take part in this process. As illustrated in figure 1, he tried to visualize and explain that there is no simple relationship between the cell and gene expression and the possible outcomes. Epigenetics is understood as the result of many cascades of interactions throughout the process that can lead to many possible outcomes.
Consequently, Conrad Waddington defined epigenetics as “the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being” (Waddington, 1947). For example, in nature it can be appreciated the fact that having the same genotype does not always lead to the same outcome. For example, most of the cells in the human body share the same genotype but clearly do not all have the same phenotype (e.g. skin cells are quite different from the lungs cells and they maintain the same phenotype throughout divisions).
Some old experiments in frogs (Briggs and King, 1952) and some more recent in mammals (Wilmut et al., 1997 and Wakayama et al., 1998), demonstrated the basic principle that the nucleus of a differentiated cell contains the complete genetic information for a new cell/organism and can, at least in theory, be reprogrammed from scratch (for review see McCarrey 2011). The phenotypic difference observed in multicellular organisms is explained by the term plasticity. Plasticity in this cellular context is understood as the ability of a cell to differentiate or develop into every cell type of that organism (a particular characteristic of stem cells) (Price et al., 2003).
Plasticity is easily appreciated in colonies of honeybees where the development of the queen bee is quite different from the development of the worker bees (although they share exactly the same genetic information) and it is solely dependent on the way they have been fed at larvae stage. This difference in phenotype is a plasticity example brought on by environmental factors and mediated through epigenetics rather than differences in genotype.
Modern usage of the term epigenetics differs significantly from Waddington’s original definition. In 2001 it was redefined in Science as “The study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence” (Wu et al., 2001). Nevertheless, the definition and term are still nowadays under continuous consideration and discussion (Reviewed in Jablonka and
Lamb 2002).
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An obvious deduction from Waddington’s theory that has since interested the scientific community is the possible involvement of epigenetics in evolution. Do epigenetics have any theoretical implications for the neo-Darwinian theory of evolution? Given the fact that epigenetic modifications occurring from environmental adaptations could be inherited between generations (Ashe et al., 2012) maybe Lamark was not so wrong after all.
The scientific community disagrees when it comes to epigenetic inheritability. It is clear that epigenetic changes are decisive for differentiation in an organism, but for many specialists it is consider unlikely that this heritability of phenotype is frequently transmitted in a transgenerational manner. Nevertheless, this statement is not true for some species such Caenorhabditis elegans (Henderson & Jacobsen, 2007; Ashe et al.,
2012), and it is accepted nowadays that heritable epigenetic variation can lead to evolutionary changes (Jablonka and Lamb, 2002; McCarrey 2011; Ashe et al., 2012;
Chong and Whitelaw, 2004).
It has also been identified that when the chromatin modifications are inherited, they form an epigenetic memory (Bird 2007; Paschos and Allday 2010). Furthermore, epigenetics is not only important or crucial for differentiation of tissues and organs, since the epigenetic mechanisms have also been found in unicellular organisms. Here, epigenetics play a different role where the environment is a major issue in epigenetics inheritance (Jablonka and Lamb, 2002; Ashe et al., 2012). Therefore, the environmental component also has to be taken into account in evolution and adaptation; as Lamark once stated, “environment is a stimulator in addition to a selector of variation and specialization”. There are some examples where organisms such Caenorhabditis elegans for instance, can under certain conditions inherit epigenetic modifications through several generations at a selective advantage (see Ashe et al., 2012; Jablonka et al., 1995).
A study from the University of Michigan in 2013 showed that tadpoles grow larger tails when they are under stress because of the nearby presence of predators, such as dragonfly larvae. By these means, they achieve an advantage over tadpoles with smaller tails, since they can swim faster and hence, they are more difficult prey for the dragonfly. It has been proposed that this is due to plasticity responses to the environment that allows this body change (Maher et al., 2013).
Another example comes from a study by Chandler and Stam in 2004; they identified cases where paramutation (explained later) is affected by environmental temperature.
The change in temperature alters the transitions between the active and inactive forms of an allele (Mikula, 1995). Thus, paramutation and paramutation-like events supply a mechanism in which gene expression can change rapidly due to adaptation.
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2.CHROMATIN STRUCTURE
Understanding how the chromatin is organized and knowing how proteins interact with the DNA to form this peculiar structure is vital to the study and comprehension of the mechanisms behind epigenetics. In eukaryotes, chromosomal DNA is organized inside the nucleus of the cell. Condensed DNA and associated proteins constitute the chromatin (Bernstein et al., 2007). The organization of chromatin takes place at several levels. The first stage consists of approximately 147 base pairs of double stranded DNA wrapping approximately 1.65 times around the core of a histone octamer. This represents the basic level of organization. The histones that form the octamer are two copies of each H2A, H2B, H3 and H4. The next level of organization involves the nucleosomes aligning along the DNA and a fifth type of histone (H1 and isoforms) which serves as a linker between them. H1, H1-isoforms, RNAs and other non-histones proteins assist in a higher level of condensation forming the 30nm fiber. Scaffold proteins together with chromatin remodeling complexes maintain the chromatin structure and allow dynamic movements from euchromatin (easily accessible DNA) to heterochromatin (DNA that is difficult to access) (Bierne et al., 2012). Euchromatin is the loosely packed chromatin where active genes are found transcribed, whereas the heterochromatin contains highly compact DNA in two different varieties, facultative and constitutive heterochromatin. Note that the core histones (H2A, H2B, H3 and H4) are the histones found in the S-phase of the cell cycle and for a long time they were considered the only core histones (Kornberg and Lorch, 1999). Later studies revealed that many other histone variants are found outside the S-phase in various organisms and are introduced into the chromatin in a DNA-replication independent manner (review in
Kamakaka & Biggins, 2005). They are referred as non-canonical histones. Differences between the canonical and non-canonical histones are found in the histone tails or between key amino acid residues (Bönisch & Hake, 2012).
Histones are characterized by long C or N-terminal tails subject to several posttranslational modifications which in turn are connected with biological functions. Posttranslational modifications in histone tails are diverse and often the same type of chemical modification can result in different responses. Such observation indicates that cross-talk must exist between histone modifications (see figure 6). Cross-talk is defined as the influence of post-translational modifications on the same or separate histones tails that can result in different signaling cascades leading to chromatin changes. For instance, methylation on H3-K9 seems to cause inhibition of acetylation of residues
K14, K18, and K23 in the H3 tail, thereby preventing transcription (Fischle et al.,
2003).
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Figure 2: Representation of the chromatin organization in the nucleus. The DNA is condensate into chromatin, firstly organized into nucleosomes (11nm diameter) with histones H2A, H2B, H3 and H4. The second level of organization is set when the histone H1 binds nucleosomes into the fiber of 30nm of diameter. Higher level of condensation is set by the scaffold proteins that grant different levels of condensations or loops to allow or repress transcription. Figure from CliffsNotes, Structure of
Chromatin, 2013.
The chromatin not only functions to keep the DNA tight up together inside the nucleus, but also to regulate gene expression and regulate DNA processes such as transcription, replication, recombination, DNA repair, DNA control, formation of the chromosomes, mitosis, meiosis, etc.
As explained before, the definition of epigenetics includes every mechanism that can control gene expression in a potentially inherited way without a change in the DNA sequence. Thus, DNA can be modified without disruption of its sequence by epigenetic marks that alter the chromatin structure and the affinity of proteins to chromatin
(Miranda, & Jones 2007). But, how do these changes work? What makes them potentially inheritable? To answer these questions, we need to look at the modifications that the chromatin and DNA undergo in order to determine inheritable changes in gene expression.
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3.DNA METHYLATION
Epigenetic marks at DNA level include methylation and hydroxymethylation of the cytosine bases. For instance, in mammals, cytosines preceding guanines (CpG) are known to be susceptible to methylation and almost all CpGs in the genome are found methylated (Bird, 2002; Goll and Bestor, 2005). However, only 3% of the cytosines in the human genome are methylated. In addition to mCpG a small fraction is found as mCpNpG, where N can be any nucleotide (Hum Mol Gen, 4 th
Edition).
Methylation of the 5 C of the cytosines has a crucial role for the survival and the correct function of the cell and is found in almost all vertebrates. However, cytosines that are methylated are chemically unstable and more prone to deamination.
Deamination of methylated cytosines results in conversion to the nucleotide thymine which is a normal base not recognized by the DNA repair machinery and consequently, over time, C pG sequences in highly methylated regions are converted to T pG unless the presence of C is selected for. Spontaneous deamination of non-methylated cytosines gives rise to uracil that is an abnormal base in DNA and is recognized and efficiently removed by the DNA repair machinery (see Weiner and Toth, 2011; Hum Mol Gen, 4 th
Edition). The conversion of CpG dinucleotides to TpG dinucleotides has been analysed by the Human Genome Sequencing Consortium which have concluded that CpG dinucleotides in the human genome only occur at 21% of the expected frequency
(International Human Genome Sequencing Consortium, et al. 2001).
Methylated DNA has been demonstrated to attract methyl-DNA-binding proteins that in turn recruit chromatin modifying complexes which condensate the chromatin around the methylated DNA silencing the region to a repressive state (Bird and Wolffe, 1999;
Urnov and Wolffe, 2001).
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Figure 3: CpG methylation reaction. A) Representation of a CpG islands where the cytosine is preceded by a guanine base in a DNA sequence. B) Methylation reaction of
CpG islands. The reaction is catalyzed by a DNA methyltransferases (DNMTs) that use the methyl group from S-adenosyl methionine (SAM-CH3) and transfer it to the cytosine base. Figure from Maxwell et al., 2009.
Cytosine methylation (along with the derived variant hydroxymethylation) is the only covalent DNA modification known in mammals and provides heritable epigenetic information that is not encoded in the DNA sequence (Bernstein et al., 2007). Passage of heritable information through cell division is possible because the CpG methylation is not reset but copied to the nascent DNA strands.
Covalent modifications of the protein component of chromatin can be classified according to the function of the enzymes that carry out such modification. There are basically two main functions, adding or removing covalent modifications. Examples of enzymes that add the covalent modifications are histone methyltansferases (HMTs), histone acetyltransferases, kinases, etc. On the other hand, enzymes that remove these modifications are histone demethylases (HDMs), histone deacetylases (HDAC), and phosphatases (see Bierne et al., 2012). Histone deacetylases catalyse deacetylation of acetylated lysine residues in histone tails. They are divided into HDAC class I, II and III where class I and II are Zinc-dependent enzymes and class III is NAD-dependent
(Paschos and Allday 2010).
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3.1.
DNA methyltransferases in mammals:
In humans, DNA methyltransferases are classified into de novo methyltransferases and maintenance methyltransferases. The de novo DNA methyltransferases in humans are named DNMT3A and DNMT3B and the single maintenance DNA methyltransferase is known as DNMT1 (Bestor, 2000; and Okano et al., 1999).
De novo DNA methyltransferases carry out the methylation reaction by the transfer of a methyl group (-CH3) from S adenosyl-L-methionine to the carbon 5 of the cytosine
(Doerfler, 1983) (see figure 4).
There is another methyltransferase in humans known as DNMT3L that allows targeting to specific sequences. Finally, a related enzyme named TRDMT1 has similar structure to the other DNA methyltransferase and was therefore classified as such and named DNMT2. It has however shown to be RNA-methylating enzyme and has therefore been renamed (Human Molecular Genetics, Garland Science, 4 th
Edition).
Figure 4: A schematic figure of de novo and maintenance methylation in mammals.
DNMT3a and DNMT3b introduce methyl groups to DNA in cytosine bases preceding guanines. DNMT1 maintain the state of methylation when replication occurs. It follows the pattern of the original strand and copies it to the daughter strand. Picture adapted from Reik and Walter 2001.
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Maintenance of the CpG methylation is only possible thanks to the enzyme DNMT1 recognizes the newly synthesized DNA strand (recently methylated) and methylates according to the pattern on the parent strand. Recent studies have shown that DNMT methyltransferases are brought to specific location of the genome through interactions with proteins such as the polymerase clamp PCNA, or transcription factors suggested to play a role in repression of transcription (see Rodríguez et al., 2004).
Table 1: Human DNA methyltransferases. Adapted from Human Molecular Genetics.
Associated proteins Enzyme OMIM no.
Major functions
DNMT1 126375 Maintenance methylase PCNA, HP1, methyl-DNAbinding proteins
DNMT2 602478 Methylation of cytosine-38 in tRNA, no
DNA methylation activity
DNMT3A 602769 De novo methylase
DNMT3B 602900 De novo methylase
DNMT3L 606588 Binds to chromatin with unmethylated H3K4 and stimulates activity of DNMT3A/3B
Histone methyltransferases; histone deacetylases
Histone methyltransferases; histone deacetylases
DNMT3A, DNMT3B; histone deacetylases
3.2.
CpG islands
Even though the frequency of the CpG dinucleotides in the genome is low, there are regions of CpG dinucleotides that are rarely or never methylated. These regions, named
CpG islands, are rich in CG content and have CpG frequency that would be expected by chance, unlike the rest of the genome. Furthermore, they also contain about 7% of all
CpG dinucleotides in the genome (see Bernstein et al., 2007).
Gardiner-Garden and Frommer defined CpG islands as “regions of at least 200 bases with a (C+G) content of at least 50% and a ratio of observed CpG frequency to be expected of at least 0.6” (Gardiner-Garden and Frommer, 1987). Methylation is found in gene bodies, introns and exons as well as in intergenic sequences with the CpG dinucleotide being the least common dinucleotide (Hum Mol Gen, 4 th
Edition) while on the other hand, CpG islands are found at gene promoters at high frequency, without methylation, which allows gene expression. It has also been found that CpG islands are differently methylated depending on the developmental stage of the organism that they are found in (Fazzari and Greally, 2004). For instance, globin genes, where the CpG islands are demethylated in all tissues of the adult organism are found silenced during embryonic development (Bird et al., 1987). Additionally, differential CpG island methylation is also present in imprinted genes.
Interestingly, DNA methylation is found to be more frequent within gene-bodies than in promoter regions, and somewhat surprisingly, methylation in gene-bodies appears to be positively correlated with gene expression in contrast to the repressive effect that promoter methylation has on transcription. The reasons for this paradox have yet to be
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resolved, and contrary to what was firstly thought, human genome-wide methylation studies have shown that genes with middle level of expression exhibit the highest levels of methylation. However, genes with low and high gene expression levels show low methylation patterns as illustrated by the bell-shaped plots in figure 5 (Jjingo et al.,
2012).
Figure 5: Representation of the total percentage of methylation seen within gene bodies against gene expression. Graph A shows a human lymphoblastoid cell line (GM12878).
Graph B is from a human myelogenous leukemia line (K562) and Graph C is from a liver hepatocellular cell line (HepG2). A positive correlation between gene expression and percentage of methylation exists until a maximum where methylation starts to decrease (bell-shape correlation). Adapted from Jjingo et al., 2012.
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4.HISTONE MODIFICATIONS
In addition to DNA cytosine methylation, histones can be epigenetically modified.
Histone modifications are defined as the second level of epigenetic marks that are achieved by covalent modifications. Like DNA methylation these modifications can be inherited through successive cell-divisions.
Chemical histone modifications occur at the amino-terminal tails of histone proteins.
Histones are modified by specific enzymes and modifications include: acetylation of lysines, methylation of lysines and arginines, phosphorylation of serines and threonines,
ADP ribosylation of lysines, ubiquitination of lysines, and sumoylation of lysines (see figure 4). This list is however by no means exhaustive and it is not unlikely that yet undescribed types of covalent histone modifications remain to be discovered.
Figure 6: Nucleosome modifications. All eight histones of the nucleosome have Nterminal tails exposed on the nucleosomal surface. The figure shows the sequences of human H3 and H4 N-terminal histone tails, in single letter amino acid code and the potential modifications. Red K stands for acetylatable lysines; sites of potential phosphorylation are represented in purple and methylation sites in blue. In addition, the interplay between different post-translational modifications is shown . Acetylated (Ac),
Methylated (Me), Phosphorylated (P) residues are represented by those letters. Positive and negative effects are indicated.
From Zhang and Reinberg, 2001.
Epigenetic modification at histone tales gives the nucleosome surface chemical information and “memory” (Turner, 2007 and Li et al., 2007). Such modifications have been observed when studying the role of the chromatin in transcription (Reviewed in Li et al., 2007). For example, acetylation of histone 3 and 4 or di- or tri-methylation of
H3K4 is known as euchromatin modifications because they are implicated with sites of active transcription. On the other hand, heterochromatin domains are characterised by
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methylation on H3K9 and H3K27 residues which are correlated with transcription repression (Strahl and Allis, 2000). Although it is often suggested as a “common rule” that acetylation opens up the chromatin for transcription and that methylation does the contrary (repression of transcription), this is by no means a universal truth. For instance, acetylation of histone H2A at position K119 results in repression of transcription while methylation of H3 K4 results in activation of transcription. For more detail see table 2.
The majority of the modifications are found in regions within the upstream region, the core promoter, the 5’end of the open reading fragment (ORF) and the 3’end of the ORF, and this location is of great importance for transcription (Li et al., 2007).
Table 2: Histone modifications associated with transcription. Li et al., 2007.
Ever since the discovery of histone modifications, scientists have been wondering whether these modifications have specific functions and what those functions are. This has led to the introduction of a new term, the epigenetic code. It is known that all histone modifications, except for methylation, result in a change in the overall charge of the nucleosomes, leading to changes in inter- and intranucleosomal interactions (Li et al., 2007). Histones that have been acetylated are easier to remove from the DNA in vivo (reviewed in Li et al., 2007). Furthermore, it has been proposed that such
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modifications could have an effect on the higher order chromatin structures. For example, acetylation of H4K16 is responsible for inhibiting the formation of 30nm chromatin fibbers (Shogren-Knaak et al., 2006).
One possible model of how histone modifications can interfere with transcription initiation was suggested by Li et al., 2007.
Figure 7: Schematic representation of chromatin regulation during transcription. In the silent promoter nucleosomes are found flanking a ~200bp NFR (nucleosome free region). Prior to UAS targeting, co-activation recruitment occurs (e.g. recruitment of
SAGA and mediator). At this point, histones are found to be acetylated at promoter regions mobilizing the nucleosomes in order to prepare the chromatin for transcription.
In the model on the left, called histone eviction, the acetylated histones and the chromatin remodeling complexes collaborate to remove the HTZ-1 histone variant from chromatin. Thus, the promoter is now free to engage the GTFs and RNA Pol II, and with the help of SAGA and mediator, enable the formation of a pre-initiation complex
(PIC). The partial PIC model on the right shows the assemblage of the PIC without loss of HTZ-1. In this case, the binding of Pol II and the transcription factor TFIIH are responsible for histone eviction and therefore the assembly of PIC at the promoter (Li et al., 2007).
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5.NON-CODING RNAs AS
EPIGENETICS REGULATORS
The third class of epigenetic modifications is mediated by non-coding RNAs.
Growing evidence supports the role of non-coding RNA and RNA-binding proteins in chromatin development, modifications and epigenetic control.
Non-coding RNAs are generated from every part of the genome, and they have been underestimated for years. It has not been until recently that whole genome approaches using high-throughput transcript sequencing have shown that almost all the genome (85 to 90%) is actually transcribed, contrary to what was previously believed (Human
Molecular Genetics). This led to the discovery that non-coding RNAs can be transcribed from introns, exons, long intergenic regions, and enhancer regions (Mattick and
Makunin 2005). Moreover, they play a fundamental role in the survival of the cell by mediating gene silencing repressing the mobilization of transposable elements, as well as for defence mechanisms against virus attack (Matzke et al., 2000). Several kinds of non-coding RNAs have been identified to have a relationship with chromatin modifications and regulation. Examples of them are microRNA (miRNA), piwi-binding
RNA (piRNA), endogenous short interfering RNA (endo-siRNA) and long non coding regulatory RNA. Their major functions include gene regulation in development, repression of transposon activity, gene imprinting, X-inactivation and antisense regulation (Hum Mol Gen, 4 th
Edition).
Mechanisms of silencing mediated by RNAi can be grouped into two different categories:
PTGS: when RNAi leads to post-transcriptional gene silencing.
TGS: when RNAi leads to transcriptional gene silencing, including mechanisms that epigenetically modify chromatin (see Tomari and Zamore 2005).
RNAi-like mechanisms are often found to co-operate in gene silencing, dosage compensation, DNA elimination in protists, and centromere silencing (Reviewed in
Bernstein and Allis 2005).
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Figure 8: Illustration showing some examples of non-coding RNA roles in histone modifications in different kingdoms. From Bernstein and Allis 2005.
Several different examples of how RNAs play an important role in genetic regulation in the four different kingdoms of life are shown. An example from the kingdom of
Fungi is the yeast S. pombe where siRNA influence methylation of H3K9 and H3K27 to repress transcription. The fission yeast silences genes in centromeric and telomeric regions as well as in the mat loci using RNAi dependent mechanisms (see Forsburg,
2005). Representing the Protist kingdom is Tetrahymena thermophila which in the vegetative state (upper cell) contains a micronucleus (MIC). The micronucleus is transcriptionally inactive (silent) and a macronucleus (MAC) which is transcriptionally active (represented in green). When conjugation occurs, represented in the lower panel, the MIC divides to form the new MAC and the old MAC is marked with H3K9me and
K27me for destruction by DNA an elimination process initiated by siRNA. In the Plant kingdom siRNAs regulates heterochromatic knobs (Arabidopsis, maize) by H3K9me and in the Animal kingdom the mechanisms of dosage compensation in both flies and mammals involve regulatory RNAs (roX and Xist, respectively). Whereas mammals silence one of the X chromosomes in females, flies up regulate expression of genes on of the male X chromosome by two fold. In flies the process is control by the roX RNAs and the chromosome is marked by H416ac and H3K4me while the centromere that is transcriptionally inactive is marked by H3K9me. In mammals, the silencing of the Xchromosome is achieved by the Xist RNAs and the chromosome is marked by chromatin modifications such as H3K9me, H3K29me and H4K20me (for more details see figure 10).
Recent publications have also identified a combined role of the spliceosome
(complex of snRNA and proteins which function is to remove introns) and chromatin where some chromatin modifications influence spliceosomal functions. For instance,
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HDAC (histone deacytalase) inhibition modifies the splicing patterns of around 700 human genes. Moreover, the spliceosome has been seen to interact with chromatin regulators such HP1 or MRG15 (reviewed in Bierne et al., 2012). Likewise, it has been revealed that in the genome of Arabidopsis thaliana there is a strong correlation between DNA methylation, DNA repeats, non-coding RNA and H3K9 methylation in the heterochromatin knobs (see figure 8) (reviewed in Bernstein and Allis, 2005).
Amplification of siRNA responses to dsDNA in the worm C. elegans by RNA dependent RNA polymerase leads to a strong response that can persist for at least two generations. Additionally, Ashe et al., 2012 have reported a transgenerational epigenetic memory generated by piRNAs in C. elegans . piRNA or piwi-protein-interacting RNAs are different from siRNAs in size and biogenesis. Their major role is to avoid transposition of retrotransposons and they have been found in several animals (Hum
Mol Gen, 4 th
Edition; Ashe et al., 2012). A co-operation between chromatin factors and
RNAi which is involved in passing on the signal has been identified. piRNAs can mediate stable long-term silencing for at least 20 generations, depending solely on the nuclear RNAi and chromatin silencing pathways. The exact mechanisms involved in transgenerational inheritance are still unclear.
6.THE EPIGENETIC CODE
Ever since the discovery of epigenetics, scientists have sought to understand the mechanisms involved in the chromatin changes that lead to changes in gene expression.
The notion of an “epigenetic code” was originally coined as the code that hypothetically defines the specific modifications that occur in the histones of the nucleosomes as a consequence of external factors and how these changes are in principle, inherited through a series of cell divisions. Unlike the genetic code, it is apparent that the epigenetic code is both tissue and cell specific, and differs significantly between organisms. In 2007 an issue about the epigenetics code was published in Nature Cell
Biology “Defining an epigenetic code” where the author Brian M. Turner tried to settle the bases of a possible epigenetics code. He established that the primarily the code needs to be arbitrary, meaning that the code does not imply a direct relation between the sign and the outcome (e.g. red traffic light means stop and there is not direct relationship between the red light and the fact that we have to stop the car).
For example, as Crick already stated: “The genetic code describes the way in which a sequence of twenty or more things is determined by a sequence of four things of a different type”
(Crick, 1963). Something similar could be used to determine the epigenetic code, although it has turned out to be difficult to decide what the epigenetics code would actually code for. It would be more useful and correct to talk about an epigenetic code when possible inheritable modifications are involved in long term effects. Turner proposes the following definition: “The epigenetic code describes the way in which the potential for expression of genes in a particular cell type is specified by chromatin modifications put in place at an earlier stage of differentiation” (Turner,
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2007). Contrary to the genetic code, the epigenetic code has many possible numbers of signs (more than one outcome is possible) and the number of outcomes is likely to be a large one (see figure 1). Hence, for the code to be meaningful, it must be combinatorial
(Turner, 2007). In addition, many other factors such as non-coding RNAs can alter the chromatin status which in turn can affect the epigenetic code (Bernstein and Allis,
2005). Thus, the context of the cell and the DNA surroundings matter and has to be taken in consideration when deciphering the epigenetic code (Weiner and Toth 2011).
Bearing this complexity in mind it is not surprising that little progress has been made in deciphering the code thus far, and to many experts the goal seems unattainable. It is at least clear that a universal epigenetic code, similar to the genetic code does not exist.
Figure 9: Environmental factors or conditions cause histone modifications at the amino acid tails that can define short-term and long-term output. Depending on the stimuli, the result can be a short-term responses or heritable traits. From Turner, 2007.
7.X-CHROMOSOME INACTIVATION IN
MAMMALS
The X-chromosome inactivation is a common phenomenon in mammalian organisms. It occurs as a consequence of the need to balance gene expression levels between females and males due to the different sex chromosomes. While females carry two copies of the same sex chromosome (XX), males only carry one (XY) (Lyon,
1961). The process of silencing is the best example of epigenetic regulation through
DNA methylation along an entire chromosome that subsequently gets converted into heterochromatin along most of its length (Heard et al., 1997) forming a structure called the Barr body. As mentioned above, non-coding RNAs also play an important role in
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this process (see figure 8). Shortly after implantation of the nascent embryo, the X chromosome to be silenced is chosen at random in each cell. The silencing is then passed on to the progeny of that cell. Notably, the X chromosome silencing in human is not entirely complete as some genes escape from the inactivation and are express to some degree (Carrel et al., 1999). X-inactivation starts with the expression of the XIST gene (X inactive specific transcript). XIST is localized in the middle of the inactivation centre of the chromosome X (XIC, X inactivation centre in Xq13). The XIST gene is in a demethylated state when the X chromosome is silenced but is methylated on active X chromosomes (see Rodríguez et al., 2004). There are several features that differentiate the inactive X chromosome from the active one. Among them are different distribution of histone variants, delayed replication in S-phase and different covalent histone modifications. Furthermore, the inactivated human X chromosome contains two different kinds of non-overlapping heterochromatin. One of these two domains in the Xi is characterized by the presence of Xi-specific transcript RNA, histone variant macroH2A and histone H3 trimethylated at lysine 27 (H3TrimK27) while the other domain includes histone H3 trimethylated at lysine 9 (H3TrimK9), heterochromatin protein 1 and histone H4 trimethylated at lysine 20 (H4TrimK20) (see Chadwick and
Willard 2004).
Figure 10: X-chromosome inactivation. Chadwick and Willard proposed this scheme for the specialized packaging of the inactivated X chromosome into the Barr body. The two different types of heterochromatin that can be found in the X inactivated chromosome are distinguished by macroH2A, H3TrimK27 (represented in red) or XIST
(yellow) and H3TrimK9, H3TrimK20 (represented in green) and HP1 (blue). When the chromosome is packaged into the Barr body at metaphase, the XIC (X inactivation centre) synthesizes XIST RNA that associates with macroH2A and H3TrimK27 in cis along the chromosome. On the other hand, HP1 associates with H3TrimK9 and
H3TrimK20. Figure from Chadwick and Willard 2004.
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8.STEM CELLS
Epigenetic mechanisms are responsible for the maintenance of pluripotency in stem cells through cell divisions. This is achieved by histone modifications, Polycomb group proteins, non-coding RNAs and chromatin remodeling complexes leading to the expression of specific regulatory genes (Lunyak and Rosenfeld 2008).
Stem cells are defined cells that are able to divide “endlessly” conserving the same phenotype and give rise to differentiated somatic cells (Plews et al., 2012). Stem cells can be classified into three groups, embryonic, postnatal, and induced pluripotent stem cell (iPS). The embryonic cells are considered to be pluripotent, meaning that they can differentiate into every cell of the organism (excluding placenta and adjacent tissues).
Postnatal are cells found in some organs in the body and contribute to regeneration and renewal of that particular organ or tissue, whereas induced pluripotent stem cells are stem cells derived from a non-pluripotent cell by experimental manipulation (see
Williams et al., 2011). Pluripotency of somatic cells decrease as they differentiate caused by gene silencing due to specific expression of transcription factors (TFs), chromatin modifications, DNA methylation, and histone modifications (Lunyak and
Rosenfeld 2008). The TFs most important for pluripotency are Oct-4, Nanog, Sox-2 and some others such Stat3, Tbx3, FoxD3, Myc, p53. Their common function is to repress transcription of genes that lead to differentiation (reviewed in Lunyak and Rosenfeld
2009; Szutorisz and Dillon 2005). Cells from the blastula are characterized by unmethylated DNA and genes involved in pluripotency at early stages of embryonic development are secure from chromatin remodeling complexes and therefore from silencing, while the rest of the genome is known to be subject to compaction and relaxation of chromatin (Williams el al., 2011).
Differentiated cells can already be re-programmed into a stem cell state and the next step is to be able to direct these cells toward specific fates or phenotypes. The use of adult cells instead of embryonic stem cells is also less ethically controversial.
Understanding and being able to manipulate the epigenetic mechanisms involved in the maintenance of cellular pluripotency will lead to important medical advantages.
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9.IMPRINTED GENES
In mammal cells, autosomal genes are present as alleles, two copies of the same gene, one inherited paternally and the other one inherited from the mother. Both are normally evenly expressed in the cell. Nonetheless, there is a small subset of genes that does not follow this rule. These genes which are transcribed unequally depending on their paternal origin are known as imprinted genes (Davies et al., 2005). This is due to epigenetic mechanisms that control their silenced or transcribed state. Defects in imprinting are linked to several known syndromes (Reik and Walter, 2001; Davies et al., 2005).
The methylation pattern of the vast majority of genes is reprogrammed shortly after fertilization of the embryo. In other words the methylation patterns present in the egg and sperm are deleted (Kafri et al., 1992; Morgan et al., 2005). However, some genes retain a specific methylation pattern of alleles that is conserved and passes through to the embryo. These genes are known as imprinted genes. In the first stages of germ cells development imprinted genes undergo total erasure of the methylation patterns.
Methylation marks are then reset during spermatogenesis or oogenesis so that both alleles become identically imprinted (Reik and Walter, 2001). Some imprinted methylation patterns can be maintained throughout development and tissues or they can change through development and acquire specific methylation patterns in specific tissues (Shemer et al., 1997). Nevertheless, how these epigenetically imprinted marks are linked with methylation remains a mystery. It has been shown that most imprinted genes are found in clusters (around 80% of all known) and unusually contain regions rich in CpG (Paulsen et al., 2000). In addition, no histone or chromatin modifications that are independent of methylation seem to be involved in the development of imprinted genes (Reik and Walter, 2001).
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Figure 11: Imprinted clusters on human chromosome 15 and the orthologous regions on mouse chromosome 7. Maternally expressed genes are shown in red while paternally expressed genes are shown in black and imprinted genes with intermediate expression patterns are represented in green. a) Represents the Beckwith–Wiedemann (BWS) cluster and b) Prader-Willi syndrome/Angelman (PWS/AS) syndrome cluster (15q11q13. Adapted from Reik and Walter 2001.
As explained above, imprinting can be understood as the expression of a determined gene in a parent-of-origin. Methylation of imprinted genes has been studied in CpG islands of three differentially imprinted genes, the H19, the IGF-2 and the IGF2receptor (for review see Obata et al., 1998). These three genes present a specific methylation state according to maternal or paternal inheritance. Using knock-out mice for the receptor IGF-2 gene it was demonstrated that when the mice inherited the nonfunctional IGF-2r maternally, they displayed a larger body size, 20-30% more than the control mice, and died at birth (Lau et al, 1994). However, when the paternal allele was deleted, the embryo developed normally to adult state (Kono, 1996). Many experiments have been performed after the discovery that DNA methylation is required to ensure proper fetal development. Before 2010, the number of known imprinted genes was around 50 in the mouse, most of them having a counterpart homologue in the human. It was around 2010 when a science paper signed by Gregg et al. claimed to have found
1300 possible loci by performing a genome-wide characterization in the mouse brain.
The method used relied on whole-transcriptome sequencing (mRNA-seq) of SNPs and robust statistical methods to determine false negatives (Gregg et al. 2010). Studies by two separate groups suggest that results obtained by genome-wide high-throughput
25
screens must be carefully studied and alternative methods are necessary to validate data
(DeVeale et al., 2012; Okae et al., 2012).
As stated previously, the study of genomic imprinting is of great importance because it affects many diseases, gene aberrations and syndromes. One of the best studied cases involves the two well-known syndromes, Prader Willi and Angelman.
9.1.
Prader Willi syndrome
Prader Willi is a syndrome caused by the loss of function of imprinted genes on chromosome 15 (region 15q11-q13), that are imprinted and expressed paternally. The loss of function can be due to deletion or silencing of the genes involved. The syndrome affects about 1 in 10,000 to 30,000 people worldwide (Bittel and Butler, 2005).
Figure 12: Picture showing facial phenotypic characteristics from Prader Willi syndrome. Clinical symptoms include hypotonia (severe weak muscle tone), feeding difficulties at early infancy, short stature, undeveloped genitalia (mostly sterile) and delayed language and learning skills, and motor development. In childhood patients show an insatiable appetite that if not controlled leads in the majority of the cases to morbid obesity. PWS is also characterized by a peculiar behavior phenotype; individuals suffer temper tantrums, stubbornness, manipulative behavior, and obsessive-compulsive characteristics (Driscoll et al., 1998).
The genetics behind this syndrome are explained by genomic imprinting and usually caused by deletion of a segment of chromosome 15. In around 70% of cases the syndrome is caused by the deletion of a segment of the paternal chromosome 15 in each cell. Sometimes the cause is the inheritance of two copies of the chromosome 15 from the mother (maternal uniparental disomy) (25% of the cases). More rarely the Prader
Willi syndrome can be due to chromosomal translocations or silencing of the region
(inactivation of the genes) (see Driscoll et al., 1998). In those cases where the syndrome is caused by silencing of the gene, the paternal chromosome contains a maternal pattern of imprinting. However, it has been claimed that other epigenetic phenomena than
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silencing can be involved in the development of the syndrome (see Rodríguez et al.,
2004).
9.2.
Angelman syndrome
The genetics behind this syndrome show that it is due to the loss of function of the
UBE3A gene located within the same 15q11.2-q13 chromosomal region that is involved in Prader Willi syndrome. Both alleles paternally and maternally inherited alleles are active in almost every tissue of the body except in the brain where only the allele inherited from the mother is active. The fact that only one copy is actively expressed in the brain means that Angelman syndrome will develop if the active copy is defective.
The loss of function is predominantly caused by deletion of the gene (in 70% of the cases). In about 11% of cases, the syndrome is caused by a mutation in the maternal
UBE3A copy. A small minority of cases are caused by paternal uniparental disomy or chromosomal translocations but curiously the causes of 10-15% of cases still remain unknown and other genes or chromosomes are likely to be involved (Genetics home reference, 2011).
Angelman syndrome is present every 1 in 12,000 to 20,000 people, a lower incidence than Prader Willi syndrome, although both syndromes are caused by defects in the same chromosomal region (see figure 11). Like Prader Willi, Angelman syndrome is a clear example of genomic imprinting (Dagli and Williams, 1998).
1 2
Figure 13: Picture showing the typical facial characteristics of Angelman syndrome.
Individuals possess coarse facial features (1 and 2). Pictures A to F clearly demonstrate the distinct happy, excitable behavior, characteristic of Angelman syndrome, with frequent smiling and laughing including uplifting of arms. Some other characteristics include ataxia (hard and unbalanced movements), epilepsy, delay in development, severe retardation, hyperactivity and water fascination, Source: psychnet-uk (1 and 2) and Dagli and Williams, 1998 (A to F).
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10.
OTHER SYNDROMES CAUSED
BY EPIGENETIC MISFUNCTION
10.1.
Rett syndrome
Rett syndrome is a neurological defect caused by mutations in Methyl-CpG-binding protein 2, (MeCP2) which is encoded on the X chromosome (Xq28). It affects mainly women, with a prevalence of 1 in 10 000. Due to the random nature of X inactivation
50% of cells in the body of heterozygous females heterozygous express only the mutated allele. It has recently been reported that MeCP2 binds strongly to 5-
Hydoxymethylcytosine (5-hmC), an epigenetic mark predominantly found in the brain, as well as to conventional methylcytosine (5-mC). MeCP2 binding to 5-hmC results in transcription of highly expressed genes. The Rett syndrome mutation MeCP2, R133C has been demonstrated to specifically inhibit binding of MeCP2 to 5-hmC while binding to 5-mC still occurs. This likely explains why symptoms are neurological (Mellén et al.,
2012).
10.2.
Fragile-X syndrome
Fragile X syndrome is associated with the FRAXA region on the X-chromosome
( fragile site, X chromosome, A site - Xq27.3). It is due to a massive trinucleotide repeat amplification within the gene FMR1 ( fragile X mental retardation-) . The 5’ region of the first exon contains a CGG trinucleotide repeat which is between 7-60 fold in normal individuals. Affected individuals possess repetitions that are larger than 230 repeats and usually hyper-methylated. Therefore, the gene experiences silencing and the product is missing.
Alleles containing between 60 and 230 CGG repetitions are considered permutated, i.e.
the gene is normally transcribed due to normal methylation profiles but highly unstable during female meiosis and can easily give cause amplification and subsequent methylation in the next generation (reviewed in Jin and Warren 2000).
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11.
MODEL SYSTEMS TO STUDY
EPIGENETICIS
Currently, there are many examples and model species available in laboratories to study epigenetics. Some of them present better advantages over others depending on the features of interest. From unicellular fungi, plants, mammals, etc, it has been shown possible to study epigenetics marks one way or another.
For instance, yeast has been widely used to study chromatin structure and function.
Yet they do not present methylation in their genome, the fission yeast
Schizosaccharomyces pombe presents siRNA regulation on histone modifications. In turn, Saccharomyces cerevisiae also known as the budding yeast has been best studied for the annotation of the chromosome structure, cell cycle, telomere silencing and mating type switching ( mat loci) (Forsburg 2005; Nakayama et al., 2001). Neurospora crassa is the fungus chosen to study methylation patterns in the DNA given the fact that yeast lack DNA methylation profiles (Allis et al., 2007).
Caenorhabditis elegans is another key model organism well suited for the study of epigenetics. The worm has a fairly short generation time (around 3 days), is easy to manipulate and maintain (feeds on bacteria, e.g. E.coli
) and easy to control and follow under experimental conditions. It has been used to study epigenetic-mediated cellular differentiation, dosage compensation of X-chromosomes, and RNAi, epigenetic inheritance, etc (see Ashe et al., 2012).
The protozoan Tetrahymena thermophilia is another example of an organism use for studies of epigenetics. T. thermophilia contains a micro nucleus, MIC that is inactive during vegetative life and is only active during reproduction (for more details see figure
8) (Bernstein and Allis 2005; Allis et al., 2007).
The fruit fly Drosophila melanogaster has been the classical genetic model for epigenetic investigation. The epigenetic mechanisms studied in Drosophila include the
PEV or position variegation effect, the Polycomb group (PcG) and the Trithorax group
(TrxG) that will be explained later. One of the disadvantages of Drosophila is that cytosine methylation is virtually absent and hence, DNA methylation cannot be studied in this model organism (Hum Mol Gen, 4 th
Edition; Kraus and Reuter, 2011). More recently the fly has been used to study transgenerational epigenetic inheritance
(reviewed in Chong and Whitelaw, 2004)
In the plant kingdom Arabidopsis thaliana is the most intensely studied plant by far.
Paramutations that were firstly discovered in maize (see below) are currently studied in
Arabidopsis (Chong and Whitelaw, 2004). It also serves as a sophisticated tool for the study of RNAi pathways, DNA methylation, environmental studies, histone modifications and many more (Henderson and Jacobsen 2007).
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12.
PLANT EPIGENETICS
One of the best examples of epigenetics inheritance is found in plants. Some plants exhibit a phenomenon called paramutation that does not follow Mendelian rules of inheritance. Paramutation is defined as the interaction between two alleles of the same locus where one of them changes the gene expression of the other, making this change heritable without involving mutation (Chong and Whitelaw, 2004).
An example of paramutation involves a silenced allele that acts in trans on a homologous allele generating heritable and stable inheritance, then this new silenced allele can also induce a silenced state in other alleles as paramutations (Stam and
Scheid, 2005).
Paramutation was first identified in the maize ( Zea mays ) where it plays an important role determining pigmentation. Paramutation has also been discovered in polyploid plants where this feature is of great importance for plant specialization (Scheid and
Paszkowski 2003).
12.1.
Arabidopsis thaliana
Figure 14: Picture of Arabidopsis thaliana.
A. thaliana is one of the best model plants for studying epigenetics. Available and small sequenced genome allows forward and reverse genetics.
Arabidopsis thaliana was the first polyploid plant (tetraploid) where paramutationlike effects have been reported (Scheid and Paszkowski 2003). Scheid and Paszkowski identified that diploid A. thaliana homozygous for a hygromycin phosphotransferase transgene (HPT) maintain uniform hygromyzin resistance over generations while
30
crossing with tetraploid plants resulted in plants with reduced HPT activity. This change was discovered to be due to epigenetic silencing activated by changes in ploidy.
Another well studied epigenetic regulatory system in A. thaliana is the Polycomb system FLC . FLC stands for Flowering Locus C and it is responsible for flowering. It is an example of how the environment triggers the epigenetic switch for flowering which depends on how the cold or warm the winter was (Kim and Sung, 2013). During early development of the plant, FLC is maintained repressed preventing flowering. In addition, its expression can also be found silenced if the adult plant has been exposed to long periods of cold weather, leading to a late flowering. The FLC is found to be regulated solely by the Polycomb system that mediates histone methylation and not by
DNA methylation (Sung and Amasino, 2005).
DNA methylation is also widespread in plant genomes both on Cytosines preceding
Guanines (CpG) and in Cytosines not directly preceding Guanines (CpNpG) identified in both symmetric and asymmetric sequence context (Vaughn et al., 2007). It is known that the cytosine patterns in eukaryote genomes are inherited from mother cell to daughter cell in a process involving methyltransferases enzymes. Methyltransferases are known to act symmetrically on CG and CNG nucleotides. Nevertheless, the process is not entirely perfect and possible misses leadings to loss of methylation are corrected by de novo methyltransferases (explained previously) , although the mechanisms of action are still unknown (Bender, 2004). In mammalian cells maintenance and de novo methyltranferase mutants are lethal owing to the broad number of genes that are miss regulated, being this issue a strong evidence of how important imprinted genes are for mammalian genomes (Klose and Bird 2006). However, while mammals do not allow methylation mutation studies, plants like Arabidopsis can in fact tolerate them, creating a great advantage for methylation genetic analysis.
13.
INSECT EPIGENETICS
Phenotypic plasticity is a mechanism where epigenetic modifications allow organisms to adapt their phenotypes to a changing environment without genetic change.
A good example of genome plasticity is found in the group of Hymenoptera which includes social insects such as ants, bees and wasp, all of them showing behavioural plasticity. Social insects are frequently found organized in complex levels of organization (castes) presenting different phenotypes that are derived from the same genetic input (e.g. worker bees are sterile but not the genetically identical queen bee).
This is mainly due to environmental reasons that affect the genomic plasticity either by inferring in alternative splicing, posttranslational modifications, transcription regulation or epigenetic modifications (see Weiner and Toth, 2011). DNA methylation has been proposed to be the main epigenetic modification to cause the development of castes, although some other epigenetic modification might have to be taken into consideration
(Drewell et al., 2012). DNA methylation is important in this organism to suppress mobile elements, transposons being the most methylated regions found within gene
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bodies. It has been suggested that DNA methylation in social insects affects caste determination and knock-down experiments of DNA methyltransferases have lent support to this hypothesis (see Weiner and Toth, 2011). For example, dmnt3 knockdown in worker bees at larval stage will produce adult honeybees presenting queen-like features (larger size, larger ovaries) and methylation patterns characteristics of the queen (see Weiner and Toth, 2011).
Nevertheless, not all insect show DNA methylation patters as strong as social insects.
For instance, one of the best studied model organisms in epigenetics is the fruit fly
Drosophila melanogaster which presents very little amount of DNA methylation throughout its genome. Thus, it has lost almost all of its DNA methyltransferases
(Krauss and Reuter, 2011). However, Drosophila melanogaster is highly useful to study other epigenetic features such as chromatin modifications, plasticity of the nervous system, RNA silencing, and for the study of the Polycomb and Trithorax systems.
13.1.
The Polycomb system
The Polycomb system is a group of proteins responsible for chromatin remodeling.
One of the best studied examples involved Polycomb regulation are the Hox genes. The
Hox genes modulate the chromatin structure through development in multicellular organisms where methylation of cytosines regulates gene expression in in cooperation with the Polycomb complex to achieve development of an organism (Terranova et al.,
2006). Even though a lot of work has been done in Drosophila on the study of the polycomb complex and Trithorax system, it is still unclear exactly how these systems are regulated.
In humans, the contribution of the Polycomb complex to development is poorly understood. Lee and colleagues found that the Polycomb Repressive Complex 2
(PRC2) subunit SUZ12 in human stem cells is spread over 200 genes containing trimethylated nucleosomes at lysine 27 (transcription repressor). Among the genes found were stem cell transcription factors like Oct4, Sox2 and Nanog (see Lunyak and
Rosenfeld 2009; Szutorisz and Dillon 2005). PRC2 targeted genes are mostly activated upon stem cell differentiation. The authors conclude that the repression by H3K27 methylation is important to maintain pluripotency of ES cells (Lee et al., 2006).
Moreover, Polycomb group proteins have been reported to be associated with aberrant methylation patterns in cancer because they interact directly with DNMTs directing them to chromatin with Polycomb target genes being more prone to be abnormally methylated than others (see Paschos and Allday 2010). Mitotic inheritance of geneexpression specific profiles is also catalyzed by the Polycomb group and Trithoraxgroup complexes by methylation of histone H3K26 and H3K4. Suggesting a close cooperation between the Polycomb group and methylated histones in the chromatin that could guide them back to destination sites after cell division, although the mechanisms involved are still unknown (Bernstein et al., 2007).
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14.
YEAST AND EPIGENETICS
Saccharomyces cerevisiae and Schizosaccharomyces pombe are the two eukaryotic unicellular organisms belonging to the fungi kingdom that are most used in laboratories to answer biological and molecular questions. Although neither of the two yeast methylate DNA, they serve as great tools for the study of epigenetics. Examples of epigenetics processes studied in yeast include the RNA interference (RNAi) machinery that is important in the fission yeast Sc. pombe but conspicuously absent in S cerevisiae.
This system makes the fission yeast especially useful because it has single-copy genes for components of the RNAi pathway (argonaute, dicer and RNA-dependent RNA polymerase (RdRP)) (Martienssen et al., 2005).
Another epigenetic mechanism studied is the position effect variegation (PEV). It is a rare event observed in genes that are located close to boundaries between euchromatin and heterochromatin. These genes can represent a mosaic pattern of gene expression as a result of silencing or activation. One example is found in Sc. pombe , where the gene
CLR4 (a SET domain containing histone methyltransferase enzyme 6) homologue of the Drosophila gene Su( var )3-9 (suppressor of variegation effect) is responsible for silencing of the mating type by methylation of the lysine residues at position 9 in histone 3 (H3K9) (Nakayama et al., 2001).
Yeast lack DNA methylation, instead they maintain the heterochromatic state over cell divisions through hypoacetylated histones and SIR proteins (in the case of
S.cerevisiae
) and between the interplay of H3K9 methylated histones and the Swi6 chromodomain (in the case of S. pombe ), both to conserve the heterochromatic state during cell division (Grewal and Moazed, 2003; Nakayama et al., 2001).
Furthermore, in S. pombe , RNAi are found to act in transcriptional gene silencing silencing of repetitive regions especially through H3K9 methylation (Bernstein and
Allis 2005). RNAi-like mechanisms can be seen acting on repetitive sequence as the centromeres, the mating type loci and at telomeres repetitions (see figure 8) (Bernstein and Allis 2005).
However, S. cerevisiae has been widely chosen over S. pombe for the study of chromatin remodeling complexes, telomere silencing, etc. For instance, in S. cerevisiae the telomere position effect has been elucidated. The telomere position effect is an epigenetic mechanism which silences all the genes close to telomeres and is dependent on SIR proteins. Besides telomere silencing the mating-type loci (HML and HMR) and tandem rDNA arrays, are silenced by conversion to heterochromatin (Loney et al.,
2009). Furthermore, S. cerevisiae has been used to study histone modifications like acetylation, phosphorylation, etc .
Other phenomena originally identified in budding yeast include the role of chromatin complexes like the INO80 which mobilizes nucleosomes throughout the
DNA and where it has also been found that the Rvb proteins are necessary for the functional integrity of the complex by recruiting the Arp5p (Jónsson et al., 2004).
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Studies in the field of epigenetics using yeast as model organisms have yielded countless discoveries, making them very suitable tools for assisting basic molecular genetics and cellular approaches.
15.
CLINICAL APPLICATIONS OF
EPIGENETICS
15.1.
Mitochondria in epigenetics
Being derived from a prokaryote, the mitochondrial DNA is not associated with histones and lacks introns (except for a few self-splicing ones). However DNA methyltransferases have been identified in mammalian mitochondria. These include mitochondrial DNA methyltransferase 1 (mtDNMT1) which gives rise to both 5-mC and 5-hmC (Shock et al., 2011). As already explained above, 5-mC stands for methylation at carbon 5 of cytosine residues and 5-hmC stands for hydroxymethylation
(Kriaucionis and Heintz, 2009). Defective mitochondria will cause dysfunction in redox state and in S-adenosyl methionine (SAM-CH
3
) production. SAM-CH
3
is precursor that donates a methyl group to methylate cytosines (see figure 3). Such dysfunction can cause changes in methylation profiles in the nuclear genome leading to genomic instability and pathologies (Minocherhomji et al., 2012).
15.2.
Bacterial and virus infections
Recent publications have provided evidence that bacteria and viruses can promote infection through epigenetics means. They can alter chromatin modifications, DNA methylation and affect the activity of chromatin remodelling complexes in the host cells for their own benefit.
One of the best examples of how viruses can influence host responses to infection is found in viruses called latent (e.g. herpesviruses) that remain “silent” within genome of the host. Other examples include the Hepatitis B virus, which has been linked to abnormal DNA methylation of the host genome by inducing high levels of methyltransferase (DNMT1, DNMT3A and DNMT3B) expression (reviewed in
Paschos and Allday, 2010).
The well-known HIV virus that infects CD4+ T cells has also been reported to mediate epigenetic response by increasing DNA methylation in the infected lymphocyte. As a result, general repression of transcription is localized at the gamma interferon (IFN-γ) promoter leading to reduced IFN-γ expression (Mikovits et al, 1998).
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Figure 15: Schematic description of the increase methylation in T lymphocytes infected by the HIV virus. CD4+ T cells from the same donor were used in this study where one set was infected with HIV-1 and the other was uninfected. Note the peak of maximum methyltranferase activity at day 7 for the HIV-1 infected cells compared to the control sample (healthy cells). Figure from Mikovits et al, 1998.
Other pathogens have also been linked different kinds of epigenetics responses such as chromatin remodeling complexes, the Polycomb protein group (e.g. Paramecium bursaria), etc. Bacteria can also disrupt the epigenetic environment of the infected host cell. Bacteria mostly produce signalling cascades (phosphorylation/acetylation and dephosphorilation/deacetylation) in the host cell though their components (metabolites), bringing by activation of transcriptional factors, controlling chromatin remodeling complexes, DNA methylation, etc. (Reviewed in Bierne et al., 2012). Furthermore, an increasing number of bacteria having SET domains that have linked functions with chromatin remodeling like in Chlamydophila pneumoniae (Paschos and Allday, 2010) have been described.
Some of the main effects caused by bacterial infections are illustrated in table 3 from
Bierne et al., 2012.
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Table 3: Bacterial species, bacterial factors and effects caused. Bierne et al., 2012.
As many studies have linked virus infection with up-regulation or down-regulation of methylation patterns of the host cell they infect, it is of great importance to continue research on this topic in order to understand the role of epigenetics in pathogenesis. For instance, the use of DNMT inhibitors could be a major advantage in the treatment of many of these diseases. The same way, the full knowledge of how bacteria influence and disrupt the epigenome of a cell can be useful to find better treatments for infectious diseases.
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16.
CANCER AND EPIGENETICS
It is a well-known fact that mutations are one of the bases of cancer development.
However, recently the fact that underlying epigenetic modifications also play an important role in the maintenance and development of cancer has become apparent.
DNA methylation is essential for mammalian cells, and is necessary for many important processes such as telomere length regulation, establishment of gene expression, development, embryogenesis, X chromosome inactivation, cell differentiation, etc. (Reviewed in Bernstein et al., 2007). As explained in the previous sections, 5-methylcytosine is prone to deamination to thymine because of its chemical instability, leading to point mutations that can result in genetic disorders such as cancer.
It is well-known that DNA methylation profiles change drastically in cancer. Changes observed in cancerous cells include hypomethylation (loss of methylation in sequences that are usually methylated) and hypermethylation of sequences usually unmethylated, particularly CpG islands (Rodriguez et al., 2003; Wilson et al., 2007; Gal-Yam et al.,
2007) whereby methylation causes permanent silencing. Furthermore, high activity of methyltransferases has been observed in several cancer types suggesting a role in the proliferation of malignant cells. In particular, de novo methyltranferase activity which produces permanent methylation is propagated during mitosis to descendent cells (Bird,
2002).DNA methylation changes in cancer seem to be even more prevalent than mutations, and have been identified even in precancerous cells (Rodríguez et al., 2004).
16.1.
Epigenetic therapy
Nowadays, new drugs targeting epigenetic modifications are being developed and some of them are even approved for clinical trials. Some examples are DNA methyltransferases inhibitors like 5-aza cytidine and 5-aza 2’ deoxyazacytine. 5-aza cytidine works by “kidnapping” methyltransferases at nucleoside incorporation sites
(Gal-Yam et al., 2007). These two drugs, 5-aza cytidine and 5-aza 2’ deoxyzacitine have been recently approved by the U. S. Food and Drug Administration and are commonly used in patients with myelodysplactic syndromes (MDS).
In other types of cancer, aberrant epigenetic histone modifications are implicated, and therefore, inhibitors of histone deacetylases are used instead, such as vorinostat in cutaneous T-cell lymphoma. Remarkably, vorinostat induces cell cycle-arrest, and/or apoptosis of transformed cells in vitro and in vivo (see Pratap et al., 2010). Pratap and colleagues showed that vorinostat treatment in mice with breast and prostate cancer cells reduces tumour growth effectively.
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17.
EPIGENETIC RESEARCH
METHODOLOGY
Comparison of the epigenetically modified regions to their unmodified counterparts can be done by common molecular biology techniques such as chromatin immunoprecipitation, protein-protein interaction assays, protein-chromatin interaction assays, etc. or by Next-Generation whole genome sequencing approaches.
Modifications include DNA methylation, histone modifications, RNA, nucleosome, etc.
The following sections name a few examples of approaches that are used to study genome-wide DNA methylation patterns:
17.1.
High resolution tiling oligonucleotides arrays:
Tiling oligonucleotides are a set of overlapping oligonucleotide probes that represent a contiguous specific known region in the DNA. They are used to characterize genomic regions bound by specific factors or modifications. Another methodology is the WGA
(whole genome array) where array-based hybridization is used to scan the complete genome of an organism (Gal-Yam et al., 2007).
17.2.
Shotgun bisulfite sequencing:
This method is used to profile the methylation of cytosines in genomic DNA. The assay consists on treating the DNA with sodium bisulfite or metabisulfite (Na2SO3 or
Na2S2O5) which will deaminate cytosine to uracil leaving the 5-methylcytosine unchanged. For identifying the bases that have changed, PCR is performed and compared with the sequence before and after treatment with Na2SO3. Because uracil is read as thymine during the PCR process it is possible to identify which cytosines where methylated. There are several methods used to reveal the CpG dinucleotide methylation status in the genome, and one of them is by sequencing after the PCR, another method is to digest the DNA with restriction enzymes or by arrays containing specific oligonucleotides that only match the unchanged sequence or the treated one can also be used (see Human Molecular Genetics, Garland Science 4th Edition).
17.3.
Digestion with restriction endonucleases:
Some restriction enzymes are methylation sensitive and cut the DNA at specific sequences depending on the methylation state, for example the enzyme TaqI recognizes
38
the cut-site sequence only when cytosine is unmethylated. Another example is the restriction enzyme McrBC that has been used to discriminate symmetric CpG and
CpNpG methylation versus asymmetric methylation in Arabidopsis in combination with tiling microarrays that can also profile polymorphisms (Vaughn et al., 2007). Other restriction enzymes used to identify methylation include HpaII which cuts unmethylated
CCGG and MspI that indistinctly cuts CCGG methylated or not allowing the comparison of different size restriction fragments (Hum Mol Gen, 4th Edition).
17.4.
Chromatin immunoprecipitation followed by
CHIP-sequencing:
This approach can be used to detect histone modifications by using antibodies specific for the various kinds of modified histones. First, the specific modified histone, cross-linked to its cognate DNA is identified by chromatin immunoprecipitation (ChIP).
After this treatment, the samples are sonicated to reach ideal DNA sizes. Finally, oligonucleotides adapters are added to the DNA in order to allow whole genome DNA sequencing. This method also helps quantifying the amount of specific histone modifications at given sites (Illumina, Inc. 2013).
17.5.
MeDIP and next generation sequencing:
MeDIP stands for methylated DNA immunoprecipitation. This assay uses a specific antibody raised against 5-methylcitosine (5mC). The purified immunoprecipitated methylated DNA can then be identified and quantified by next generation sequencing or, PCR / microarray hybridization approaches (Weber et al., 2005).
17.6.
Cobra assays (Combined Bisulfite
Restricted Assay):
The cobra assay is a quantitative molecular technique that employs restriction enzyme digestions of sodium bisulfite-treated DNA (as described above) to discover the
DNA methylation levels at specific gene loci in small quantities of genomic DNA (see
Xiong and Laird, 1997).
Many more methodologies can be used coupled to the previous approaches in order to study epigenetics utilizing PCR, DNA sequencing, quantitative PCR, Southern and
Northern blotting, HPLC, primer extension, MALDI-TOF MS, etc. (Esteller, 2008;
Suzuki, 2008).
39
Figure 16: Schematic representation of some approaches used for the detection of epigenetic marks. DNA methylation can be identified by Bisulfite conversion, using specific restriction enzymes to cut methylated or non-methylated DNA or by immunoprecipitation using antibodies to 5’mC. In addition, quantification of genes activated after treatment with 5-aza-CdR (a demethylating agent) can be done by qPCR.
Histone modifications are unraveled by ChIP (chromatin immunoprecipitation) using modification-specific antibodies Picture taken from Gal-Yam et al., 2007.
18.
CONCLUDING REMARKS
Epigenetics is a relatively new and complex branch of biology and scientist have just started scratching the tip of the iceberg with respect to understanding it. It is still not clear how most epigenetic marks are inherited over generations or what are all the factors controlling the epigenetic modifications are. Furthermore, a whole new global network has been opened with the discovery of the non-coding RNAs in the regulation of epigenetics that in combination with DNA methylation and histone modifications make possible the development, differentiation and adaptation of the cell to its environment.
Better understanding of the molecular basis of epigenetics in cell differentiation, gene expression and regulation will be beneficial for the identification, diagnosis and treatment of syndromes, illness, cancer, infections as well as explaining syndromes that are not explained by classical Mendelian inheritance.
Although the idea of an epigenetics code is enticing, the scientific community is currently far away from unravelling the basis of the code due to the broad number of combinations of the modifications that are involved.
Nevertheless, the increasing improvements of the new molecular methodologies are currently allowing great discoveries in this new field and will help to unravel the mysteries of epigenetics, hopefully within the near future.
40
41
19.
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