epigenetics today and tomorrow

ISSN 2079�0597, Russian Journal of Genetics: Applied Research, 2014, Vol. 4, No. 3, pp. 168–188. © Pleiades Publishing, Ltd., 2014.Original Russian Text © B.F. Vanyushin, 2013, published in Vavilovskii Zhurnal Genetiki i Selektsii, 2013, Vol. 17, No. 4/2, pp. 805–832.

168

INTRODUCTION

“Genetics proposes, epigenetics disposes”

Peter Medawar, Nobel laureate

It always seems that all scientists and expertsunderstand what epigenetics is, but everyone stillunderstands it differently. The term “epigenetics” wasproposed by K. Waddington as the study of the causal

mechanisms of realization of the genome (genes) inthe phenotype. He termed the phenotypic changesoccurring from cell to cell during the development of amulticellular organism “phenotypic landscape.” Infact, this is a very common and very indefinite concept(definition), which unites and represents the entiredevelopment of the organism, including all ontoge�netic mechanisms. D.L. Nanney considers epigenetics

Epigenetics Today and TomorrowB. F. Vanyushin

Belozersky Institute of Physicochemical Biology, Moscow State University, Moscow, 119992 Russiae�mail: [email protected]

Received June 3, 2013; in final form, November 1, 2013

Abstract—Epigenetics is the science of the heritable properties of the organism that are not associated withchanges in the DNA nucleotide sequence but can be indirectly encoded in the genome. The most well�knownepigenetic mechanisms (signals) are enzymatic DNA methylation, the histone code (various enzymatic his�tone modifications including acetylation, methylation, phosphorylation, ubiquitination, etc.), and genesilencing mediated by small RNAs (miRNA, siRNA). All these processes are usually interconnected and evenpartially interchangeable. This is apparently required for the reliable implementation of epigenetic signaling.Anyway, these processes are closely associated with chromatin structural and functional organization. DNAmethylation in plants and animals, performed by site�specific enzymes, cytosine DNA�methyltransferases,produces 5�methylcytosine (m5C) residues in DNA sequences such as CG, CNG, and CNN. Adenine DNAmethylation also occurs in plants. The resulting m5C residues in DNA substantially affect the interaction ofDNA with different proteins, including regulatory proteins. DNA methylation often prevents DNA bindingto such proteins and inhibits gene transcription, but sometimes it is required for binding to other regulatoryproteins. Specific m5CpG DNA�binding proteins were described. The binding of such proteins to DNAorchestrates the whole protein ensemble of the transcription machinery and induces its activity. Thus, DNAmethylation can serve as a signal of positive and negative control for gene activities. DNA methylation ineukaryotes is species� and tissue�specific. It is regulated by hormones, changes with age, and is one of themechanisms controlling cellular and sex differentiation. DNA methylation controls all genetic processes:DNA replication, repair, recombination, transcription, etc. Distortions in DNA methylation and other epi�genetic signals cause premature aging, cancer, diabetes, asthma, severe mental dysfunctions, etc. Changes inthe DNA methylation profile accompany carcinogenesis and are a reliable diagnostic marker of various typesof cancer even at the early stages of tumorigenesis. Epigenetic parameters are very important for understand�ing the somaclonal variation mechanisms; characterization of clones and cell cultures, including stem cellsat various differentiation stages; and determination of their differentiation directions. Directed change in theDNA methylation profile is an efficient biotechnological tool for activation of transcription of seed storageprotein genes in cereals and it is used, in particular, for an inheritable increase in protein content in wheatgrain. The inhibitor of DNA methylation with 5�azacytidine is used for treatment of skin cancer. Various reg�ulators of enzymatic modifications of histones are already used in clinical practice for the treatment of somehuman and animal diseases. The use of specific small RNAs in the therapy of cancer and other diseasesappears to be particularly promising, especially in connection with directed inhibition of transcription of thegenes responsible for cell malignization and metastasis. The therapeutic effect of many small biologicallyactive peptides can be largely determined by their action at the epigenetic level. Thus, the phenotype is theproduct of combined realization of the genome and epigenome. In this regard, P. Medawar’s well�knownexpression “genetics supposes, epigenetics disposes” sounds quite correct and very impressive. Epigenetics isa quickly developing and very promising science of the 21st century that is already ingrained in biotechnolo�gies, medicine, and agriculture.

Keywords: apoptosis, histone, DNA methyltransferase, DNA�binding proteins, genomics, genosystematics,gene silencing, cell differentiation, DNA methylation, mitochondria, development, replication, cancer, aging,transcription, chromatin, evolution, endonucleases, epigenetics, 5�methylcytosine, N6�methyladenine

DOI: 10.1134/S2079059714030083

RUSSIAN JOURNAL OF GENETICS: APPLIED RESEARCH Vol. 4 No. 3 2014

EPIGENETICS TODAY AND TOMORROW 169

as a field of knowledge explaining how and why cells(organisms) with an identical genotype can differ ininheritable phenotype. Others (e.g., D. Gotchling andA. Riggs), describe epigenetics as the science of heri�table changes that are not associated with mutations inthe DNA itself. S. Ellis defines epigenetics as inherited“cellular memory” associated with structural changesin chromatin. This definition, though too general andvague, is in fact correct, because many things in ontog�eny are ultimately determined by the modulations ofthe chromatin structure. Renowned geneticist RobinHolliday considers epigenetics as “the study of thecontrol of gene activity in space and time in the courseof development of complex organisms.” He is amongthe first to point out the possible biochemical nature(DNA methylation) of heritable epigenetic signals.These signals accompany and are often crucial duringthe realization of genetic information. Epigenetics isconsidered as a set of knowledge about changes in genetranscription as a result of the modulation of chroma�tin organization without changing the DNA sequence.In principle, perhaps, this would be enough. I wouldonly like to add that epigenetics is the science of heri�table properties of the organism that are not associatedwith changes in the DNA nucleotide sequence but canbe indirectly encoded in the genome. This rapidlydeveloping field of knowledge has already gained itsfooting, turned into an independent science, and issubstantially ingrained in modern biotechnology andmedicine. The epigenetic phenomena also include, inparticular, the phenomenon of lysogeny in bacte�riophages, the so�called gene position effect in Droso�phila, prion diseases, X chromosome inactivation insexual differentiation in animals, etc. Anyway, even atthe early stages of its inception, epigenetics was asso�ciated with chromatin reorganization (remodeling,rearrangements) and modifications of chromatin pro�teins, including histones. In 1930, H. Muller describedmutations in Drosophila, which led to a change in phe�notype and were caused by movement rather thanchange in genes—rearrangement of chromosomes(“eversporting displacements”). Thus, the activity of agene depends on its position in the genome, chromo�some, and chromatin.

The classic studies of Nobel laureate John Gurdonon the transplantation of nuclei into fertilized anu�clear Xenopus eggs showed that the realization ofgenetic information of nuclear DNA (nDNA) andembryo development are not associated with anymutations in nDNA but are triggered and controlledby some competent epigenetic elements (signals) in thecytoplasm. Interestingly, the siliation pattern of in theprotozoan Paramecium is transmitted clonally. It wasestablished that transposons can determine the geneexpression profile in somatic cells. On the other hand,the great diversity of antibodies is mainly due to DNArearrangements in somatic cells. In 1975, Arthur Riggsand R. Holliday reported that X chromosome inacti�vation and, consequently, sexual differentiation in

mammals is associated with DNA methylation. InRussia, tissue (cell) heterogeneity of DNA methyla�tion was discovered and the idea that DNA methyla�tion is a mechanism of regulation of gene expressionand cell differentiation was formulated (Vanyushinet al., 1970). In fact, it was the first material chemi�cally identified and interpreted epigenetic signal.Today, the idea of the epigenome has appeared, and itis clear that the phenotype is the product of combinedrealization of the genome and epigenome. The con�cept and term epimutations are already entered into theuse. Along with genetic diseases, there exist epigeneticdiseases. Abnormalities in the epigenome cause can�cer, diabetes, asthma, and many mental and other dis�orders. Of course, this largely depends on the environ�ment. The so�called epigenetic profile of the body isthe basis for creating the digital pathology. Epigenomesignificantly changes with age. In particular, we dis�covered the age specificity of DNA methylation. Fur�thermore, according to our understanding, epimuta�tions caused by DNA methylation may underlie pro�grammed aging and determine the lifespan.Fundamental epigenetic studies have changed ourunderstanding of the genetic identity of homozygoustwins and clones of plants and animals. It was foundthat they can significantly differ in epigenetic profiles.Somaclonal variation is often largely determined bythe changes in epigenetic parameters and signals.

Despite the great advances in molecular biologyand molecular genetics in the last century, manyimportant problems of general biological significanceremain unsolved. Among them, the most importantare cell differentiation and gene activity regulation. Westill do not understand completely how the normaldevelopment of the organism proceeds and how cellswith initially the same genetic information make theirown (different) ways during development, with accu�rate and correct realization in space and time of spe�cific regions of the genome to form a specific pheno�type. How does the cell decides when it is time todivide and differentiate? Epigenetics offers a new wayto look at these issues and find a solution to burningmysteries of biology, such as cell identity (specificity),carcinogenesis, stem cell plasticity, regeneration ofanimal and plant cells and tissues, aging, and pro�grammed cell death.

Epigenetics is often recalled when the effect of theenvironment on gene expression (diet, hormones, andother factors and environmental conditions) are con�sidered. Epigenetics represents a new comprehensiveand promising horizon of our knowledge in the post�genomic era. Indeed, we inherit something more thanjust the sum of the genes and, according to Nobel lau�reate D. Watson, “something else besides the DNAsequences.” All this emphasizes once again that, with�out epigenetics, it is impossible to solve the main prob�lem of biology—to establish the driving mechanismsof regulation of gene expression and cell differentia�tion under different environmental conditions.

170


VANYUSHIN

In humans, genetic information is written in23 pairs of chromosomes comprising approximately25000 genes. The human genome contains approxi�mately 3 × 109 base pairs, or 1 × 107 nucleosomes. Thelength of DNA in higher eukaryotes is about 2 m, andit is condensed in the nucleus by a factor of approxi�mately 10000. As much as approximately 96% of themammalian genome is represented by noncoding andrepetitive DNA sequences. There is a great diversity ofnumerous elements and factors for regulating geneexpression, such as DNA binding to different proteins,including the regulatory ones; proteins methyl�CpGDNA�binding and other proteins; different modifica�tions of histones; nucleosome remodeling and chro�matin rearrangement in general; and hormone–receptor complexes, DNA methylation, and interac�tion with short noncoding RNAs. Gene expressionrequires large complex systems and assemblies of morethan 100 proteins involved in transcription initiationand elongation from one promoter and in messengerRNA processing. Most likely, a transcription�compe�tent gene state cannot be ensured by only one trigger(e.g., a particular protein modification) and requiresthe sum and cumulative effect of many factors to cre�ate a proper active epigenetic state in a certain chro�matin region. Epigenetic control can promote(enhance) the primary signal (promoter stimulation)or mediate gene silencing. Epigenetic memory is oftenassociated with specific histone modifications in chro�matin. This unique chromatin configuration appar�ently can be transmitted from cell to cell in a series ofcell divisions. In particular, this can be observed ingene silencing, and this ban (conformational “lock”)in a particular chromosomal region can become evenstronger and more significant as a result of additionalDNA methylation induced (permitted) by this chro�matin state. In this case, DNA itself can be consideredas a self�organizing polymer with an ordered structurein chromatin, able to respond and perceive differentepigenetic signals.

Environmental factors can markedly affect theactivity of enzymes (and their cofactors) performingmodifications of histones and DNA. Such cofactorsinclude ATP for kinases, acetyl coenzyme A for acety�lases, and S�adenosyl�L�methionine (SAM) for vari�ous methyltransferases. Of course, the content of thesecofactors in the cell may significantly vary and dependon the environment, including the diet. An adequateepigenetic control in the cell is based on the balance ofmany factors, which is not always accurately transmit�ted during cell division. In view of this, an importantquestion arises as to how information about the chro�matin structure is transmitted from the maternal cellto the daughter cells. In principle, this could take placeas follows. It is known that the synthesis of core his�tone proteins is strictly regulated in the cell cycle. Thetranscription of the genes encoding these proteinstakes place in the S phase of the cell cycle and is wellcoordinated with the nDNA replication. Upon the

assembly of the newly formed chromatin, its specificproteins, depending on the degree and nature of theirenzymatic modifications, may or may not bind toDNA to form sites inaccessible or accessible, respec�tively, for transcription. In this process, DNA againserves as a self�organizing matrix. Recently it wasestablished that mutations in the genes encoding theenzymes involved in histone modifications lead tonucleosome remodeling and are accompanied byimpaired development of organisms and neoplasia.The development of tumors in the mutant mice withthis pathology was traditionally attributed to the cate�gory of genetic diseases. In fact, the changes in the his�tone and DNA methylation patterns and the structuralchanges in nucleosomes are not caused directly by themutated gene and, therefore, should be considered asepigenetic aberrations. This equally applies to themutations in the genes involved in the SAM synthesisand utilization: the absence of SAM leads to disrup�tion of many transmethylation reactions in the celland inactivation of many enzymes that use SAM as anallosteric effector. This is completely consistent withthe idea that epigenetics mostly deals with heritablephenomena (properties of living organisms) that areindirectly, rather than directly, encoded in thegenome. Nevertheless, epigenetics and genetics aretwo closely related phenomena or areas of knowledge.The genome may contain valuable genes; however,depending on the particular specific epigenetic signal,they can remain unrealized at all. At the same time,although the epigenetic changes are not inherited, thisis not infinite. Cells often try to return to the originalepigenetic status; if they succeed, these epigeneticchanges are erased in a series of generations. There aremany such examples. It is still unclear whether andhow epigenetic parameters or features can be some�how reflected in the germline itself (i.e., in DNA).

Today, the central dogma of biology (DNA ↔RNA ↔ protein) is supplemented with new informa�tion about prions. Similarly to DNA and RNA, theseproteins are able to replicate and are inherited withoutRNA and DNA templates.

It has long been known that proteins of chromatinhistones inhibit transcription: the histone�free DNA istranscribed much more effectively than the DNAassociated with histones in chromatin. It may seemthat, for efficient transcription, it is necessary to stripDNA from histones. However, B. Olfry and A. Mirskyshowed many years ago that the transcription of inac�tive chromatin can be activated by acetylation of his�tones, which significant weakens the associationbetween these proteins and DNA. The common ideaabout the existence of the histone code has alreadybeen formed.

Histone modifications (acetylation, phosphoryla�tion, ubiquitination, ADP�ribosylation, biotinylation,sumoylation, isomerization of the proline residue,etc.) have been discovered and described.



If, indeed, the histone code does exist, it is not uni�versal, unlike the genetic code. Every organism has itsown histone code.

Epigenetic signals in the cell and organism arehighly numerous and diverse, and much in this arearemains obscure. Nevertheless, many of them havealready been materialized and described. For example,the most important epigenetic signals known today aregiven below:

—DNA methylation;—Various enzymatic modifications of histones

(histone code);—Genomic and chromosomal rearrangements;—Small noncoding RNAs (siRNAs, or small

interfering RNAs);—Other.These signals, their detailed nature, the interaction

between them, the physical and chemical effectscaused by them, as well as their resulting biologicaleffects in the cell in different functional states of theorganism and under different internal and externalenvironmental conditions are the main study subjectof epigenetics.

Unfortunately, a short review does not allowdescribing all aspects of epigenetics in detail. Let usconsider only some of them.

DNA METHYLATION—MECHANISM OF EPIGENETIC CONTROL FOR THE GENETIC

FUNCTIONS OF AN ORGANISM

“Methylation helps life, but it may takeit away. In fact, without methylationlife would be altogether impossible.”

Craig CooneyOver half a century ago, Professor Andrei Belozer�

sky offered me, a student of the Department of PlantBiochemistry, Faculty of Biology, Moscow State Uni�versity, to study the nucleotide composition of theDNA and RNA of several bacteria. Analysis of thecomposition of these DNA at that memorable timeclearly showed that the GC content in DNA is spe�cies�specific and can serve as an important taxonomictrait in bacteria (Spirin et al., 1957). In fact, this workwas one of those that laid the foundations of genosys�tematics. The world of eukaryotic DNA was almostuntouched at that time. For example, informationabout the DNA structure in representatives of theentire plant kingdom was only represented by the dataon the wheat germ DNA. One of our goals at that timewas to answer the question, at least in part, as to whatare the DNAs of eukaryotes. The first systematic stud�ies of DNA of higher plants were performed in Russia,they covered the DNAs of archegonial (mosses, clubmosses, horsetails, ferns, and gymnosperms) andflowering (angiosperms, both monocot and dicot)plants.

Even then it was found that the characteristic fea�ture of DNA of all higher plants is the relatively highcontent of the additional base, 5�methylcytosine(m5C) (Table 1). Later, it was shown that, similarly tobacteria, plant DNA contains N6�methyladenine(m6A).

The origin of these bases in DNA remainedunknown for a long time. The enzymes that selectivelymethylated certain cytosine and adenine residues inDNA strands in the presence of the donor of methylgroups S�adenosyl�L�methionine were found only in1963, first in bacteria and then in eukaryotes. Itbecame clear that the minor bases (m5C and m6A)found in the DNA molecule are not incorporated intothe DNA strands in the finished form but emerge as aresult of enzymatic modification (methylation) of therespective conventional bases in the formed or emerg�ing DNA strands (Fig. 1). The enzyme DNA methyl�transferase deftly deals with DNA: it forms a

Table 1. Minor methylated bases in DNA

OrganismsMinor bases, %

m5C m6A

Bacteria 0.01–1.53 0.02–0.70

Algae 0.20–3.50 0.10–0.60

Fungi + 0–0.5

Protozoa 0.3–1.0

Plants 2.0–10.0 0.5–1.0

Invertebrates 0.1–2.5 ?

Vertebrates 0.7–3.5 +

CH3

AdenineThymine

To stra

nd

Cytosine Guanine

To strand

2.80 Å

3.00 Å

11.1 Å

50° 51°

CH3

To st

rand

To strand

2.90 Å

3.00 Å

10.8 Å

52° 54°

2.90 Å

Fig. 1. Canonical WC base pairs in DNA.

172


VANYUSHIN

covalently bound complex with it, turning the modi�fied base out of the double�stranded DNA helix, andthen methylates this base (Fig. 2). Then, the covalentbond between the enzyme and DNA breaks, the com�plex decomposes, and the methylated base (m5C)returns to its original place in the DNA structure.

The specificity and functional significance of enzy�matic DNA methylation remained unknown for manyyears. Moreover, there was a general belief that theseminor bases do not play any role, either in the DNAstructure itself or in its functioning. The favorite objectof classical genetics, Drosophila, was often used as acompelling argument in such concepts. In the genomeof this insect, nobody could find minor bases, includ�ing m5C, for a long time. This fact gave many scien�tists, including Nobel laureate William Gilbert, a rea�son to postulate that, since Drosophila lives withoutDNA methylation, this modification of the genome isnot significant in the life of eukaryotic organisms. Thiscooled the interest of many biochemists and molecu�lar biologists in studying DNA methylation for manyyears and allowed us to study DNA methylation in dif�ferent organisms step�by�step for a fairly long time ina more or less quiet atmosphere. As a result, it wasfound that the Drosophila genome is characterized bya substantial deficit of CpG sequences, which usuallyserve as the main site of in vivo DNA methylation ineukaryotes. In our opinion, such a pronounced CpGsuppression in the Drosophila genome could only bedue to the methylation of cytosine residues in it. Sincewe could not detect DNA methyltransferase activityper se in Drosophila at that time, we called this possibleDNA modification in this insect an obsolete DNAmethylation. Today, other researchers have clearlydemonstrated that, DNA of Drosophila is methylated,this genome modification is important for the devel�opment of this insect, and DNA methyltransferaseactivity is clearly detected in the early stages of devel�opment.

We have always been convinced that the minorbases in DNA and the enzymatic modification of thegenome as such cannot be traceless in the genomestructure and must affect its biological functions.

METHYLATION AND ITS EFFECT ON THE STRUCTURE OF DNA

AND ITS INTERACTION WITH PROTEINS

We have found an unusual double�stranded naturalDNA (Bac. brevis bacteriophage AR9 DNA) that con�tains uracil, a characteristic RNA base, instead ofthymine, a common base in DNA. Thus, uracil ceasedto exist as a principal characteristic feature of RNA.Roughly speaking, uracil represents thymine lackingthe methyl group. The uracil�containing DNA of bac�teriophage AR9 melted (denatured) at a much lowertemperature than the normal thymine�containingDNA of a similar composition. It became clear thatmethylation of cytosine residues is not indifferent tothe DNA structure. This was the first reliable indica�tion that the methyl groups of pyrimidine bases inDNA stabilize its secondary structure. Even moreattractive was the fact that DNA methylation signifi�cantly affects its interaction (binding) with variousproteins. In particular, we have identified proteins inplant nuclei that specifically bind to the regulatory ele�ments of rRNA genes (135 bp subrepeat element) anddemonstrated that the binding of some of thesenuclear proteins is modulated by in vitro methylationof cytosine residues in DNA. In many cases, the meth�ylation of DNA at cytosine residues prevents its bind�ing to the specific DNA�reactive nuclear proteins(factors) that mediate various genetic processes,including DNA transcription, replication, and repair.For example, preliminary methylation of the secondcytosine residue at the SSGG site in the wheat rRNAgene fragment eliminated its ability to bind to one ofthe wheat nuclear proteins (Fig. 3). On the otherhand, there exist m5CpG DNA�binding proteins thatspecifically arrange on DNA the whole ensemble ofsophisticated protein complexes that control andmediate gene expression.

NONENZYMATIC DNA METHYLATION

If DNA is incubated with S�adenosyl�L�methion�ine (SAM, AdoMet) labeled in the methyl group in theabsence of any proteins, the radioactivity is thendetected in the DNA in the form of newly formed5�methylcytosine and thymine residues. Thus, thenonenzymatic DNA methylation was discovered (Fig. 4).Interestingly, the labeled thymine was detected inDNA in much higher quantities than m5C. Thus, itwas found that nonenzymatic methylation of DNA inan aqueous solution is accompanied by a rapid oxida�tive deamination of any m5C residues with their trans�formation into thymine residues. This was evidencethat the methylation of cytosine residues in DNA can

Fig. 2. Complex of cytosine DNA methyltransferase cDNA.



lead to C → T transitions (the GC base pair is replacedwith the AT base pair) and that 5�methylcytosine res�idues are mutation hotspots. This phenomenon itselfunderlies the significant partial disappearance (sup�pression) of some CpG sequences from the genes andgenomes of various organisms and is one of the majorroutes of natural mutagenesis and evolution.

SPECIFICITY OF ENZYMATIC DNA METHYLATION

The existence of the potential of DNA methylationin nature, apparently, was used by the specific enzymesDNA methyltransferases, which appeared in thecourse of evolution and, in contrast to the chaoticnonenzymatic DNA methylation, modify cytosine oradenine residues in strictly defined nucleotidesequences. We have decoded one of the first suchmethylated nucleotide sequences in bacterial DNA. InBac. brevis cells, cytosine DNA methyltransferasemethylates cytosine residues in the (5')GCTGC(3')sequence. Later, this was confirmed in the studies ofNobel laureate R. Roberts. It was found that DNAmethylation in bacteria is the basis of the host restric�tion–modification. This phenomenon, in particular,means that the bacteriophage grown in the cells of acertain bacterial strain acquires the host’s specificityand can infect only the cells of this host (restriction—limiting the host range), because the bacteriophageDNA is methylated by the host’s bacterial DNA meth�yltransferases and, thus, is protected from hydrolysisby the host’s endonucleases, which are sensitive tosuch DNA methylation. To some extent, these andother data made it possible to substantiate the chemi�cal nature of the phenomenon of host restriction–mod�ification in bacteria. Before that, we demonstrated thatDNA in different bacteria is methylated in differentways and revealed the strain and species specificity ofgenome methylation in microorganisms. Furthermore,it was shown that DNA methylation in bacteria is mod�ified upon dissociation (R� and S�forms) and sporula�tion. Probably, these data were among the very first indi�cations that DNA methylation in microorganisms isassociated with cell differentiation.

It was necessary to establish the chemical and bio�logical specificity of DNA methylation in eukaryotes,including plants and animals. Before the appearanceof DNA sequencing methods, we analyzed pyrimidinesequences (blocks) cleaved from DNA and showedthat the plant genome contains 5�methylcytosine inthe sequences Pu–m5C–Pu, Pu–m5C–T–Pu, Pu–m5C–C–Pu, and Pu–m5C–m5C–Pu (Kirnos et al.,1981). This finding is consistent with the later dataobtained by A. Razin (Israel) on the methylation ofcytosine residues at CG and CNG sites in plant andanimal DNAs. According to our data, a significantproportion (approximately 30%) of 5�methylcytosinein the plant genome is contained in the m5CNGsequences (Kirnos et al., 1981). The presence of m5C

in CNG sites, especially in animal DNA, has not beenrecognized for a long time, and the first reports of thisphenomenon even caused sharp distrust and rejection.Meanwhile, such DNA methylation is indeed realizedin animal cells, and it is of great biological importance.Methylation of cytosine residues in these and asym�metric sequences is mainly observed in the DNAmethylation induced by double�stranded small RNAs,which is associated with gene inactivation. Theenzyme that methylates cytosine residues in any con�text, except CpG, was identified in plants. Thus, inprinciple, the nature of the chemical specificity ofDNA methylation in plants and animals has beenestablished.

In Arabidopsis, DNA in the centromeric and peri�centromeric heterochromatin regions is stronglymethylated; this especially applies to transposons andother repetitive sequences. DNA in euchromatin ismethylated much less strongly; m5C was found both inintergenic regions and in individual genes. Approxi�mately 55% of m5C is contained in CG, 23% in CNG,and 22% in CNN sequences (Vanyushin and Ashap�kin, 2011). All three types of methylated sequences arefound in the repeats of chromatin centromericdomains, whereas gene bodies are methylated almostexclusively at CG sites. Respective coding siRNAregions of the genome contained high quantities of5�methylcytosine residues at CG, CNG, and CNNsites. Over 60% of all expressed genes remain unmeth�

→CG

DNA + methyl* – SAM → methyl* – DNA + SAH

Methylation DeaminationCytosine → 5�methyl*cytosine → (5�methyl*uracil)thymine

GC → AT (Transition)

m5C♥G →

→

→T♠G

TA GC

Fig. 4. Nonenzymatic DNA methylation.

1 2 3

DNA�protein complex

Fig. 3. Binding of wheat nuclear protein with the DCRfragment (174 bp) of the wheat rRNA gene is blocked by invitro methylation of the CCGG site with the DNA meth�yltransferase HpaII. Designations: 1—rRNA fragment ispreliminarily methylated with HpaII; 2, 3—complex ofthe rRNA gene fragment with the protein.

174


VANYUSHIN

ylated at all. The 5'� and 3'�end proximal parts of themethylated genes are relatively hypomethylated. Theunmethylated genes are usually transcribed only mod�erately, and all of them are greatly enriched in thegenes encoding transcription factors. The genes withmethylated promoter are transcribed relatively weaklyin a tissue�specific manner. In all circumstances, themethylation of the promoter is more important forgene inactivation (silencing) than the methylation ofthe gene body itself (Vanyushin and Ashapkin, 2009,2011). The DNA methylation profile in triple drm1drm2 cmt3 Arabidopsis mutants did not differ signifi�cantly from that of the wild�type plants, and over 90%of sites that can be methylated were methylated. Thus,plants have a potent compensatory system for protect�ing the genome methylation status from the loss offunctions of individual genes encoding DNA methyl�transferases.

As for the biological specificity of DNA methyla�tion in eukaryotes, we have long known that it is spe�cies�specific: in many invertebrates, the degree ofgenome methylation is very low; as mentioned above,m5C in Drosophila DNA could not be detected for along time, whereas in vertebrate DNA m5C is alwaysfound in sufficiently high quantities, and in plantDNA m5C cannot be called a minor base at all,because its content here is often comparable with thatof cytosine. For example, in Scilla satellite DNA,nearly all cytosine is represented by its methylatedderivative (m5C).

We found that animals and plants, along with thespecies specificity, also show tissue (cell) (Vanyushinet al., 1970), subcellular (organelle), and age (Berdy�shev et al., 1967) heterogeneity (specificity) of DNAmethylation. One of our American colleagues, CraigCooney, has acknowledged that Russian researchersshowed that DNA methylation in animals decreaseswith age. It was an intriguing indication that aging andreduction of DNA methylation go hand�in�hand.Does this mean that there is a correlation between theaging of cells and the reduction in the level of DNAmethylation? Most likely, yes. Back in the 1960s,B.F. Vanyushin and colleagues were the first to showthat the level of DNA methylation in salmon decreaseswith age (Berdyshev et al., 1967). They showed thatthis also applies to the majority of organs of aging ratsand cows (Vanyushin et al., 1973). Later, severalgroups of scientists in the United States and Japanfound that the level of DNA methylation in micedecreases with aging (Sooney, 1999). As a result, theage�related decrease in the DNA methylation levelbecame quite obvious, and some researchers evenbelieve that the degree of DNA methylation can serveas kind of a biological clock that makes it possible toassess the age and predict life expectancy. Distortion ofDNA methylation can lead to premature aging. In ouropinion, epimutagenesis determined by DNA methy�lation serves as a mechanism of programmed agingand phenoptosis. We found significant age�related

changes in the DNA methylation pattern in plants.The level of DNA methylation in plants varies in thecourse of ontogeny starting from seed germination andending by the final stages of plant development,including apoptosis and phenoptosis (the term wasproposed by V.P. Skulachev)—programmed death ofthe organism, which is particularly brightly and some�times even dramatically expressed in monocarpicplants. Many years ago, I saw a very sad picture at theBatumi Botanical Garden: a large multiannual bam�boo plantation that died all of a sudden quite soonafter flowering. Similarly to spawning salmon (Berdy�shev et al., 1967), the programmed death of bambooplants was accompanied by a global decrease in thelevel of DNA methylation in all organs. Most likely,this is a general biological phenomenon, which isapparently determined by the hormonal control bothin animals and plants. Thus, the florigen proposed byM.Kh. Chailakhyan, may be responsible not only theinduction of flowering but also for phenoptosis as aresult of modulation of genome methylation by phyto�hormones.

We have found that DNA is methylated differen�tially in different cells of the same organism and,therefore, methylation of the genome is associatedwith cell differentiation. This finding allowed us topostulate that DNA methylation is a mechanism ofregulation of gene expression and cell differentiation(Vanyushin et al., 1970). This and other our studieshave attracted much attention of many researchers inour country and abroad and served as an impetus forthe intensive study of DNA methylation in the world.

It was established that DNA in the mitochondriaand nucleus of the same cell is methylated differen�tially. 5�Methylcytosine was identified in bovine heartmitochondrial DNA. Along with this, we isolatedcytosine DNA methyltransferase from the mitochon�dria of animals and showed that this enzyme has a dif�ferent site specificity of action as compared to thenuclear DNA methyltransferase. Thus, the subcellular(organelle) specificity of DNA methylation was dis�covered. Unlike animals, in plants we did not find 5�methylcytosine in the mitochondrial DNA but foundN�methyladenine. Unlike the heavily methylatednuclear DNAs of higher plants, their chloroplast DNAis unmethylated. There are scarce data that DNA ofother plastids (leucoplasts, chromoplasts, and amylo�plasts) of higher plants may contain various minormethylated bases. It was assumed that DNA methyla�tion may be involved in the differentiation of plastids;however, these data and assumptions have not yet beenconfirmed.

DNA METHYLTRANSFERASES

Plants were shown to contain at least three classesof cytosine DNA methyltransferases and more than adozen genes encoding DNA methyltransferases (Table 2,Fig. 5) (Finnegan et al., 2000; Vanyushin and Ashap�



kin, 2009). This is much greater than in all knowneukaryotes.

CLASS METI CYTOSINE DNA METHYLTRANSFERASES

These enzymes catalyze the maintenance methyla�tion of CpG sites during DNA replication, ensuringgeneral preservation and inheritance of the overallmethylation pattern at these sites in the genome.Therefore, it is not surprising that MET1 genes areexpressed in all organs (Fig. 6), but the body of thesegenes itself remains almost unmethylated, both inwild�type plants and in different transgenic Arabidop�sis plants (Fig. 7). This is also characteristic of DNAmethyltransferase CMT3—the main methyltrans�ferase maintaining the methylation of CpNpG sites.The expression of this gene is highly conserved: in thetransgenic Arabidopsis lines with the antisense MET1

construct under an inducible promoter, the expressionof this gene did not change significantly depending onthe induction of the construct.

FUNCTIONAL ROLE OF DNA METHYLTRANSFERASES OF DIFFERENT CLASSES

Although the question about the involvement ofindividual methylases in DNA methylation of a par�ticular type has been largely cleared in recent years,the role of the existence of different types of DNAmethylation remains largely obscure. To a lesserextent, this applies to the methylation of the CpG typeand, respectively, to the class MET1 enzymes. There isno doubt that MET1 itself is the major maintenanceDNA methyltransferase in plants. This is confirmednot only by its high homology with animal mainte�nance methyltransferase Dnmtl but also by the expres�

Table 2. Cytosine DNA methyltransferases of plants

Family Abbreviated name

Syn�onyms

Locus, coordinates,

nts

Presence of domains Expression Function

MET MET1 METI,DMT1,DDM2

AT5G49160,19949456–19955595

BAH (2)m5C�DNA�methyltransferase

In all organs, espe�cially actively dividing ones

Major maintenance CpG�methylase

MET2a METII,MET2,DMT2

AT4G14140,8146340–8152126


Similar to MET1, though 10000 times weaker

Not established

MET2b DMT8, METIIb

AT4G08990, 5764778–5770493

BAH (2) m5C�DNA�methyltransferase

Not studied Not established

MET3 DMT3,METIII

AT4G13610,7915018–7921227


Not studied Unknown, damaged in ecotype Columbia

CMT CMT1 DMT4 AT1G80740, 30347286–30351940

BAH, Chromo, m5C�DNA�methyltransferase

Not found Unknown, damaged in ecotype Columbia

CMT2 DMT5 AT4G19020,10414537–10421211

BAH, Chromo,m5C�DNA�methyltransferase

Similar to MET1, though weaker by a factor of ~10

Second maintenance CpNpG�methylase

CMT3 DMT6 AT1G69770,26251990–26257248

BAH, Chromo,m5C�DNA�methyltransferase

In all organs, espe�cially in flower parts

Major maintenance CpNpG�methylase

DRM DRM1 DMT9 AT5G15380, 4991350–4994829

UBA (2) m5C�DNA�methyltransferase

Not found Second de novo DNA methylase?

DRM2 DMT7 AT5G14620, 4715256–4718707

UBA (2) m5C�DNA�methyltransferase

In all organs Home de novo DNA methylase

DRM3 DMT10 AT3G17310, 5909007–5913248

m5C�DNA�methyltransferase

Not found Not established

176


VANYUSHIN

sion pattern. It is expressed primarily in the dividingcells of meristematic zones. The transgenic plants car�rying antisense constructs to MET1, as well as theplants with a mutation in the conserved motif I of theMET1 gene, have a reduced level of methylation ofunique and repetitive DNA sequences. Although thedecrease in methylation is observed mostly at the CpGsites, it also affects, although to a lesser extent, themethylation at CpNpG sites.

The main, if not the sole, target of methylation byDNA methyltransferase MET1 is the symmetricalsequence CpG. The decrease in the degree of methy�lation of sequences CpNpG after the insertion intoplants of the antisense constructs METI may not be a

direct effect but an effect mediated by other methyl�transferases. In homozygous self�pollinating plantlines defective for MET1 and, respectively, for theCpG type methylation, progressive disorders of mor�phogenesis are observed. This seems a quite logicalconsequence of the gradually accumulating abnor�malities in the regulation of tissue�specific gene tran�scription as a result of the loss of methylated sites intheir regulatory regions. However, in reality the pic�ture is somewhat more complicated. The cells of suchplants, against the background of a general decline inthe degree of DNA methylation, often show localhypermethylation of some genes (e.g., the Supermangene), which is accompanied by the characteristicphenotypic manifestations (homeiotic transforma�tions of flower). The target of hypermethylation insuch plants is represented by the cytosine residues inthe asymmetric CpNpN and symmetric CpA/TpGsites, but never in the symmetric CpG sites.

In spontaneous epimutations at the Superman genein the plants with active DNA methyltransferaseMET1 (the clark kent alleles), DNA hypermethyla�tion at sites of all types is observed. The study of plantsin which the activity of MET1 methyltransferase is sig�nificantly reduced with the use of transgenic antisenseconstructs or mutations in the MET1 gene itself,showed gradual accumulation of increasingly pro�nounced developmental anomalies in a series of suc�cessive generations, which gradually disappeared

MET1

MET2a

MET2b

MET3

CMT1

CMT2

CMT3

100 aaDRM1

DRM2

DomainDNMT2BAH

DNA�methyltransferase

Chromo

UBA

Fig. 5. Family of cytosine DNA methyltransferases of Arabidopsis thaliana (Vanyushin and Ashapkin, 2009. 152 p.).

Flower Stem Leaf Root 1 2 3

Fig. 6. MET1 gene transcription in Arabidopsis thaliana(Ashapkin et al., 2011, pp. 320–331). Designations: 1—Wild type, 2—transgenic plant without induction of theantisense MET1 construct, 3—the same plant after thetreatment with Cu.



when MET1 activity was restored by backcrosses withthe wild�type plants. Hence, the anomalies werecaused by the gradual accumulation of abnormalhypomethylation in the genes that regulate develop�ment rather than by the suppression of DNA methyl�transferase.

It is now established that MET1 is the major main�tenance CpG DNA methyltransferase that is directlyor indirectly involved in the regulation of transcriptionof many genes. The analysis of several Arabidopsis nullmutants for the CMT3 gene (obtained as suppressorsof epimutations at the Superman gene) showed thatthey almost completely lost genomic DNA methyla�tion at the symmetrical CpNpG sites, whereas themethylation at the CpG sites remained practicallyunaffected. A similar picture was observed in the anal�ysis of DNA methylation in the maize mutant for theZmet2 gene, a homologue of Arabidopsis genes CMT1and CMT3. Apparently, CMT3 is a maintenanceCpNpG methyltransferase. The degree of reduction inthe methylation of asymmetric sites varies widely indifferent lines of mutant plants and is, apparently, anindirect effect. The comparison of the phenotype ofnull mutants for the genes encoding methyltrans�ferases MET1 and CMT3 and the patterns of expres�sion and methylation of several genes showed that themethylation at CpNpG but not CpG sites is necessaryfor the suppression of Superman gene activity, whereasfor the fwa gene, the picture was just the opposite. Inother words, the expression of each specific gene in

plants may depend on methylation of any two mainte�nance DNA methyltransferases. However, it should benoted that, in general, the phenotypic consequencesof MET1 inactivation are much more pronouncedthan the effects of CMT3 inactivation (Vanyushin andAshapkin, 2009).

The obvious candidates for the role of enzymesresponsible for the methylation of asymmetric DNAsequences in plants are methyltransferases of theDRM family. Indeed, the conservation of the methy�lation sites of this type implies their constant methyla�tion de novo, and DRM methyltransferases are thehomologues of de novo methyltransferases of animals.In addition, the methylation of asymmetric sites ispractically unaffected in many plant lines with nullmutations for MET1 and CMT3 genes. Arabidopsisdouble null mutants drm1 and drm2 completely lackthe de novo DNA methylase activity, which is requiredfor the inactivation of transgenes. However, directanalysis of methylation of a number of individualDNA sequences in these mutants showed that themethylation of asymmetric sites is completely elimi�nated in some loci and only partially eliminated inothers. On the other hand, the methylation at CpNpGsites in some loci is also partially eliminated. In the tri�ple mutants drm1 drm2 cmt3, the methylation at theasymmetric sites and CpNpG sites is completely elim�inated, but the methylation at CpG sites is almostunaffected. It should be also noted that noticeablemorphological abnormalities were observed neither in

X(–9.5)

WT, 34�7066�3 + Cu, pYc2�768�5,

B(–7.7)

P(–6.5)

R(–2.5)H(–2.6)

E(–1.5)

P(0.4)(1.1)

B(0.9)

P(2.3)(3.3)X(3.2)(3.5)(3.9)

B(2.3)

H(2.0)H(1.7) H(2.9)

R(3.7)

P(5.8)

B(5.6)

P(9.2)X(10.1)

E(6.8)H(8.2)

R(13.2)B(14.6)

34�7066�3, pYc2�7066�4

pMAT34 probepNc2 cDNA probe

MET1 promoters probe pNc1 cDNA probe

1 kbp

CCGC sites

GCGC sites

pYc8�7066

pYc2�768�5 + Cu, pYc8�7066�1 + Cu,pYc2�7066�4 + Cu

Fig. 7. Cytosine methylation profile of the MET1 gene in Arabidopsis thaliana (Ashapkin et al., 2011. pp. 320–331). Designations:WT—wild�type plants (others were our transgenic plants (mutants) carrying the antisense construct of the MET1 DNA methyl�transferase gene under an inducible promoter); +Cu—plants treated with an inducer (Cu); closed circles—methylated sitesfound; gray circles—partially methylated sites; open circles—unmethylated sites.

178


VANYUSHIN

the double mutants drm1 drm2 nor in the singlemutants cmt3, whereas the triple mutants drm1 drm2cmt3 had numerous morphological abnormalities.This confirms that the functions of DRM and CMT3are interdependent and locus�specific.

In the studies of the biological role of differentDNA methyltransferases, their mutual influence bothat the level of transcription of the coding genes and atthe level of DNA methylation reactions has not beentaken into account. However, the existence of sucheffects is indisputable. Firstly, the transcription ofmany genes, including the genes encoding DNAmethyltransferases themselves, depends on DNAmethylation. Secondly, all enzymatic reactions ofDNA methylation depend on the methylation at vari�ous sites, i.e., DNA methylation by one methyltrans�ferase, can and should affect its subsequent methyla�tion by other methyltransferases. Moreover, the cellsof higher plant (and other eukaryotes) apparently havea single complex system of epigenetic regulation ofgene activity. All three types of DNA methylation areclosely associated not only with each other but alsowith two other global epigenetic systems—the histonemodification system and the system of gene expressionregulation by small RNAs. For example, the activity ofLys�9 methyltransferase of histone H3 is required forthe methylation of CpNpG sites by methyltransferaseCMT3 and the methylation on histone H3 at the Lys�9residue depends on the methylation of CpG sites byDNA methyltransferase MET1. It was also shown thatthe small RNAs are the long�sought element of site�specific recognition that ensures accurate targeting ofthe cytosine de novo DNA methyltransferase at spe�cific DNA sequences.

For an in�depth study of the role of DNA methyl�transferases and genome methylation in plants, we setout to obtain transgenic Arabidopsis plants whosegenomes contain antisense constructs expressed undervarious inducible promoters (copper�, ethanol�, andsteroid�dependent) for all known genes of plant DNAmethyltransferases. In principle, this could give us areally unique opportunity to switch off each of thesegenes in any order and any combination almost at anystage of plant development at our discretion. As aresult, we obtained a whole collection of such trans�genic Arabidopsis plants. Indeed, under the influenceof inducers, we were able to selectively inactivate thegenes encoding respective DNA methyltransferases,including MET1 (Fig. 6). It was found that differenttypes of DNA methylation affect each other. Forexample, the presence of methylated CG site increasesthe probability of methylation of adjacent CNG sites,and the presence of methylated CNG site increasesthe probability of methylation of adjacent CNN sites.In the null mutants met1, the promoters of some geneswere often hypermethylated at CNG and CNN sitesagainst the background of an overall reduction inDNA methylation.

In the combined mutants met1�drm1�drm2, CNGmethylation in the structural part of many genes ismarkedly increased, as if partly replacing the disap�peared CpG methylation. Thus, the multiplicity ofDNA methyltransferases and their genes in plants isapparently of mutual compensatory importance, pro�viding reliable genome modification under various—including unfavorable—environmental conditionsand during the possible inactivation of certain ele�ments of enzymatic epigenetic control.

In studying the obtained transgenic Arabidopsisplants, we encountered a number of unforeseen cir�cumstances and problems. For example, the trans�genic plants carrying active antisense constructs ofgenes encoding cytosine DNA methyltransferases,morphological changes may not be observed immedi�ately or they may appear in subsequent generations.During the selection of stable homozygous transgenicArabidopsis lines, their phenotypic characteristics canvary considerably. The breeding of such plants is oftenimpossible because of the disturbances in seed forma�tion and ripening. Contrary to our expectations, theinactivation of target genes by the antisense constructscan be irreversible, i.e., be retained in the absence ofinducer. Finally, the effects of gene silencing may bethe result of the effect on other epigenetic mechanismsrather than the consequence of the lack of variousDNA methyltransferases.

FUNCTIONAL SIGNIFICANCE OF DNA METHYLATION

Since we were among the first researchers whowondered about the biological role of DNA methyla�tion on the background of a general skepticism aboutthe role of this genome modification, we had to chooseand use most diverse biological models to confirm theexceptional role of DNA methylation in the life oforganisms. Initially, we proceeded from the principlethat, if DNA methylation has any biological functions,it most likely cannot be indifferent to these functionsand should at least be more or less specifically modi�fied during their induction. Thus, we came to induc�ible models, such as hydrocortisone–liver and training(memory)–neuron. And indeed, it was found that, afterthe administration of hydrocortisone to animals, theDNA methylation pattern in their liver drasticallychanges, which is associated with the induction of vari�ous genes in it. We have also found that learning changesthe DNA methylation pattern in neurons but not inother brain cells. The identified changes in the DNAcaused by learning are one of the first indications thatthe genome is involved in memory formation.

In plants, DNA methylation dramatically changesduring germination and transition to flowering, as wellas upon infection with various fungi and viruses andinvasion with parasitic plants. It became clear thatinfectious agents can subtly affect the host plant, sub�



jecting it to their whims by modulating the host DNAmethylation.

In 1977, Russian researchers compared DNAmethylation patterns in the blood cells of normal cowsand cows with lymphocytic leukemia. In general, thelevel of DNA methylation in the animals with this typeof blood cancer was lower. This was one of the first evi�dence that DNA methylation is involved in this dis�ease, either as its cause or as its effect (Cooney, 1999).Indeed, the genome methylation pattern in bovineperipheral blood lymphocytes dramatically changes inchronic lymphocytic leukemia. In the background of avery high DNA methyltransferase activity, the degreeof total DNA methylation in cancerous (leukemia)animal cells is considerably lower, whereas the degreeof methylation of palindromic sequences, conversely,is much higher than in the normal cells. It was foundthat the nuclei of blood lymphocytes of leukemic cowscontain at least two DNA methylase activities, one ofwhich greatly differed in the site specificity of theaction from the DNA methylase activity from the cellsof healthy cows. All this has allowed us to be amongthe first to reasonably postulate that the disturbance ofDNA methylation is a pathway to cancer. Today, thispostulate is acknowledged to be true, which was con�firmed and developed by America researchers S. Beilin,R. Enish, P. Jones, P. Pfayfer, and M. Ehrlich, as wellas by Ya.I. Bur’yanov, F.L. Kiselev, and many others,and information about the gene methylation patternhas become an early diagnostic sign of cancer.

In plants, DNA methylation is regulated by differ�ent phytohormones and specific of plant growth regu�lators. It was shown that, under the influence of differ�ent phytohormones, global DNA methylation in thecell cycle in plants is significantly reduced (Vanyushin,2009). Furthermore, phytohormones inhibit methyla�tion of the newly synthesized DNA strands withoutaffecting the methylation of Okazaki fragments (Table 3).For example, it was shown for the first time that phy�tohormones influence the genome of plants by modu�lating its methylation. Moreover, we believe that themodulation of DNA methylation is one of the keyaspects of the effect of hormones in plants and ani�mals. It cannot be ruled out that hormone–receptorcomplexes may compete for binding sites and genomemethylation with the respective DNA methyltrans�ferases.

We have always regarded DNA methylation as a asa pathway of negative or positive control of gene activ�ity. In most cases, DNA methylation inactivates genes;however, there are already examples that methylationof individual genes induces their activity both inmicrobes (gene mom) and plants. For example, meth�ylation of the pMADS3 gene of petunia stimulates itsexpression. The mechanisms of this stimulation ofgene expression are unknown. It is assumed thatmethylation of the CG site apparently prevents thesite�specific binding of a certain repressor to a silencer,and thereby stimulates gene expression. Methylation

of 12 CG sites in the triple tandem repeat in the Arabi�dopsis gene PHERES1 is a prerequisite for the paternalallele expression. It is assumed that the maternal allelesilencing with the PHERES1 gene in the central cell ofthe female gametophyte is performed through deme�thylation catalyzed by glycosidase DME. These areonly a few examples illustrating that DNA methyla�tion has a positive effect on the transcription of genesin plants. Usually, the suppression of DNA methyla�tion in mutants defective in genome methylation orcaused by DNA methyltransferase inhibitors isaccompanied by heritable phenotypic changesinduced by ectopic reactivation of various silent genes.

Methylation of cytosine residues in plant DNA isinvolved in the silencing of repetitive transgenes(Matzke, M.A. and Matzke, A.J.M., 1995) and vari�ous transposable elements. This allows such DNAmethylation to be considered as a mechanism forselective inactivation of embedded foreign genes intothe genome, including viral elements of differentnature.

DNA methylation inhibitor 5�azacytidine sup�presses the formation of adventitious shoots in petu�nia, whereas the methylation of cytosine residues inCCGG and CGCG sites in MADS�box and CDC48genes is positively correlated to the induction ofadventitious shoots. The treatment of plants with5�azacytidine causes heritable dwarfism in rice andincreases protein content in wheat grains (Vanyushinet al., 2009). In transgenic rice plants, the bar geneexpression induced by 5�azacytidine disappears inapproximately 50 days. This means that plants in timecan more or less efficiently restore the original methy�lation status of their genome, disturbed by this chemi�cal DNA�demethylating agent. A similar picture wasobserved by us in wheat, in which 5�azacytidine�induced increase in protein content of seed wasretained only in several generations.

DNA methylation controls the flowering of plants(Finnegan et al., 1995). For example, the treatmentwith 5�azacytidine, as well as an antisense MET1 geneinactivation, makes the vernalization of cold�sensitiveArabidopsis plants unnecessary. DNA methylationregulates the expression of the flowering repressor

Table 3. Degree of methylation of newly synthesized DNAin tobacco cell suspension culture and mouse L�cells(Kirnos et al., 1993)

Cells and culturing conditions

100 × m5C/ C + m5C

Replicative DNA fragments

≤5S ≥5S

Tobacco cells 17.0 ± 0.4 40.2 ± 0.3

Tobacco cells + 2,4�D (5 mg/L) 20.2 ± 0.6 20.1 ± 0.5

Mousr L�cells 2.8 ± 0.2 4.2 ± 0.1

180


VANYUSHIN

FLC. In fact, cold treatment (stress) leads to a partialdemethylation of DNA in many plants, which is mostlikely associated with the cold induction of specificproteins. It was found that DNA of winter wheat vari�eties is methylated more heavily than the DNA ofspring wheat varieties.

DNA methylation is a mechanism of genomicimprinting and regulation of the entire developmentprogram. In the maternal inheritance type, certainDNA regions in the endosperm are hypomethylated,whereas in the paternal inheritance type their degreeof methylation was the same as in the embryo andleaves. The well�known somaclonal variability in plantcell and tissue cultures is caused not only by mutationsbut also by epimutations, including the significantchanges in the DNA methylation profile.

DNA methylation can be significantly modulatedby various biological (viruses, bacteria, fungi, and par�asitic plants) and abiotic (stress) factors. Interestingly,the increased level of radiation as a result of the Cher�nobyl disaster drastically increased the global genomemethylation in many plants. The DNA methylationprofile may vary considerably under the influence ofthe environment. Usually it differs in the stress�toler�ant and stress�sensitive plants. For example, the DNAof salt�tolerant mangrove trees growing in salt marshesis hypomethylated compared to the DNA of thesetrees growing in freshwater river areas. Thus, the epi�genetic variability in natural plant populations isapparently important for the adaptation of plants toenvironmental conditions.

The damage of cotton plants caused by wilting isaccompanied with a distortion of the methylation pat�ter of repetitive, but not unique sequences in the plantgenome. The methylation pattern of the total DNA inthe host plant (alfalfa) damaged with the parasite Cus�cuta sp. is considerably modified. Thus, fungi, viruses,and other infectious agents can switch the work pro�gram of the host genes in their favor by modulatingDNA methylation. On the other hand, plants are ableto modify viral DNAs that are not integrated into thehost genome. For example, the unencapsulated DNAof the cauliflower mosaic virus in turnip leaves rapidlybecomes methylated at all HpaII/MspI sites.

Correct methylation may stabilize free foreignDNA in the host plant cells. The study of the transfor�mation of barley cells showed that the viral DNA withcompletely methylated CG sites was most stable,whereas the same DNA methylated only at the ade�nine residues rapidly degraded. Thus, barley cells def�initely have a certain system for recognition of incor�rectly methylated DNAs, which ensures their rapidremoval from proliferating cells. These intriguing datamay indicate the existence of host restriction–modifi�cation in plants. This assumption agrees with our dataon the identification of specific endonucleases recog�nizing the DNA methylation status in plants (Fedo�reyeva et al., 2007).

DNA methylation in plants and animals has muchin common; however, in plants it has a number of spe�cific features. For example, the proportion of methy�lated CNG and asymmetrical DNA sequences in theplant genomes is much higher than in animals. In gen�eral, plants have a much more complex genome meth�ylation system compared to animals. First of all, itshould be noted that plants have a much larger spec�trum of DNA methyltransferases (more than 12). Thismay provide them with a more robust genome modifi�cation system compared to animals. In plants, even tri�ple null mutants (for genes of three different DNAmethyltransferases) can survive, whereas in animals theknockout of a gene encoding even one DNA methyl�transferase is lethal. Some plant DNA methyltrans�ferases have no analogs in the animal world. They areunique and, unlike the animal DNA methyltrans�ferases, contain a conserved ubiquitin�binding domain,and their ubiquitination can affect the enzyme locationin the cell depending on various extracellular signals,cell cycle, and transposon or retrovirus activities.Interestingly, the activity of plant DNA methyltrans�ferases can directly depend on plant growth regulators.In addition, unlike animals, plants have specificorganelles—plastids (chloroplasts, chromoplasts,amyloplasts, and leucoplasts), which have own DNAmodification (methylation) systems distinct from thenuclear ones. These systems can play an importantrole in the differentiation and function of plastids.DNA methylation in plant mitochondria differs fromthat in the nucleus. It was shown that plant mtDNAscontain N6�methyladenine rather that 5�methylcy�tosine, which is characteristic of the animal mtDNAs.Therefore, in general, the DNA modification systemsin the cytoplasmic organelles in animal and plant cellsare quite different. In contrast to animals, plantsapparently have a genome restriction�modificationsystem; at least, we have found that plants have S�ade�nosylmethionine�dependent endonucleases that aresensitive to the DNA methylation status. Based onthese characteristics, the plant endonuclease foundand studied by us are to a certain extent similar to thetypical bacterial restriction endonucleases.

Plants are unique systems or model organisms thatprovide unusual and diverse opportunities for inter�preting and understanding the intimate mechanismsand functional role of DNA methylation and genomefunctioning in eukaryotes. Each time, when analyzingthe level and pattern of genome methylation, includ�ing the methylome, it should be remembered that thisis only usually a snapshot rather than a comprehensivepicture of the genome modification in ontogeny. Infact, this is a very dynamic process resulting DNAmethylation and demethylation.

There is no doubt that DNA methylation is associ�ated with the evolution and taxonomy of organisms.We have long pointed out that, in general, DNAs ofarchegonial plants and gymnosperms are methylatedto a lesser extent than the DNAs of angiosperms. The



analysis of the genome methylation profiles in 30 gen�erations of ten A. thaliana lines derived from the sameprecursor showed that approximately 30000 cytosineresidues in the DNA of these strains (lines) are meth�ylated differently; i.e., the epigenomes of these plantsare very different and peculiar. This particularlyapplies to the methylation pattern of transposons andelements coding small interfering RNAs.

When speaking about this genome modification, itshould be borne in mind that, in fact, we are dealingwith at least three components of this complex reac�tion or even a system: the reaction substrate as such(DNA), the enzyme (DNA methyltransferase), andthe donor of methyl groups (S�adenosylmethionine).Of course, the control of DNA modification and theefficiency of this process is exercised at the level of allthese components and with the involvement of theother most diverse cell metabolism components.Along with this, the methylation status of the genomein a differentiated cell at a certain stage of ontogenyalso depends on the activity of DNA demethylationenzymes. One of these animal DNA demethylases,which immediately cleaves the methyl group from the5�methylcytosine residues in DNA, was discoveredrecently; it was isolated as an individual protein, andits gene was cloned. Thus, the degree and pattern ofgenome methylation may actually represent certaindynamic traits that at every moment are largely deter�mined by the ratio of activities of DNA methylatingand demethylating enzymes.

However, even in the presence of active enzymesand a sufficient amount of S�adenosylmethionine(donor of methyl groups and modulator of enzymeactivities) and in the absence of respective inhibitors,these reactions in the nucleus are often impossiblebecause the substrate (DNA) in chromatin is inacces�sible for enzymes. Here, the most important factor isthe chromatin organization as such. In addition to theaforementioned numerous histone modifications,which markedly modulate chromatin organizationand DNA accessibility for enzymes, many other pro�teins compete for the binding and interaction of DNAmethyltransferases with DNA. In particular, these arethe proteins of hormone–receptor complexes. This,apparently, largely explains the discovered regulationof DNA methylation in plants and animals by hor�mones and the effects of hormones in the cell. Anyway,further progress in the genome methylation researchstrongly depends on a detailed study of the fine chro�matin structure and its various functional fluctuationsin the nucleus.

Thus, cytosine DNA methylation, indeed, controlsthe growth and development of plants (Vanyushin,2006) and animals (Holliday and Pugh, 1975). It isinvolved in the regulation of all genetic processes,including transcription, replication, DNA repair, celldifferentiation, genomic imprinting, and gene trans�position.

REPLICATIVE DNA METHYLATION AND INHERITANCE OF GENOME

METHYLATION PATTERN

We have long been interested in the question ofwhen and to what extent plant and animal DNAs aremethylated in the cell cycle. It is known that one of theDNA strands during the replication of double�stranded DNA is synthesized continuously, whereasthe synthesis of the other strand proceeds intermit�tently, with the formation of relatively short fragments(Okazaki fragments), which are then linked into onecontinuous strand (Fig. 8). We decided to isolate thesefragments upon the synthesis (replication) of DNAand determine whether or not they are methylated. Itappeared that, when plant and animal cells are grownin a medium at a high concentration of cells, DNAsynthesis in them is limited to the formation of shortintact fragments without their ligation. We isolatedthese fragments in appreciable quantities and studiedtheir methylation. They represented the Okazaki frag�ments, which were ligated in a Hershey�Chase exper�iment to form long normal DNA strands. It appearedthat the Okazaki fragments are methylated (Table 3,Fig. 8). This is how the replicative DNA methylationin plant and animals was discovered and documented.It was assumed that DNA methyltransferases may beincluded in the replication complex (Aleksandrush�kina et al., 1991; Kirnos et al., 1986, 1993).

In the degree and specificity of methylation, theOkazaki fragments formed in vivo in wheat seedlingsdiffered from the ligated replication intermediates ofmature DNA (Table 3). In contrast to the methylationof ligated DNA, the methylation of Okazaki fragments

3'5'

5'5'

5'

5'

5'

5'

5'

5'

3' 3'3'

3'

3'

3' 3' 3'

3'

Leading strand

Lagging strand

Newly

Newly methylated site

Initial methylated site

with Okazakifragments

synthesizedstrand

Fig. 8. Replicative DNA methylation.

182


VANYUSHIN

is resistant to various methylation inhibitors (S�isobu�tyladenosine, etc.) and is not inhibited by hormones(auxins in plants). We came to the conclusion thatthere are several DNA methyltransferases in thenucleus and that DNA methylation at different repli�cation stages can be catalyzed by enzymes with differ�ent specificity of action. This is entirely consistentwith the modern information about the plurality ofnuclear DNA methyltransferases in plants and ani�mals. Then, we have discriminated between the repli�cative and postreplicative DNA methylation in plants.These processes differ in the specificity of methylatedsequences in DNA and in their sensitivity to differenthormones and inhibitors.

We proposed and described the mechanism of nat�ural regulation of DNA replication through methyla�tion (Fig. 9). This was made possible due to the amaz�ing gift of nature—the existence of the synchronousdevelopment of cereals. It appeared that, during thedevelopment of wheat seedlings under standardizedconditions, a natural pronounced synchronous andperiodic synthesis (replication) of DNA proceeds intheir first leaf and coleoptile (Kirnos et al., 1993).Such a degree of synchronization of cell division can�not be obtained even in bacteria, not to mention theanimal and plant cell cultures, including those grownwith the use of most modern methods of synchroniza�tion. In the leaves of seedlings, we have identified atleast five distinct peaks of synthesis (replication) ofnuclear DNA. Thus, in terms of DNA, a whole intactplant organ (leaf) can be considered as a single cell.Moreover, unlike the artificial cell culture suspen�sions, all signaling systems of the organism function inthis intact organ on the plant. We only had to isolateDNA from the first leaves at the beginning, during,and after replication and determine how their individ�ual strands are methylated. For this purpose, we grewseedlings in the presence of 5�bromouracil to make thenewly synthesized DNA strands heavier, isolatedDNA, and divided the light original parental strandand the heavy DNA strand newly synthesized in the

cell cycle. First, it was found that, as a result of repli�cation, hemimethylated DNA duplexes are formed inplants, and the DNA molecules are strongly asymmet�rical in the degree of methylation of the complemen�tary strands. This asymmetry of strands decreasestowards the end of the cell cycle, and before a new rep�lication the hemimethylated sites become fully meth�ylated again. Replication of hemimethylated DNA incells is apparently prohibited, since it would lead to theloss of the epigenetic signal. Much later than in ourwork, it was shown that such regulation of replicationis realized through the dam�methylation in bacteria.Now it is more or less clear how this signal (DNAmethylation pattern) is inherited.

As noted above, replication is accompanied by theoccurrence of hemimethylated DNAs; apparently, insuch a state, most of the genes in the interphasenucleus seem to work. Before the next round ofgenome replication and cell division, DNA methyl�transferase of the maintenance type, in particular dmt1(they maintain the ancestral genome methylation sta�tus), methylate the hemimethylated sites to form fullymethylated sites. Methylated cytosine in one strandserves as a signal for the methylation by this enzyme ofthe cytosine residue in the opposite complementarystrand. During this process, genes are inactivated and,in principle, are ready to replicate when the cell com�pletes its cycle (Fig. 9). Thus, the DNA methylationpattern is inherited. For example, it was found that thetreatment of wheat plants with the DNA methylationinhibitor 5�azacytidine causes a strong (sometimes morethan 30%) increase in the protein content in grains,which is inherited in several generations (Fig. 10). Usu�ally storage proteins genes are strongly repressed, andthe storage protein contained in the grain is sufficientfor germination and initial development of the plant;an excess of the protein is not required. In the presenceof 5�azacytidine, DNA in plants highly demethylated,and the expression of the genes encoding storage pro�teins in the grain increases. This fact can be used inbiotechnology.

Cycle I Cycle IIReplication

allowed proceeds prohibited

Methylated siteUnmethylated site

allowed proceeds prohibitedprohibited

allowed

Fig. 9. Regulation of DNA replication by methylation in the cell cycle (Vanyushin, 2009).



Hence, the targeted regulation of gene expressionin plants can serve as a very effective biotechnologicaltool. Here, the wonderful words of Jonathan Swift,written in 1726, come to mind: “… whoever couldmake two ears of corn, or two blades of grass, to growupon a spot of ground where only one grew before,would deserve better of mankind, and do more essen�tial service to his country, than the whole race of poli�ticians put together” (Jonathan Swift. The King ofBrobdingnag to Gulliver, in Gullivers Travels, “A Voy�age to Brobdingnag”). These are truly just words,which should be grasped by our politicians. They con�tain a noble task addressed to us from time immemo�rial. Maybe, nowadays it also can be further solved in asomewhat different way (for example, by at least dou�bling the protein content in the grains). Bread is valu�able for the protein, whereas starch can be obtainedfrom potato. Our modest experience shows that it ispossible to achieve this, in particular with the use ofthe knowledge of epigenetics.

Genome demethylation. We have already noted thatDNA replication is accompanied by the occurrence ofunmethylated sites in the newly formed strand in theDNA duplex. It is often erroneously referred to as pas�sive DNA demethylation. Actually, this is either unm�ethylation or hypomethylation of DNA, occurred dur�ing replication as a result of a certain blockade of themaintenance DNA methyltransferase DMT1. Theissue about DNA demethylation has long remainedcontroversial, although all the time there appearedirrefutable facts that it does exists, particularly inembryogenesis. In principle, the 5�methylcytosineresidues can be cleaved from DNA and then replacedwith the cytosine residues during subsequent repair.According to M. Schiff, a direct cleavage of the methylgroup from the 5�methylcytosine residues with its oxi�dation to methanol is also possible.

Plants have the DEMETER (DME) gene, which isexpressed in the female gametophyte with the induc�tion of the maternal alleles with the imprinted geneMEDEA. DME encodes DNA glycosidase, a lyase thatactivates the MEA gene by excising certain 5�methyl�cytosine residues in MEA in two compact regionsupstream and downstream of the coding sequence.The paternal allele is not affected because DME isexpressed in the central cell only before fertilization.Another m5C�specific DNA glycosidase, ROS1(repressor of silencing 1), was detected in the trans�genic Arabidopsis plants mutant for ROS1. Glycosi�dases DME and ROS1 preferentially excise the 5�methylcytosine residues from the m5CG sites. Theycan also excise the thymine residues from the TG mis�matches. Two other proteins from the DME family,DEMETER�LIKE 2 and 3 (DML2 and DML3), arealso m5C�specific DNA glycosidases, which arerequired to control the correct methylation of variousgenes. During fertilization, one sperm fertilizes thehaploid egg to form a diploid embryo, and the otherenters the diploid central cell, which gives rise to the

triploid endosperm. Before fertilization or at the earlystages of development of the endosperm, the DNA init is subject to strong demethylation at many and mostdiverse sites. The majority of m5CG sites are demeth�ylated by the maternal DME. The simultaneousremoval of both m5C residues from a symmetricallymethylated site can lead to a fatal rupture of the dou�ble�stranded DNA helix. At the terminal stages ofdevelopment of the female gametophyte, the MET1gene expression is strongly suppressed before the lastsyncytial division and is very weak in the central cell.Maybe, this is a neat beneficial evolutionary adapta�tion in plants. The last round of DNA replication inthe absence of MET1 activity should lead to theoccurrence of DNA with hemimethylated sites, whichthen can be demethylated by DME without fatal dou�ble�stranded DNA breaks.

The question is often asked as to what is better—when DNA is strongly or weakly methylated? Myanswer is neither one nor the other. It should be meth�ylated normally, as a normal cell can do and maintainit with all the strengths and possibilities of a normalcell, following the principle of maintaining homeosta�sis. Indeed, when my colleague L. Poirier from theU.S. National Poison Control Centre excludes theamino acid methionine (source of methyl groups)

Wheat grain protein

C

5�aza Cytidine

N

NH

N

NH2

O

34

5672

Fig. 10. Increase in the protein content in wheat grainsunder the influence of DNA methylation inhibitor 5�aza�cytidine. C—control. Other lanes show the protein iso�lated from grains of plants treated with 5�azacytidine indifferent ways.

184


VANYUSHIN

from the diet, all experimental rats inevitably developliver cancer (hepatoma) within two weeks. Cancer alsodevelops in transgenic mice with an activated geneencoding human DNA methyltransferase, which leadsto the hypermethylation of their genome. As a result ofknocking out only one of the genes encoding DNAmethyltransferases, the embryo development in ani�mals stops and the programmed cell death (apoptosis)is triggered. Changes in DNA methylation should beregarded with caution.

Be that as it may, the skeptics who were mentionedearlier in this article are defeated, and today it isknown for sure that DNA methylation in the cell is animportant issue: it controls all genetic processes,including transcription, replication, recombination,gene transposition, repair, and X chromosome inacti�vation (sexual differentiation). It is not surprising thattoday the study of this relatively small enzymaticgenome modification arouses great interest of manyresearchers around the world.

DNA METHYLATION AND OTHER EPIGENETIC SIGNALS

The discovery and description of the crucial role ofDNA methylation in the life of organisms entailed theformation and materialization of epigenetics. For along time epigenetics was not recognized, and oftenresearchers shamefully or even deliberately kept silentabout it. This is largely because the nature of epige�netic signals and the ways of their realization in theorganism in the body were very vague. Now it hasbecome clear that one of these epigenetic signals in thecell is the enzymatic modification (methylation) of thegenetic matrix itself (Vanyushin, 2006).

There are also other systems of epigenetic signals inthe cell; they are numerous and highly diverse. Insome of them, the crucial role is played by proteins(including the chromatin proteins) rather than byDNA. Today, the histone code is widely discussed.This code significantly expands the potential of thegenetic code of DNA. Various histone modificationschange the chromatin structure, which causes herita�ble changes in gene transcription. The back and forthhistone modification (methylation, phosphorylation,acetylation, and ubiquitination with or without theprefix “de”) often determines whether genes are activeor not. Histone modifications are complemented by

numerous specific nonhistone regulatory proteinsforming intricate complexes on DNA, which silencegenes or, conversely, trigger their functioning. We arenow close to understanding these curious signals.

There is already ample evidence that there is a rela�tionship between DNA methylation and histone mod�ification. In Neurospora, the methylation of lysine 9 inhistone H3 is critical for the cytosine methylation ofDNA and normal fungus development. In otherwords, histones can function as signal transducers forthe genome methylation. On the other hand, in Arabi�dopsis thaliana, the CpG methylation in DNA directsand precedes the methylation of lysine 9 in histoneH3. Interestingly, the histone H3 tail nonmethylatedon lysine 4 (K4) serves as an allosteric activator ofDNA methyltransferase Dnmt3a. The stage of inter�action of the unmethylated histone tail is a peculiarcheckpoint for the methylation of DNA by thisenzyme.

In animals and plants, there is a relationshipbetween the DNA methylation and histone deacetyla�tion. For example, the histone deacetylase gene isrequired for the DNA methylation induced by smallRNAs (dsRNAs). Indeed, “methylation meets acety�lation.” On the other hand, it is known that knockingout the histone demethylase gene inhibits de novoDNA methylation and distorts mouse embryo devel�opment. Without demethylation of the methyllysineresidue K4 in histone H3, de novo DNA methylationby DNA methyltransferase dmt3a is not observed inmice.

RNA�DIRECTED DNA METHYLATIONAND GENE SILENCING

Of particular interest today is the study of the mecha�nisms and biological role of DNA methylation directedby small RNAs (RNA�directed DNA methylation),which trigger specific gene silencing (Matzke, M.A. andMatzke, A.J., 1995).

It is believed that site�specific DNA methyltrans�ferases in the presence of a small signal RNA (Fig. 12)perform de novo DNA methylation on CNG andother sites in the DNA nucleotide sequence that is rec�ognized by the small RNA; this methylation mobilizescorresponding enzymes that, in particular, modify his�tones.

Histone modification results in the induction orpotentiation of methylation of CNG sites, which isthen maintained without the RNA trigger (Fig. 11). Inthe chain of these events, DNA methylation may beboth the cause and effect of the “silencing” of thegenes. It is not surprising that particular progress in thefield of RNA�directed gene methylation was madethrough the study of DNA methylation in plants ratherthan in animals. This can be largely explained by thefact that plants are characterized by the well�expressedmethylation of CNG and asymmetrical sites in DNA,which is largely involved in the RNA�directed genome

K9Me Ac

Inactive chromatin

Me Me Me

Active chromatin

Acetylase

Fig. 11. Chromatin activation as a result of DNA demeth�ylation and histone acetylation.



methylation. Recently, there are many indications thatsuch RNA�directed DNA methylation in the RNA–DNA triplex plays a crucial role in silencing genes withsmall RNAs; in the organisms or mutants with defec�tive cytosine DNA methylation, RNA silencing ofgenes is ineffective.

The discovery of specific small RNAs (siRNAs,miRNAs) has sharply increased the understanding ofthe molecular mechanisms of gene expression regula�tion and played a crucial role in the recognition andstrengthening of epigenetics. Typically, these RNAsare encoded in inverted repetitive genome sequences,as a result of reading and subsequent processing ofwhich they appear as short 12–14�nt oligonucleotides.These regulatory fragments were first detected in petu�nia; it was shown that they control the expression ofgenes responsible for the synthesis of pigments anddetermine the nature of the flower color. They recog�nize the corresponding complementary sequences inDNA or mRNA, contact them, and thereby can blockthe expression of the respective genes at the transcrip�tional and/or translational level. It is believed that themajority of such palindromic repeats in the eukaryoticgenome has a viral origin and, in principle, can protectcells from viral genetic invasions.

CONCLUSIONS

One of the fundamental epigenetic signals in a cellis DNA methylation. In higher plants, DNA is heavilymethylated at the cytosine residues; 5�methylcytosineis located primarily in the CG and CNG sequences.Global DNA methylation is species�, tissue�, andorgan�specific; it changes during seed germination,upon the transition of plants to flowering, and in vari�ous viral and fungal infections and decreases with age.Usually, the DNA of archegonial plants is methylatedless heavily that the DNA of flowering plants. Species�specific differences of phylogenetic significance withrespect to the frequency of the occurrence of methy�

lated CNG sequences in plant genomes were found.The tissue specificity of DNA methylation was firstidentified in animals (Vanyushin et al., 1970) and thenin plants. Different genes in different tissues of thesame organism are methylated differently. The contentof m5C in DNA of different tissues is also associatedwith the so�called flowering gradient. The first map�ping of the methylation pattern of the whole genome(methylome) of Arabidopsis thaliana has shown thatDNA of the pericentromeric heterochromatin, repet�itive sequences, and zones of formation of small inter�fering RNAs are heavily methylated; in the transcribedregions, approximately one�third of genes are methy�lated; and in the promoter region only approximately5% of genes are methylated.

The methylated genes in the transcribed regions arecharacterized by constitutive transcriptional activity,whereas the methylated genes in the promoter regionsare characterized by a strong tissue specificity ofexpression. For example, we have found that thedegree of methylation of the patatine gene promoter invarious tissues of potato was inversely correlated to thedegree of expression of this gene: it is noticeablyhypomethylated in potato tubers, where this protein ismainly synthesized.

Specific changes in the DNA methylation patternoccur throughout the life of the plant, from seed ger�mination until its death, either programmed orinduced by different agents, as well as biotic and abi�otic factors. The ontogeny of plants and animals, ingeneral, is not possible without genome methylation,because DNA methylation is involved in controllingall genetic functions, including transcription, replica�tion, DNA repair, gene transposition, genomicimprinting, and cell differentiation. Cold�inducedflowering of plants is regulated by DNA methylation.During cold treatment (vernalization), DNA is par�tially demethylated, which leads to the activation ofgenes responsible for the induction of flowering.

CG CNG CNN dsRNA

Inverted DNA repeats

or

RDR2

DCL3

DRD1

MET1 DRM2 AGO4

m

De novo methylation(RNA�dependent)

CG CNG CNNm m

Fig. 12. Gene silencing by small interfering RNAs (siRNAs).

186


VANYUSHIN

Genome methylation is involved in gene silencingwith small RNAs (siRNAs). Knockouts and knock�downs of genes encoding respective DNA�methyl�transferases are accompanied by serious changes in thephenotypic properties and disturbed growth anddevelopment. 5�Azacytidine�induced DNA demethy�lation during the formation of grains causes a heritableincrease in the protein content of wheat. Thus, theselective regulation of DNA methylation is a neweffective tool for biotechnologically controlling plantproductivity.

We found that the replication of the genome isaccompanied by the occurrence of hemimethylatedsites in DNA; the resulting asymmetry of methylationof DNA strands significantly decreases or disappearsby the end of the cell cycle. Based on this, we proposeda model for the regulation of DNA replication bymethylation.

We found that the primary replicative genomic ele�ments (Okazaki fragments) are methylated. Thus, theactual replicative DNA methylation in plants and ani�mals was discovered and documented, and it wasshown that DNA methyltransferases may be compo�nents of the replication complex. We came to the con�clusion that there are several DNA methyltransferasesin the nucleus and that DNA methylation at differentstages of replication can be catalyzed by enzymes withdifferent specificity of action. This assumption wasconfirmed by the recent data on the multiplicity ofnuclear DNA methyltransferases. We managed to dis�criminate between the replicative and postreplicativeDNA methylation in plants. These processes differ inthe specificity of methylated sites in DNA and by theirsensitivity to different hormones and methylationinhibitors.

It was found that DNA methylation is controlledby phytohormones. Phytohormones significantlyreduce the global DNA. Furthermore, they inhibit themethylation of the newly synthesized DNA strandswithout affecting the methylation of the Okazaki frag�ments. Thus, it was shown for the first time that phy�tohormones affect the genome itself by modulating itsmethylation. We believe that the modulation of DNAmethylation is one of the key mechanisms of theaction of hormones in plants and animals. It cannot beruled out that the hormone–receptor complexes com�pete for the binding and genome methylation siteswith the corresponding DNA methyltransferase.

There is no doubt that DNA methylation is associ�ated with the evolution and taxonomy of plants. Wehave long pointed out that, in general, DNA of arche�gonial plants and gymnosperms is methylated to alesser extent than the DNA of angiosperms. The anal�ysis of genome methylation profiles of 30 generationsof ten A. thaliana lines derived from the same precur�sor showed that approximately 30000 cytosine resi�dues in the DNA of these strains (lines) are methylateddifferently; i.e., the epigenomes of these plants arevery different and peculiar. In particular, this applies to

the methylation patterns of transposons and the ele�ments encoding small interfering RNAs.

Speaking of this genome modification, we shouldbe aware that we are dealing with at least three compo�nents of this complex reaction or even a system: thereaction substrate as such (DNA), the enzyme (DNAmethyltransferase), and the donor of methyl groups(S�adenosylmethionine). Of course, the control ofDNA modification and the efficiency of this processare exercised at the level of all these components andwith the involvement of other most diverse cell metab�olism components. Along with this, the methylationstatus of the genome in a differentiated cell at a certainstage of ontogeny also depends on the activity of DNAdemethylation enzymes. One of these animal DNAdemethylases, which immediately cleaves the methylgroup from the 5�methylcytosine residues in DNA,was discovered recently; it was isolated as an individualprotein, and its gene was cloned. Thus, the degree andpattern of genome methylation may actually representcertain dynamic traits that at every moment are largelydetermined by the ratio of activities of DNA methylat�ing and demethylating enzymes.

However, even in the presence of active enzymesand a sufficient amount of S�adenosylmethionine(donor of methyl groups and modulator of enzymeactivities) and in the absence of the respective inhibi�tors, these reactions in the nucleus are often not possi�ble because the substrate (DNA) in chromatin is inac�cessible to enzymes. Here, the most important factoris the chromatin organization. In addition to theaforementioned numerous histone modifications,which markedly modulate chromatin organizationand DNA accessibility for enzymes, many other pro�teins compete for the binding and interaction of DNAmethyltransferases with DNA. In particular, these arethe proteins of hormone–receptor complexes. This,apparently, largely explains the discovered regulationof DNA methylation in plants and animals by hor�mones and the effects of hormones in the cell. Anyway,further progress in the genome methylation researchstrongly depends on a detailed study of the fine chro�matin structure and its various functional fluctuationsin the nucleus.

Adenine methylation in plants was discovered;N6�methyladenine (m6A) was found in the mitochon�drial and nuclear DNA. In Arabidopsis thaliana, thegene encoding cytosine DNA methyltransferase timesDRM2 is methylated both at cytosine (CCGG) andadenine (GATC) residues. The induction of the anti�sense constructs of the cytosine DNA methyltrans�ferase (METI) in transgenic Arabidopsis plantschanges the methylation pattern of adenine residues inthe DRM2 gene. Therefore, plants have an interdepen�dent control between the methylation of adenine andcytosine bases. This is a new mechanism of the fineepigenetic control of the genome functions in eukary�otes through coupled DNA methylations.



The first adenine DNA methyltransferase of highereukaryotes was isolated from the mitochondrion�richfraction of vacuolar vesicles occurring during apoptosisin aging wheat coleoptiles. In the presence of S�adeno�syl�L�methionine (SAM), this Ca2+/Mg2+�dependentenzyme (wadmtase) de novo methylates the adenineresidue in the middle of the TGATCA sequence in sin�gle� and double�stranded DNA, preferring the single�stranded structures. Apparently, wadmtase modifiesmtDNA and is involved in the regulation of mitochon�drial replication; possibly; it also methylates thenuclear DNA.

SAM�dependent endonucleases WEN1 andWEN2, sensitive to the DNA methylation status, wereisolated from the wheat coleoptiles. Earlier, theseproperties have not been known for the endonucleasesof higher eukaryotes and were characteristic of onlysome bacterial restriction endonucleases, which isindicative of the possible existence of a restriction�modification system in plants. Competitive inhibitorsof DNA methylation S�adenosyl�L�homocysteineand S�isobutyladenosine also modulate DNA hydrol�ysis by these enzymes. Thus, a new type of regulationof the activity of eukaryotic (plant) endonucleasesbased on their modulation by the methyl group donorSAM and its analogs, methylation reaction inhibitors,was discovered. Apparently, enzymes WEN1 andWEN2 are involved in the degradation of nuclearDNA during the programmed death of the plant cell.In some properties, endonuclease WEN1 resemblesthe animal endonuclease G, which performs DNAfragmentation during apoptosis. Another uniqueendonuclease isolated from wheat coleoptiles, WEN2,similarly to WEN1, is also sensitive to the DNA meth�ylation status and is modulated by SAM. Plants have asystem of a coupled regulation of the SAM�dependentendonuclease activities (including those sensitive tothe DNA methylation status) with the oppositelydirected effect of the effector (modulator) SAM onthem. In addition, the action of these endonucleases ismodulated by histone H1, which can significantlyaffect the internucleosomal fragmentation of nuclearDNA by these enzymes during apoptosis.

The genome and histone modifications describedin this review and small interfering RNAs are very del�icate and effective natural mechanisms of regulation ofgene activity, status, replication, and repair of thegenome in the cell. Genome modifications arestrongly associated with other intricate epigenetic sig�nals and with the transformations in the structural andfunctional organization of the genome and the cell,which together determine the life, its core, and itsquality. It is crucial to note that the multiple epigeneticsystems and signals existing in the cell are often interde�pendent. In many respects, this has a certain securingsense to ensure a guaranteed reliability of the regulatorysignal. The specific chains, individual connections, andsophisticated network of these interdependent signalsare very far from a complete understanding. However,

there is no doubt that DNA methylation and histonemodifications, as well as selective gene silencing bysmall RNAs, play a very important role in the life ofthe cell and organism. Therefore, further exhaustivestudies in this exciting field of knowledge will be a veryimportant and fruitful task in this century, which isrightly called the era of epigenetics. Epigenetics playsthe crucial role in addressing many biological prob�lems. According to the biotechnology newsletter ofMassachusetts Institute of Technology (MIT, UnitedStates), epigenetics belongs to the top�ten new tech�nologies that in the next decade may turn the worldupside down. The development and improvement ofcellular technologies (stem cells), reliable diagnosis,prevention and treatment of various types of cancer,and the prevention of premature aging are not possiblewithout the knowledge of epigenetics. Epigeneticsunderlies the effective ways of combating many infec�tious (including viral) diseases of humans, animals,and plants. Undoubtedly, epigenetics will also serve toimprove the quality of yield of different crops and pro�ductivity of animal breeds. In other words, theprogress of biology, medicine, agriculture, and bio�technology is unthinkable without epigenetics.

REFERENCES

Aleksandrushkina, N.I., Rotaenko, E.P., Nikiforova, I.D.,et al., Two�stage methylation of wheat genome in cellculture and in seedling, Biokhimiya, 1991, vol. 56,pp. 361–368.

Ashapkin, V.V., Kutueva, L.I., and Vanyushin, B.F., Is thecytosine DNA methyltransferase gene MET1 regulatedby DNA methylation in Arabidopsis thaliana plants?,Russ. J. Genet., 2011, vol. 47, no. 3, pp. 279–289.

Berdyshev, G.D., Korotaev, G.K., Boyarskikh, G.V., andVanyushin, B.F., The nucleotide composition of DNAand RNA of somatic tissues of the pink salmon and itschange during spawning, Biokhimiya, 1967, vol. 32,pp. 988–993.

Epigenetika (Epigenetics), Ellis, S.D, Ed., Moscow: Tekh�nosfera, 2010.

Epigenetika (Epigenetics), Zakiyan, S.M, Vlasov, V.V, andDement’ev, E.V., Eds., Novosibirsk: Izd. SO RAN,2012.

Fedoreyeva, L.I. and Vanyushin, B.F., N6�Adenine DNA�methyltransferase in wheat seedlings, FEBS Lett., 2002,vol. 514, pp. 305–308.

Fedoreyeva, L.I., Sobolev, D.E., and Vanyushin, B.F.,Wheat endonuclease WEN1 dependent on S�adenosyl�L�methionine and sensitive to DNA methylation sta�tus, Epigenetics, 2007, vol. 2, pp. 50–53.

Finnegan, E.J., Genger, R.K., Kovac, K., et al., DNAmethylation and the promotion of flowering by vernal�ization, Proc. Natl. Acad. Sci. USA, 1998, vol. 95,pp. 5824–5829.

Holliday, R. and Pugh, J.E., DNA modification mecha�nisms and gene activity during development, Science,1975, vol. 187, pp. 226–232.

188


VANYUSHIN

Kirnos, M.D., Aleksandrushkina, N.I., and Vanyushin, B.F.,5�Methylcytosine in pyrimidine DNA sequences ofplants and animals: the specificity of methylation,Biokhimiya, 1981, vol. 46, pp. 1458–1474.

Kirnos, M.D., Aleksandrushkina, N.I., and Vanyushin, B.F.,Two�stage methylation of replicating genome in cells ofhigher plants, Usp. Biol. Khim., 1993, vol. 33, pp. 148–172.

Matzke, M.A. and Matzke, A.J.M., Homology�dependentgene silencing in transgenic plants: what does it reallytell us?, Trends Genet., 1995, vol. 11, pp. 1–3.

Sooney, C., Methyl Magic, Kansas City: McMeel, 1999. Spirin, A.S., Belozerskii, A.N., Shugaeva, N.V., and Vany�

ushin, B.F., Study of the species specificity of nucleicacids in bacteria, Biokhimiya, 1957, vol. 22, pp. 744–754.

Vanyushin, B.F., DNA methylation and epigenetics, Gene�tika, 2006, vol. 42, pp. 1–14.

Vanyushin, B.F., Methylation in plants: mechanisms andbiological role, in Timiryazevskie chteniya (Timiryazev

Memorial Lectures), Kuznetsov, V.V., Ed., Moscow:Nauka, 2009, vol. 69.

Vanyushin, B.F. and Ashapkin, V.V., DNA Methylation inPlants, New York: Nova Biomedical Books: Nova Sci�ence, 2009.

Vanyushin, B.F. and Ashapkin, V.V., DNA methylation inhigher plants: past, present and future, Biochim. Bio�phys. Acta, 2011, vol. 1809, pp. 360–368.

Vanyushin, B.F., Tkacheva, S.G., and Belozersky, A.N.,Rare bases in animal DNA, Nature, 1970, vol. 225,pp. 948–949.

Vanyushin, B.F., Nemirovsky, L.E., Klimenko, V.V., et al.,The 5�methylcytosine in DNA of rats. Tissue and agespecificity and the changes induced by hydrocortisoneand other agents, Gerontologia, 1973, vol. 19, pp. 138–152.

Translated by M. Batrukova

epigenetics today and tomorrow

Documents