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123-463: Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome . The field is analogous to genomics and proteomics , which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance

246-415: A 3-week diet supplemented with soy. A decrease in oxidative DNA damage was also observed 2 h after consumption of anthocyanin -rich bilberry ( Vaccinium myrtillius L.) pomace extract. Damage to DNA is very common and is constantly being repaired. Epigenetic alterations can accompany DNA repair of oxidative damage or double-strand breaks. In human cells, oxidative DNA damage occurs about 10,000 times

369-471: A catalytically active site called the Jumonji domain (JmjC). The demethylation occurs when JmjC utilizes multiple cofactors to hydroxylate the methyl group, thereby removing it. JmjC is capable of demethylating mono-, di-, and tri-methylated substrates. Chromosomal regions can adopt stable and heritable alternative states resulting in bistable gene expression without changes to the DNA sequence. Epigenetic control

492-416: A change that is not erased by cell division, and affects the regulation of gene expression . Such effects on cellular and physiological phenotypic traits may result from environmental factors, or be part of normal development. Epigenetic factors can also lead to cancer. The term also refers to the mechanism of changes: functionally relevant alterations to the genome that do not involve mutation of

615-463: A chromodomain (a domain that specifically binds methyl-lysine) in the transcriptionally repressive protein HP1 recruits HP1 to K9 methylated regions. One example that seems to refute this biophysical model for methylation is that tri-methylation of histone H3 at lysine 4 is strongly associated with (and required for full) transcriptional activation (see top Figure). Tri-methylation, in this case, would introduce

738-513: A chromosome without alterations in the DNA sequence" was formulated at a Cold Spring Harbor meeting in 2008, although alternate definitions that include non-heritable traits are still being used widely. The hypothesis of epigenetic changes affecting the expression of chromosomes was put forth by the Russian biologist Nikolai Koltsov . From the generic meaning, and the associated adjective epigenetic , British embryologist C. H. Waddington coined

861-432: A chromosome without alterations in the DNA sequence," was made at a Cold Spring Harbor meeting. The similarity of the word to "genetics" has generated many parallel usages. The " epigenome " is a parallel to the word " genome ", referring to the overall epigenetic state of a cell, and epigenomics refers to global analyses of epigenetic changes across the entire genome. The phrase " genetic code " has also been adapted –

984-477: A combined bisulfite conversion with third-generation single-molecule real-time sequencing, it is called single-molecule real-time bisulfite sequencing (SMRT-BS), which is an accurate targeted CpG methylation analysis method capable of a high degree of multiplying and long read lengths (1.5 kb) without the need for PCR amplicon sub-cloning. First mathematical models for different nucleosome states affecting gene expression were introduced in 1980s [ref]. Later, this idea

1107-456: A complex interplay of at least three independent DNA methyltransferases , DNMT1, DNMT3A, and DNMT3B, the loss of any of which is lethal in mice. DNMT1 is the most abundant methyltransferase in somatic cells, localizes to replication foci, has a 10–40-fold preference for hemimethylated DNA and interacts with the proliferating cell nuclear antigen (PCNA). By preferentially modifying hemimethylated DNA, DNMT1 transfers patterns of methylation to

1230-446: A conventional DNA methyltransferase; this would suggest that other mechanisms other than DNA methylation are also involved. Within an organism, DNA methylation levels can also vary throughout development and by region. For example, in mouse primordial germ cells , a genome wide de-methylation even occurs; by implantation stage, methylation levels return to their previous somatic values. When DNA methylation occurs at promoter regions ,

1353-511: A day and DNA double-strand breaks occur about 10 to 50 times a cell cycle in somatic replicating cells (see DNA damage (naturally occurring) ). The selective advantage of DNA repair is to allow the cell to survive in the face of DNA damage. The selective advantage of epigenetic alterations that occur with DNA repair is not clear. In the steady state (with endogenous damages occurring and being repaired), there are about 2,400 oxidatively damaged guanines that form 8-oxo-2'-deoxyguanosine (8-OHdG) in

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1476-494: A fixed positive charge on the tail. It has been shown that the histone lysine methyltransferase (KMT) is responsible for this methylation activity in the pattern of histones H3 & H4. This enzyme utilizes a catalytically active site called the SET domain (Suppressor of variegation, Enhancer of Zeste, Trithorax). The SET domain is a 130-amino acid sequence involved in modulating gene activities. This domain has been demonstrated to bind to

1599-660: A functional group that can serve as a nucleophile in the reaction: the hydroxyl groups of serine , threonine , and tyrosine ; the amine forms of lysine , arginine , and histidine ; the thiolate anion of cysteine ; the carboxylates of aspartate and glutamate ; and the N- and C-termini. In addition, although the amide of asparagine is a weak nucleophile, it can serve as an attachment point for glycans . Rarer modifications can occur at oxidized methionines and at some methylene groups in side chains. Post-translational modification of proteins can be experimentally detected by

1722-936: A given loci is determined by the relative intensity ratios of the two dyes. Adaptation of next generation sequencing to DNA methylation assay provides several advantages over array hybridization. Sequence-based technology provides higher resolution to allele specific DNA methylation, can be performed on larger genomes, and does not require creation of DNA microarrays which require adjustments based on CpG density to properly function. Bisulfite sequencing relies on chemical conversion of unmethylated cytosines exclusively, such that they can be identified through standard DNA sequencing techniques. Sodium bisulfate and alkaline treatment does this by converting unmethylated cytosine residues into uracil while leaving methylated cytosine unaltered. Subsequent amplification and sequencing of untreated DNA and sodium bisulphite treated DNA allows for methylated sites to be identified. Bisulfite sequencing, like

1845-491: A global level has been made possible only recently through the adaptation of genomic high-throughput assays. Genomic modifications that alter gene expression that cannot be attributed to modification of the primary DNA sequence and that are heritable mitotically and meiotically are classified as epigenetic modifications. DNA methylation and histone modification are among the best characterized epigenetic processes. The first epigenetic modification to be characterized in depth

1968-430: A global level, similar to the study of the complete set of DNA in genomics or the complete set of proteins in a cell in proteomics. The logic behind performing epigenetic analysis on a global level is that inferences can be made about epigenetic modifications, which might not otherwise be possible through analysis of specific loci. As in the other genomics fields, epigenomics relies heavily on bioinformatics , which combines

2091-535: A higher rate of read-through of stop codons , an effect that results in suppression of nonsense mutations in other genes. The ability of Sup35 to form prions may be a conserved trait. It could confer an adaptive advantage by giving cells the ability to switch into a PSI+ state and express dormant genetic features normally terminated by stop codon mutations. Prion-based epigenetics has also been observed in Saccharomyces cerevisiae . Epigenetic changes modify

2214-619: A large scale was first made possible through the Restriction Landmark Genome Scanning (RLGS) technique. Like the locus-specific DNA methylation assay, the technique identified methylated DNA via its digestion methylation sensitive enzymes. However it was the use of two-dimensional gel electrophoresis that allowed be characterized on a broader scale. However it was not until the advent of microarray and next generation sequencing technology when truly high resolution and genome-wide DNA methylation became possible. As with RLGS,

2337-408: A large variety of biological functions in plants and animals. So far, in 2013, about 2000 miRNAs have been discovered in humans and these can be found online in a miRNA database. Each miRNA expressed in a cell may target about 100 to 200 messenger RNAs(mRNAs) that it downregulates. Most of the downregulation of mRNAs occurs by causing the decay of the targeted mRNA, while some downregulation occurs at

2460-460: A microarray containing immobilized gDNA. Analysis of the relative signal intensity allows the sites of histone modification to be determined. ChIP-chip was used extensively to characterize the global histone modification patterns of yeast . From these studies, inferences on the function of histone modifications were made; that transcriptional activation or repression was associated with certain histone modifications and by region. While this method

2583-412: A newly synthesized strand after DNA replication , and therefore is often referred to as the 'maintenance' methyltransferase. DNMT1 is essential for proper embryonic development, imprinting and X-inactivation. To emphasize the difference of this molecular mechanism of inheritance from the canonical Watson-Crick base-pairing mechanism of transmission of genetic information, the term 'Epigenetic templating'

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2706-542: A particular genomic region. More typically, the term is used in reference to systematic efforts to measure specific, relevant forms of epigenetic information such as the histone code or DNA methylation patterns. Covalent modification of either DNA (e.g. cytosine methylation and hydroxymethylation) or of histone proteins (e.g. lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation) play central roles in many types of epigenetic inheritance. Therefore,

2829-448: A prion. Although often viewed in the context of infectious disease , prions are more loosely defined by their ability to catalytically convert other native state versions of the same protein to an infectious conformational state. It is in this latter sense that they can be viewed as epigenetic agents capable of inducing a phenotypic change without a modification of the genome. Fungal prions are considered by some to be epigenetic because

2952-414: A protein or part of a protein attached to the cell membrane . Other forms of post-translational modification consist of cleaving peptide bonds , as in processing a propeptide to a mature form or removing the initiator methionine residue. The formation of disulfide bonds from cysteine residues may also be referred to as a post-translational modification. For instance, the peptide hormone insulin

3075-628: A role of DNA methylation in gene repression, regions that were associated with high levels of DNA methylation were not actively expressed. This method was limited not suitable for studies on the global methylation pattern, or ‘methylome’. Even within specific loci it was not fully representative of the true methylation pattern as only those restriction sites with corresponding methylation sensitive and insensitive restriction assays could provide useful information. Further complications could arise when incomplete digestion of DNA by restriction enzymes generated false negative results. DNA methylation profiling on

3198-474: A systematic and reproducible way is called the histone code , although the idea that histone state can be read linearly as a digital information carrier has been largely debunked. One of the best-understood systems that orchestrate chromatin-based silencing is the SIR protein based silencing of the yeast hidden mating-type loci HML and HMR. DNA methylation frequently occurs in repeated sequences, and helps to suppress

3321-412: A template. Each of the four DNA bases is attached to one of four different fluorescent dyes . When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and the detector detects the fluorescent signal of the nucleotide incorporation. As the sequencing occurs, the polymerase enzyme kinetics shift when it encounters a region of methylation or any other base modification. When

3444-648: A variety of techniques, including mass spectrometry , Eastern blotting , and Western blotting . Examples of non-enzymatic PTMs are glycation, glycoxidation, nitrosylation, oxidation, succination, and lipoxidation. In 2011, statistics of each post-translational modification experimentally and putatively detected have been compiled using proteome-wide information from the Swiss-Prot database. The 10 most common experimentally found modifications were as follows: Some common post-translational modifications to specific amino-acid residues are shown below. Modifications occur on

3567-555: Is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification . Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis . The study of epigenetics on

3690-552: Is a function of all kinetic steps after nucleotide binding and up to fluorophore release, and IPD is determined by the kinetics of nucleotide binding and polymerase translocation. In 2010 a team of scientists demonstrated the use of single-molecule real-time sequencing for direct detection of modified nucleotide in the DNA template including N6-methyladenosine , 5-methylcytosine and 5-hydroxylcytosine . These various modifications affect polymerase kinetics differently, allowing discrimination between them. In 2017, another team proposed

3813-445: Is a source of false positives. Further, bisulfite treatment also causes DNA degradation and requires an additional purification step to remove the sodium bisulfite. Next-generation sequencing is well suited in complementing bisulfite sequencing in genome-wide methylation analysis . While this now allows for methylation pattern to be determined on the highest resolution possible, on the single nucleotide level, challenges still remain in

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3936-545: Is accomplished through two main mechanisms: There is frequently a reciprocal relationship between DNA methylation and histone lysine methylation. For instance, the methyl binding domain protein MBD1 , attracted to and associating with methylated cytosine in a DNA CpG site , can also associate with H3K9 methyltransferase activity to methylate histone 3 at lysine 9. On the other hand, DNA maintenance methylation by DNMT1 appears to partly rely on recognition of histone methylation on

4059-456: Is affected by which of its genes are transcribed, heritable transcription states can give rise to epigenetic effects. There are several layers of regulation of gene expression . One way that genes are regulated is through the remodeling of chromatin. Chromatin is the complex of DNA and the histone proteins with which it associates. If the way that DNA is wrapped around the histones changes, gene expression can change as well. Chromatin remodeling

4182-407: Is condensed is associated with a certain transcriptional state. Unpackaged or loose chromatin is more transcriptionally active than tightly packaged chromatin because it is more accessible to transcriptional machinery. By remodeling chromatin structure and changing the density of DNA packaging, gene expression can thus be modulated. Chromatin remodeling occurs via post-translational modifications of

4305-590: Is cut twice after disulfide bonds are formed, and a propeptide is removed from the middle of the chain; the resulting protein consists of two polypeptide chains connected by disulfide bonds. Some types of post-translational modification are consequences of oxidative stress . Carbonylation is one example that targets the modified protein for degradation and can result in the formation of protein aggregates. Specific amino acid modifications can be used as biomarkers indicating oxidative damage. Sites that often undergo post-translational modification are those that have

4428-402: Is generally related to transcriptional competence (see Figure). One mode of thinking is that this tendency of acetylation to be associated with "active" transcription is biophysical in nature. Because it normally has a positively charged nitrogen at its end, lysine can bind the negatively charged phosphates of the DNA backbone. The acetylation event converts the positively charged amine group on

4551-404: Is known about the mechanism of heritability of DNA methylation state during cell division and differentiation. Heritability of methylation state depends on certain enzymes (such as DNMT1 ) that have a higher affinity for 5-methylcytosine than for cytosine. If this enzyme reaches a "hemimethylated" portion of DNA (where 5-methylcytosine is in only one of the two DNA strands) the enzyme will methylate

4674-435: Is mutagenic. Oxoguanine glycosylase (OGG1) is the primary enzyme responsible for the excision of the oxidized guanine during DNA repair. OGG1 finds and binds to an 8-OHdG within a few seconds. However, OGG1 does not immediately excise 8-OHdG. In HeLa cells half maximum removal of 8-OHdG occurs in 30 minutes, and in irradiated mice, the 8-OHdGs induced in the mouse liver are removed with a half-life of 11 minutes. When OGG1

4797-431: Is negatively charged. The basic and repeating units of chromatin, nucleosomes , consist of an octamer of histone proteins (H2A, H2B, H3 and H4) and a 146 bp length of DNA wrapped around it. Nucleosomes and the DNA connecting form a 10 nm diameter chromatin fiber, which can be further condensed. Chromatin packaging of DNA varies depending on the cell cycle stage and by local DNA region. The degree to which chromatin

4920-446: Is often associated with alternative covalent modifications of histones. The stability and heritability of states of larger chromosomal regions are suggested to involve positive feedback where modified nucleosomes recruit enzymes that similarly modify nearby nucleosomes. A simplified stochastic model for this type of epigenetics is found here. It has been suggested that chromatin-based transcriptional regulation could be mediated by

5043-420: Is present at an oxidized guanine within a methylated CpG site it recruits TET1 to the 8-OHdG lesion (see Figure). This allows TET1 to demethylate an adjacent methylated cytosine. Demethylation of cytosine is an epigenetic alteration. As an example, when human mammary epithelial cells were treated with H 2 O 2 for six hours, 8-OHdG increased about 3.5-fold in DNA and this caused about 80% demethylation of

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5166-449: Is the "trans" model. In this model, changes to the histone tails act indirectly on the DNA. For example, lysine acetylation may create a binding site for chromatin-modifying enzymes (or transcription machinery as well). This chromatin remodeler can then cause changes to the state of the chromatin. Indeed, a bromodomain – a protein domain that specifically binds acetyl-lysine – is found in many enzymes that help activate transcription, including

5289-534: Is the measure of how "accessible" or "open" a region of genome is to transcription or binding of transcription factors. The regions which are inaccessible (i.e. because they're bound by nucleosomes ) are not actively transcribed by the cell while open and accessible regions are actively transcribed. Changes in chromatin accessibility are important epigenetic regulatory processes that govern cell- or context-specific expression of genes. Assays such as MNase-seq, DNase-seq, ATAC-seq or FAIRE-seq are routinely used to understand

5412-516: Is the process of cellular differentiation . During morphogenesis , totipotent stem cells become the various pluripotent cell lines of the embryo , which in turn become fully differentiated cells. In other words, as a single fertilized egg cell – the zygote – continues to divide , the resulting daughter cells change into all the different cell types in an organism, including neurons , muscle cells , epithelium , endothelium of blood vessels , etc., by activating some genes while inhibiting

5535-799: The N-terminal tails of core histone proteins . The collective set of histone modifications in a given cell is known as the histone code . Many different types of histone modification are known, including: acetylation , methylation , phosphorylation , ubiquitination , SUMOylation , ADP-ribosylation , deamination and proline isomerization ; acetylation, methylation, phosphorylation and ubiquitination have been implicated in gene activation whereas methylation, ubiquitination, SUMOylation, deimination and proline isomerization have been implicated in gene repression. Note that several modification types including methylation, phosphorylation and ubiquitination can be associated with different transcriptional states depending on

5658-501: The PCR process. The Oxford Nanopore Technologies MinION sequencer is a technology where, according to a hidden Markov model, it is possible to distinguish unmethylated cytosine from the methylated one even without chemical treatment that acts to enhance the signal of that modification. The data are registered commonly in picoamperes during established time. Other devices are the Nanopolish and

5781-490: The SWI/SNF complex. It may be that acetylation acts in this and the previous way to aid in transcriptional activation. The idea that modifications act as docking modules for related factors is borne out by histone methylation as well. Methylation of lysine 9 of histone H3 has long been associated with constitutively transcriptionally silent chromatin (constitutive heterochromatin ) (see bottom Figure). It has been determined that

5904-441: The nucleotide sequence . Examples of mechanisms that produce such changes are DNA methylation and histone modification , each of which alters how genes are expressed without altering the underlying DNA sequence. Further, non-coding RNA sequences have been shown to play a key role in the regulation of gene expression. Gene expression can be controlled through the action of repressor proteins that attach to silencer regions of

6027-422: The transcription factor activity of the proteins they encode. RNA signalling includes differential recruitment of a hierarchy of generic chromatin modifying complexes and DNA methyltransferases to specific loci by RNAs during differentiation and development. Other epigenetic changes are mediated by the production of different splice forms of RNA , or by formation of double-stranded RNA ( RNAi ). Descendants of

6150-438: The " epigenetic code " has been used to describe the set of epigenetic features that create different phenotypes in different cells from the same underlying DNA sequence. Taken to its extreme, the "epigenetic code" could represent the total state of the cell, with the position of each molecule accounted for in an epigenomic map , a diagrammatic representation of the gene expression, DNA methylation and histone modification status of

6273-530: The 22 amino acids by changing an existing functional group or adding a new one such as phosphate. Phosphorylation is highly effective for controlling the enzyme activity and is the most common change after translation. Many eukaryotic and prokaryotic proteins also have carbohydrate molecules attached to them in a process called glycosylation , which can promote protein folding and improve stability as well as serving regulatory functions. Attachment of lipid molecules, known as lipidation , often targets

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6396-709: The 5-methylcytosines in the genome. Demethylation of CpGs in a gene promoter by TET enzyme activity increases transcription of the gene into messenger RNA. In cells treated with H 2 O 2 , one particular gene was examined, BACE1 . The methylation level of the BACE1 CpG island was reduced (an epigenetic alteration) and this allowed about 6.5 fold increase of expression of BACE1 messenger RNA. While six-hour incubation with H 2 O 2 causes considerable demethylation of 5-mCpG sites, shorter times of H 2 O 2 incubation appear to promote other epigenetic alterations. Treatment of cells with H 2 O 2 for 30 minutes causes

6519-403: The DNA becoming less accessible to transcriptional machinery, such as RNA polymerase , effectively repressing gene expression. In eukaryotes , genomic DNA is coiled into protein-DNA complexes called chromatin . Histones , which are the most prevalent type of protein found in chromatin, function to condense the DNA; the net positive charge on histones facilitates their bonding with DNA, which

6642-511: The DNA repair enzyme polymerase beta localizing to oxidized guanines. Polymerase beta is the main human polymerase in short-patch BER of oxidative DNA damage. Jiang et al. also found that polymerase beta recruited the DNA methyltransferase protein DNMT3b to BER repair sites. They then evaluated the methylation pattern at the single nucleotide level in a small region of DNA including the promoter region and

6765-401: The DNA strands unbounded by nucleosomes or other proteic factors, while the bounded pieces are sheltered, and can be retrieved and analysed. Since active, unbound regions are destroyed, their detection can only be indirect, by sequencing with a Next Generation Sequencing technique and comparison with a reference. MNase-seq utilises a micrococcal nuclease that produces a single strand cleavage on

6888-405: The DNA. These epigenetic changes may last through cell divisions for the duration of the cell's life, and may also last for multiple generations, even though they do not involve changes in the underlying DNA sequence of the organism; instead, non-genetic factors cause the organism's genes to behave (or "express themselves") differently. One example of an epigenetic change in eukaryotic biology

7011-478: The SignaAlign: the former expresses the frequency of a methylation in a read while the latter gives a probability of it derived from the sum of all the reads. Single-molecule real-time sequencing (SMRT) is a single-molecule DNA sequencing method. Single-molecule real-time sequencing utilizes a zero-mode waveguide (ZMW). A single DNA polymerase enzyme is bound to the bottom of a ZMW with a single molecule of DNA as

7134-436: The accessible chromatin landscape of cells. The main feature of all these methods is that they're able to selectively isolate either the DNA sequences that are bounded to the histones , or those that are not. These sequences are then compared to a reference genome that allows to identify their relative position. MNase-seq and DNase-seq both follow the same principles, as they employ lytic enzymes that target nucleic acids to cut

7257-443: The activation of certain genes, but not the genetic code sequence of DNA. The microstructure (not code) of DNA itself or the associated chromatin proteins may be modified, causing activation or silencing. This mechanism enables differentiated cells in a multicellular organism to express only the genes that are necessary for their own activity. Epigenetic changes are preserved when cells divide. Most epigenetic changes only occur within

7380-430: The activation of oxidative stress pathways. Foods are known to alter the epigenetics of rats on different diets. Some food components epigenetically increase the levels of DNA repair enzymes such as MGMT and MLH1 and p53 . Other food components can reduce DNA damage, such as soy isoflavones . In one study, markers for oxidative stress, such as modified nucleotides that can result from DNA damage, were decreased by

7503-580: The assembly step because of reduced sequence complexity in bisulphite treated DNA. Increases in read length seek to address this challenge, allowing for whole genome shotgun bisulphite sequencing (WGBS) to be performed. The WGBS approach using an Illumina Genome Analyzer platform and has already been implemented in Arabidopsis thaliana . Reduced representation genomic methods based on bisulfite sequencing exist as well, and they are particularly suitable for species with large genome sizes. Chromatin accessibility

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7626-491: The average mammalian cell DNA. 8-OHdG constitutes about 5% of the oxidative damages commonly present in DNA. The oxidized guanines do not occur randomly among all guanines in DNA. There is a sequence preference for the guanine at a methylated CpG site (a cytosine followed by guanine along its 5' → 3' direction and where the cytosine is methylated (5-mCpG)). A 5-mCpG site has the lowest ionization potential for guanine oxidation. Oxidized guanine has mispairing potential and

7749-449: The carbon 5 position of cytosine residues (5mC) adjacent to guanine , termed CpG dinucleotides . DNA methylation patterns vary greatly between species and even within the same organism. The usage of methylation among animals is quite different; with vertebrates exhibiting the highest levels of 5mC and invertebrates more moderate levels of 5mC. Some organisms like Caenorhabditis elegans have not been demonstrated to have 5mC nor

7872-414: The cell in which the gene was turned on will inherit this activity, even if the original stimulus for gene-activation is no longer present. These genes are often turned on or off by signal transduction , although in some systems where syncytia or gap junctions are important, RNA may spread directly to other cells or nuclei by diffusion . A large amount of RNA and protein is contributed to the zygote by

7995-566: The chromatin to be extracted and fragmented, either by sonication or treatment with a non-specific restriction enzyme (e.g., micrococcal nuclease ). Modification-specific antibodies in turn, are used to immunoprecipitate the DNA-histone complexes. Following immunoprecipitation, the DNA is purified from the histones, amplified via PCR and labeled with a fluorescent tag (e.g., Cy5, Cy3 ). The final step involves hybridization of labeled DNA, both immunoprecipitated DNA and non-immunoprecipitated onto

8118-403: The cleavage distribution can be biased, lowering the quality of the results. FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements) requires as its first step crosslinking of the DNA with nucleosomes, then DNA shearing by sonication . The free and linked fragments are separated with a traditional phenol-chloroform extraction, since the proteic fraction is stuck in the interphase while

8241-681: The constraints of requiring heritability . For example, Adrian Bird defined epigenetics as "the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states." This definition would be inclusive of transient modifications associated with DNA repair or cell-cycle phases as well as stable changes maintained across multiple cell generations, but exclude others such as templating of membrane architecture and prions unless they impinge on chromosome function. Such redefinitions however are not universally accepted and are still subject to debate. The NIH "Roadmap Epigenomics Project", which ran from 2008 to 2017, uses

8364-432: The contact between nucleosomes and by recruiting chromatin remodeling ATPases . An example of the first mechanism occurs during the acetylation of lysine terminal tail amino acids, which is catalyzed by histone acetyltransferases (HATs) . HATs are part of a multiprotein complex that is recruited to chromatin when activators bind to DNA binding sites. Acetylation effectively neutralizes the basic charge on lysine, which

8487-589: The course of one individual organism's lifetime; however, these epigenetic changes can be transmitted to the organism's offspring through a process called transgenerational epigenetic inheritance . Moreover, if gene inactivation occurs in a sperm or egg cell that results in fertilization, this epigenetic modification may also be transferred to the next generation. Specific epigenetic processes include paramutation , bookmarking , imprinting , gene silencing , X chromosome inactivation , position effect , DNA methylation reprogramming , transvection , maternal effects ,

8610-465: The disciplines of biology, mathematics and computer science. However while epigenetic modifications had been known and studied for decades, it is through these advancements in bioinformatics technology that have allowed analyses on a global scale. Many current techniques still draw on older methods, often adapting them to genomic assays as is described in the next section. The cellular processes of transcription , DNA replication and DNA repair involve

8733-482: The double-strand break, the involvement of DNMT1 causes the two repaired strands of DNA to have different levels of methylated cytosines. One strand becomes frequently methylated at about 21 CpG sites downstream of the repaired double-strand break. The other DNA strand loses methylation at about six CpG sites that were previously methylated downstream of the double-strand break, as well as losing methylation at about five CpG sites that were previously methylated upstream of

8856-403: The double-strand break. When the chromosome is replicated, this gives rise to one daughter chromosome that is heavily methylated downstream of the previous break site and one that is unmethylated in the region both upstream and downstream of the previous break site. With respect to the gene that was broken by the double-strand break, half of the progeny cells express that gene at a high level and in

8979-527: The early transcription region of the BRCA1 gene. Oxidative DNA damage from bromate modulated the DNA methylation pattern (caused epigenetic alterations) at CpG sites within the region of DNA studied. In untreated cells, CpGs located at −189, −134, −29, −19, +16, and +19 of the BRCA1 gene had methylated cytosines (where numbering is from the messenger RNA transcription start site, and negative numbers indicate nucleotides in

9102-429: The effect of small RNAs. Small interfering RNAs can modulate transcriptional gene expression via epigenetic modulation of targeted promoters . Sometimes a gene, after being turned on, transcribes a product that (directly or indirectly) maintains the activity of that gene. For example, Hnf4 and MyoD enhance the transcription of many liver-specific and muscle-specific genes, respectively, including their own, through

9225-458: The endonuclease component is retained in the method but it is coupled to new technologies. One such approach is the differential methylation hybridization (DMH), in which one set of genomic DNA is digested with methylation-sensitive restriction enzymes and a parallel set of DNA is not digested. Both sets of DNA are subsequently amplified and each labelled with fluorescent dyes and used in two-colour array hybridization. The level of DNA methylation at

9348-463: The enzyme encounters chemically modified bases, it will slow down or speed up in a uniquely identifiable way. Fluorescence pulses in SMRT sequencing are characterized not only by their emission spectra but also by their duration and by the interval between successive pulses. These metrics, defined as pulse width and interpulse duration (IPD), add valuable information about DNA polymerase kinetics. Pulse width

9471-509: The expression and mobility of ' transposable elements ': Because 5-methylcytosine can be spontaneously deaminated (replacing nitrogen by oxygen) to thymidine , CpG sites are frequently mutated and become rare in the genome, except at CpG islands where they remain unmethylated. Epigenetic changes of this type thus have the potential to direct increased frequencies of permanent genetic mutation. DNA methylation patterns are known to be established and modified in response to environmental factors by

9594-476: The expression of others. The term epigenesis has a generic meaning of "extra growth" that has been used in English since the 17th century. In scientific publications, the term epigenetics started to appear in the 1930s (see Fig. on the right). However, its contemporary meaning emerged only in the 1990s. A definition of the concept of epigenetic trait as a "stably heritable phenotype resulting from changes in

9717-437: The fight against drug-resistant bacteria. They play an important role in many biological processes, binding to mRNA and protein targets in prokaryotes. Their phylogenetic analyses, for example through sRNA–mRNA target interactions or protein binding properties , are used to build comprehensive databases. sRNA- gene maps based on their targets in microbial genomes are also constructed. Numerous investigations have demonstrated

9840-412: The following definition: "For purposes of this program, epigenetics refers to both heritable changes in gene activity and expression (in the progeny of cells or of individuals) and also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable." In 2008, a consensus definition of the epigenetic trait, a "stably heritable phenotype resulting from changes in

9963-468: The frame of one-dimensional lattice approaches. Epigenetic In biology , epigenetics is the study of heritable traits , or a stable change of cell function, that happen without changes to the DNA sequence . The Greek prefix epi- ( ἐπι- "over, outside of, around") in epigenetics implies features that are "on top of" or "in addition to" the traditional (DNA sequence based) genetic mechanism of inheritance. Epigenetics usually involves

10086-455: The histone tail and causes the methylation of the histone. Differing histone modifications are likely to function in differing ways; acetylation at one position is likely to function differently from acetylation at another position. Also, multiple modifications may occur at the same time, and these modifications may work together to change the behavior of the nucleosome . The idea that multiple dynamic modifications regulate gene transcription in

10209-502: The infectious phenotype caused by the prion can be inherited without modification of the genome. PSI+ and URE3, discovered in yeast in 1965 and 1971, are the two best studied of this type of prion. Prions can have a phenotypic effect through the sequestration of protein in aggregates, thereby reducing that protein's activity. In PSI+ cells, the loss of the Sup35 protein (which is involved in termination of translation) causes ribosomes to have

10332-696: The interaction between genomic DNA and nuclear proteins. It had been known that certain regions within chromatin were extremely susceptible to DNAse I digestion, which cleaves DNA in a low sequence specificity manner. Such hypersensitive sites were thought to be transcriptionally active regions, as evidenced by their association with RNA polymerase and topoisomerases I and II . It is now known that sensitivity to DNAse I regions correspond to regions of chromatin with loose DNA-histone association. Hypersensitive sites most often represent promoters regions, which require for DNA to be accessible for DNA binding transcriptional machinery to function. Histone modification

10455-501: The level of translation into protein. It appears that about 60% of human protein coding genes are regulated by miRNAs. Many miRNAs are epigenetically regulated. About 50% of miRNA genes are associated with CpG islands , that may be repressed by epigenetic methylation. Transcription from methylated CpG islands is strongly and heritably repressed. Other miRNAs are epigenetically regulated by either histone modifications or by combined DNA methylation and histone modification. In 2011, it

10578-622: The mechanisms of temporal and spatial control of gene activity during the development of complex organisms." More recent usage of the word in biology follows stricter definitions. As defined by Arthur Riggs and colleagues, it is "the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence." The term has also been used, however, to describe processes which have not been demonstrated to be heritable, such as some forms of histone modification. Consequently, there are attempts to redefine "epigenetics" in broader terms that would avoid

10701-503: The methylation pattern of the region. This analysis step was done by amplifying the restriction fragments via PCR, separating them through gel electrophoresis and analyzing them via southern blot with probes for the region of interest. This technique was used to compare the DNA methylation modification patterns in the human adult and hemoglobin gene loci . Different regions of the gene (gamma delta beta globin) were known to be expressed at different stages of development. Consistent with

10824-449: The mismatch repair protein heterodimer MSH2-MSH6 to recruit DNA methyltransferase 1 ( DNMT1 ) to sites of some kinds of oxidative DNA damage. This could cause increased methylation of cytosines (epigenetic alterations) at these locations. Jiang et al. treated HEK 293 cells with agents causing oxidative DNA damage, ( potassium bromate (KBrO3) or potassium chromate (K2CrO4)). Base excision repair (BER) of oxidative damage occurred with

10947-404: The mother during oogenesis or via nurse cells , resulting in maternal effect phenotypes. A smaller quantity of sperm RNA is transmitted from the father, but there is recent evidence that this epigenetic information can lead to visible changes in several generations of offspring. MicroRNAs (miRNAs) are members of non-coding RNAs that range in size from 17 to 25 nucleotides. miRNAs regulate

11070-409: The nucleosome present at the DNA site to carry out cytosine methylation on newly synthesized DNA. There is further crosstalk between DNA methylation carried out by DNMT3A and DNMT3B and histone methylation so that there is a correlation between the genome-wide distribution of DNA methylation and histone methylation. Mechanisms of heritability of histone state are not well understood; however, much

11193-399: The opposite strand of the target sequence. DNase-seq employs DNase I , a non-specific double strand-cleaving endonuclease. This technique has been used to such an extent that nucleosome-free regions have been labelled as DHSs, DNase I hypersensitive sites, and has been ENCODE consortium's election method for genome wide chromatin accessibility analyses. The main issue of this technique is that

11316-400: The other half of the progeny cells expression of that gene is repressed. When clones of these cells were maintained for three years, the new methylation patterns were maintained over that time period. Posttranslational modification Post-translational modifications can occur on the amino acid side chains or at the protein's C- or N- termini. They can expand the chemical set of

11439-458: The other half. However, it is now known that DNMT1 physically interacts with the protein UHRF1 . UHRF1 has been recently recognized as essential for DNMT1-mediated maintenance of DNA methylation. UHRF1 is the protein that specifically recognizes hemi-methylated DNA, therefore bringing DNMT1 to its substrate to maintain DNA methylation. Although histone modifications occur throughout the entire sequence,

11562-725: The pivotal involvement of long non-coding RNAs (lncRNAs) in the regulation of gene expression and chromosomal modifications, thereby exerting significant control over cellular differentiation. These long non-coding RNAs also contribute to genomic imprinting and the inactivation of the X chromosome. In invertebrates such as social insects of honey bees, long non-coding RNAs are detected as a possible epigenetic mechanism via allele-specific genes underlying aggression via reciprocal crosses. Prions are infectious forms of proteins . In general, proteins fold into discrete units that perform distinct cellular functions, but some proteins are also capable of forming an infectious conformational state known as

11685-403: The polymerase moves along the DNA molecule being sequenced. Several projects have demonstrated the ability to collect genome-wide epigenetic data in bacteria. Nanopore sequencing is based on changes of electrolytic current signals according to base modifications (e.g. Methylation). A polymerase mediates the entrance of ssDNA in the pore: the ion-current variation is modulated by a section of

11808-404: The pore and the consequently generated difference is recorded revealing the position of CpG . Discrimination between hydroxymethylation and methylation is possible thanks to solid-state nanopores even if the current while passing through the high-field region of the pore may be slightly influenced in it. As a reference amplified DNA is used which will not present copied methylationed sites after

11931-475: The progress of carcinogenesis , many effects of teratogens , regulation of histone modifications and heterochromatin , and technical limitations affecting parthenogenesis and cloning . DNA damage can also cause epigenetic changes. DNA damage is very frequent, occurring on average about 60,000 times a day per cell of the human body (see DNA damage (naturally occurring) ). These damages are largely repaired, however, epigenetic changes can still remain at

12054-634: The recruitment of chromatin remodeling complexes by the binding of activator molecules to corresponding enhancer regions. The nucleosome remodeling complexes reposition nucleosomes by several mechanisms, enabling or disabling accessibility of transcriptional machinery to DNA. The SWI/SNF protein complex in yeast is one example of a chromatin remodeling complex that regulates the expression of many genes through chromatin remodeling. Epigenomics shares many commonalities with other genomics fields, in both methodology and in its abstract purpose. Epigenomics seeks to identify and characterize epigenetic modifications on

12177-450: The repair process. This accumulation, in turn, directs recruitment and activation of the chromatin remodeling protein, ALC1, that can cause nucleosome remodeling. Nucleosome remodeling has been found to cause, for instance, epigenetic silencing of DNA repair gene MLH1. DNA damaging chemicals, such as benzene , hydroquinone , styrene , carbon tetrachloride and trichloroethylene , cause considerable hypomethylation of DNA, some through

12300-521: The same protocol for chromatin immunoprecipitation but instead of amplification of purified DNA and hybridization to a microarray, the DNA fragments are directly sequenced using next generation parallel re-sequencing. It has proven to be an effective method for analyzing the global histone modification patterns and protein target sites, providing higher resolution than previous methods. Techniques for characterizing primary DNA sequences could not be directly applied to methylation assays. For example, when DNA

12423-427: The side chain into a neutral amide linkage. This removes the positive charge, thus loosening the DNA from the histone. When this occurs, complexes like SWI/SNF and other transcriptional factors can bind to the DNA and allow transcription to occur. This is the "cis" model of the epigenetic function. In other words, changes to the histone tails have a direct effect on the DNA itself. Another model of epigenetic function

12546-531: The side-chain unless indicated otherwise. Protein sequences contain sequence motifs that are recognized by modifying enzymes, and which can be documented or predicted in PTM databases. With the large number of different modifications being discovered, there is a need to document this sort of information in databases. PTM information can be collected through experimental means or predicted from high-quality, manually curated data. Numerous databases have been created, often with

12669-407: The site of DNA repair. In particular, a double strand break in DNA can initiate unprogrammed epigenetic gene silencing both by causing DNA methylation as well as by promoting silencing types of histone modifications (chromatin remodeling - see next section). In addition, the enzyme Parp1 (poly(ADP)-ribose polymerase) and its product poly(ADP)-ribose (PAR) accumulate at sites of DNA damage as part of

12792-526: The sites of transcription initiation, it has the effect of repressing gene expression. This is in contrast to unmethylated promoter regions which are associated with actively expressed genes. The mechanism by which DNA methylation represses gene expression is a multi-step process. The distinction between methylated and unmethylated cytosine residues is carried out by specific DNA-binding proteins. Binding of these proteins recruit histone deacetylases (HDACs) enzyme which initiate chromatin remodeling such that

12915-477: The specific amino acid on the histone being modified. Furthermore, the DNA region where histone modification occurs can also elicit different effects; an example being methylation of the 3rd core histone at lysine residue 36 (H3K36). When H3K36 occurs in the coding sections of a gene, it is associated with gene activation but the opposite is found when it is within the promoter region. Histone modifications regulate gene expression by two mechanisms: by disruption of

13038-452: The term epigenetics in 1942 as pertaining to epigenesis , in parallel to Valentin Haecker 's 'phenogenetics' ( Phänogenetik ). Epigenesis in the context of the biology of that period referred to the differentiation of cells from their initial totipotent state during embryonic development . When Waddington coined the term, the physical nature of genes and their role in heredity

13161-401: The traditional restriction based methods, was historically limited to methylation patterns of specific gene loci, until whole genome sequencing technologies became available. However, unlike traditional restriction based methods, bisulfite sequencing provided resolution on a nucleotide level. Limitations of the bisulfite technique include the incomplete conversion of cytosine to uracil, which

13284-479: The transcriptionally active sites, and a less laborious sample preparation. ATAC-seq is based on the activity of Tn5 transposase. The transposase is used to insert tags in the genome, with higher frequency on regions not covered by proteic factors. The tags are then used as adapters for PRC or other analytical tools. Polymerase sensitivity in single-molecule real-time sequencing made it possible for scientists to directly detect epigenetic marks such as methylation as

13407-413: The unlinked DNA shifts to the aqueous phase and can be analysed with various methods. Sonication produces random breaks, and therefore is not subject to any kind of bias, and is also the bigger length of the fragments (200-700 nt) makes this technique suitable for wider regions, while it's unable to resolve the single nucleosome. Unlike the nuclease-based methods, FAIRE-seq allows the direct identification of

13530-470: The unstructured N-termini of histones (called histone tails) are particularly highly modified. These modifications include acetylation , methylation , ubiquitylation , phosphorylation , sumoylation , ribosylation and citrullination. Acetylation is the most highly studied of these modifications. For example, acetylation of the K14 and K9 lysines of the tail of histone H3 by histone acetyltransferase enzymes (HATs)

13653-494: The upstream promoter region). Bromate treatment-induced oxidation resulted in the loss of cytosine methylation at −189, −134, +16 and +19 while also leading to the formation of new methylation at the CpGs located at −80, −55, −21 and +8 after DNA repair was allowed. At least four articles report the recruitment of DNA methyltransferase 1 (DNMT1) to sites of DNA double-strand breaks. During homologous recombinational repair (HR) of

13776-558: The valleys where the marbles (analogous to cells) are travelling. In recent times, Waddington's notion of the epigenetic landscape has been rigorously formalized in the context of the systems dynamics state approach to the study of cell-fate. Cell-fate determination is predicted to exhibit certain dynamics, such as attractor-convergence (the attractor can be an equilibrium point, limit cycle or strange attractor ) or oscillatory. Robin Holliday defined in 1990 epigenetics as "the study of

13899-418: The word "epigenetics" is sometimes used as a synonym for these processes. However, this can be misleading. Chromatin remodeling is not always inherited, and not all epigenetic inheritance involves chromatin remodeling. In 2019, a further lysine modification appeared in the scientific literature linking epigenetics modification to cell metabolism, i.e. lactylation Because the phenotype of a cell or individual

14022-399: Was DNA methylation. As its name implies, DNA methylation is the process by which a methyl group is added to DNA. The enzymes responsible for catalyzing this reaction are the DNA methyltransferases (DNMTs) . While DNA methylation is stable and heritable, it can be reversed by an antagonistic group of enzymes known as DNA de-methylases. In eukaryotes, methylation is most commonly found on

14145-442: Was almost forgotten, until the experimental evidence has indicated a possible role of covalent histone modifications as an epigenetic code . In the next several years, high-throughput data have indeed uncovered the abundance of epigenetic modifications and their relation to chromatin functioning which has motivated new theoretical models for the appearance, maintaining and changing these patterns,. These models are usually formulated in

14268-456: Was amplified in PCR or bacterial cloning techniques, the methylation pattern was not copied and thus the information lost. The DNA hybridization technique used in DNA assays, in which radioactive probes were used to map and identify DNA sequences, could not be used to distinguish between methylated and non-methylated DNA. The earliest methylation detection assays used methylation modification sensitive restriction endonucleases . Genomic DNA

14391-458: Was demonstrated that the methylation of mRNA plays a critical role in human energy homeostasis . The obesity-associated FTO gene is shown to be able to demethylate N6-methyladenosine in RNA. sRNAs are small (50–250 nucleotides), highly structured, non-coding RNA fragments found in bacteria. They control gene expression including virulence genes in pathogens and are viewed as new targets in

14514-414: Was digested with both methylation-sensitive and insensitive restriction enzymes recognizing the same restriction site. The idea being that whenever the site was methylated, only the methylation insensitive enzyme could cleave at that position. By comparing restriction fragment sizes generated from the methylation-sensitive enzyme to those of the methylation-insensitive enzyme, it was possible to determine

14637-460: Was effective providing near full coverage of the yeast epigenome, its use in larger genomes such as humans is limited. In order to study histone modifications on a truly genome level, other high-throughput methods were coupled with the chromatin immunoprecipitation, namely: SAGE: serial analysis of gene expression (ChIP-SAGE), PET: paired end ditag sequencing ( ChIP-PET ) and more recently, next-generation sequencing (ChIP-Seq) . ChIP-seq follows

14760-494: Was first detected on a genome wide level through the coupling of chromatin immunoprecipitation (ChIP) technology with DNA microarrays , termed ChIP-Chip . However instead of isolating a DNA-binding transcription factor or enhancer protein through chromatin immunoprecipitation, the proteins of interest are the modified histones themselves. First, histones are cross-linked to DNA in vivo through light chemical treatment (e.g., formaldehyde ). The cells are next lysed, allowing for

14883-562: Was introduced. Furthermore, in addition to the maintenance and transmission of methylated DNA states, the same principle could work in the maintenance and transmission of histone modifications and even cytoplasmic ( structural ) heritable states. RNA methylation of N6-methyladenosine (m6A) as the most abundant eukaryotic RNA modification has recently been recognized as an important gene regulatory mechanism. Histones H3 and H4 can also be manipulated through demethylation using histone lysine demethylase (KDM). This recently identified enzyme has

15006-434: Was involved in stabilizing chromatin through its affinity for negatively charged DNA. Acetylated histones therefore favor the dissociation of nucleosomes and thus unwinding of chromatin can occur. Under a loose chromatin state, DNA is more accessible to transcriptional machinery and thus expression is activated. The process can be reversed through removal of histone acetyl groups by deacetylases. The second process involves

15129-525: Was not known. He used it instead as a conceptual model of how genetic components might interact with their surroundings to produce a phenotype ; he used the phrase " epigenetic landscape " as a metaphor for biological development . Waddington held that cell fates were established during development in a process he called canalisation much as a marble rolls down to the point of lowest local elevation . Waddington suggested visualising increasing irreversibility of cell type differentiation as ridges rising between

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