Paper No. : 04 Genetic Engineering and Recombinant DNA Technology Module : 06 DNA Methyltransferase
Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director The Energy and Resouurces Institute (TERI), New Delhi Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry
Jamia Hamdard University, New Delhi
Paper Coordinator: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi
Content Writer: Dr. Bhaswati Banerjee, Assistant Professor, Gautam Buddha University, Greater Noida
Content Reviwer: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi
Description of Module
Subject Name Biotechnology
Paper Name Genetic Engineering and Recombinant DNA Technology Module Name/Title DNA Methyltransferase
Module Id 06
Pre-requisites DNA Methylation, Epigenetic regulation, Restriction endonuclease
Objectives Gain Structural and Functional insight into Eukaryotic & Prokaryotic DNMTs Keywords DNMTs, CpG Islands, Restriction-Modification system
INDEX
CONCEPT NOTE
LEARNING OBJECTIVES ABOUT THIS MODULE
I. INTRODUCTION
A. DNA Methyltransferase
B. Physiological Relevance: DNMT and DNA methylation:
C. CpG Islands
II. STRUCTURE AND FUNCTION OF DNA METHYLTRANSFERASES A. Eukaryotic DNA Methyltransferases
B. Prokaryotic DNA Methyltransferases III. APPLICATIONS
A. Research Applications B. Clinical Implications IV. SUMMARY
V. REFERNCES
ABOUT THIS MODULE
Anyone who is aware of the current research trends in regulation of gene expression and fathomless phenotypic diversity among all life forms, even the closely related ones, must be aware of the concept of “epigenetics”. DNA methyltransferases have been established as one of the key players of epigenetic regulatory mechanism of gene expression. The present module attempts to provide a comprehensive theoretical overview of most of the significant information that is available about the DNMT enzymes. The opening section of the module briefly introduces DNA methyltransferase and DNA methylation. The subsequent sections outline the physiological significance of DNA methyltransferase and focus on the context of the CpG island. Thereafter a detailed structural and functional account of DNMTs is discussed and the discussion segregates between the eukaryotic and prokaryotic DNMTs with special emphasis on unique features. To conclude, the module highlights the importance of mammalian DNMTs in pathological disorders and outlines the application of bacterial DNMTs in recombinant DNA technology
I. INTRODUCTION
A. DNA Methyltransferase
To begin with, DNA Methyltransferases, also known as DNMTs, are a type of DNA modifying enzymes belonging to the “Methyltransferase” class of enzymes. As the name suggests, the “DNA-Mtases” are a family of enzymes that catalyze the transference of the S-methyl group of the co-factor S-adenosyl-L-methionine (SAM) to the 5-Carbon or 4-Nitrogen of the deoxycytosine or to the 6-Nitrogen of deoxyadenine in the genomic DNA, thus converting it to 5-Methylcytosine or 4- Methylcytosine or 6-Methyladenine respectively. Each type of reaction is mediated by its specific enzyme (Maulik & Maulik 2010; Tollefsbol 2009)
The body of any eukaryotic metazoan organism is made up of multitude of cells of different types and origin. It is remarkable that each of these cells that constitute the body of a particular organism contain the exactly similar genome, yet they display wide range of structural and functional diversity. The cells in the body of an adult organism progress through different lines and stages of differentiation and maintain the differentiated states through generations. This is true for human body as well.
Human body is made up of four basic types of tissues formed of cells of corresponding origin, each containing the same genome, yet, each cell expresses only its characteristic set of proteins while consistently does not express certain other proteins. A cardiac cell continues to perform rhythmic beating throughout its life span and never produces digestive enzymes like the gastric cells; a neuron continues to produce or transmit neuro-chemical impulse and does not suddenly begin filtering toxins out of blood like a nephron.
Have you ever wondered what could be the reason that these different cells, although governed by the same genome, behave so different from each other? What factor(s) possibly decide which genes should be expressed in which cell and which genes should remain suppressed? Further, in a broader perspective, every human being has largely the same or similar genome in all the body cells, yet why is every
Fig. 1.1: Schematic representation of DNA Methylation Source: Protein methylation (Book, 1990) [WorldCat.org]
human being phenotypically unique although they are biologically similar? Well, the answer to all these questions lies not within the genome, rather just outside the genome. The key to all these queries lies in “epigenetics” which includes all those factors and phenomena that regulate, enhance or suppress gene expression without altering the genome or the underlying DNA sequence itself. The key players of epigenetics execute their effects through histone modifications, chromatin remodeling and DNA methylation (Tollefsbol 2009; Geiman & Robertson 2002;
Bhattacharjee et al. 2016).
B. Physiological Relevance: DNA-MTases and DNA methylation The groups of enzymes which catalyze the process of DNA methylation and are responsible for maintaining the methylation patterns in the cells are collectively termed as “DNA Methyltransferases” (DNMTs). Evolutionary analyses have established that DNMTs are one of the most highly conserved enzymes occurring in almost all life forms on earth (Bestor 2000; Cabej 2013).
DNA methylation which is a significant epigenetic phenomenon, involves enzyme mediated transference and covalent addition of a methyl group from a donor onto a specific nucleotide in the genomic DNA of the cell.
The most common pattern of DNA methylation in eukaryotes occurs at the 5-Carbon position of the cytosine nucleotide of DNA thus converting it into 5- methylcytosine (5-mC). S-adenosyl-L-methionine (SAM) is the most widely active methyl donor used by the DNA methyltransferase enzymes, often known as the universal methyl donor (Lister et al. 2009; Meyers 2012). In the newly formed zygote, every gene is functionally active initially until the process of differentiation begins.
During the embryonic development, as the zygote divides to form a multicellular embryo, the DNMTs become active which, by the process of regulated DNA methylation, selectively shut down the functions of certain genes while keeping other genes active in different cells following which different precursor cells progress through their respective lines of developmental differentiation. However, the DNMT enzyme does not add methyl group to the entire DNA sequence of any gene. Rather the methyl groups are added to the 5-Carbon position of the cytosine residues only in specific –CG– regions (Fig. 1.1) of DNA known as the CpG sites (Tollefsbol 2012).
C. CpG Islands
CpG sites are specific strings of DNA which are predominantly rich in C-G dinucleotide repeats (Tollefsbol 2012). The ‘p’ in between C and G simply indicates the phosphodiester bond that links the cytosine and guanine residues directly, so that C and G appear alternately and consecutively within the CG rich sequences.
CpG islands are specific locations within genomic DNA which are exceptionally rich in CpG sites and may contain consecutive repeats of multiple CpG sites (Jabbari &
Fig. 1.3: Schematic representation of a hypothesis explaining evolution of CpG islands Source: https://en.wikipedia.org/wiki/CpG_site#/media/File:Cpg_island_evolution.svg
Bernardi 1998; Ashikawa 2001). It is noteworthy that the CpG sites have the following nucleotide sequence pattern:
–CGCG–
–GCGC–
Promoter analyses studies have revealed that almost 40% of mammalian promoters (Fatemi 2005) and nearly 70% of human gene promoters (Saxonov et al. 2006) are characterized by CpG islands while nearly 70-80% the CpG cytosine residues in mammalian promoters remain methylated (Jabbari & Bernardi 2004) as a part of epigenetic regulation (Fig. 1.4). Methyl groups are selectively added to the cytosine residues of CpG sites, initially by the de novo methyltransferases. These have similar affinity for the unmethylated and hemimethylated CpG cytosines, thus eventually both the strands get methylated in the process. Thus, by the time of DNA replication, each strand which would serve as the template for replication will contain methylated CpG sequences. But in the newly synthesized DNA, due to its semi- conservative nature, only the parent strand will continue to remain methylated at the specific CpG sites forming hemi-methylated DNA which will promote methylation of the daughter strand by another type of DNMT enzyme known as the maintenance methyltransferase enzyme which show selective affinity for the unmethylated
Fig. 1.2: Schematic representation of Methylated v/s non-methylated cytosine https://en.wikipedia.org/wiki/CpG_site#/media/File:CpG_vs_C-G_bp.svg
cytosines in hemimethylated CpG sites (Leonhardt & Bestor 1993; Jones 2000;
Subramaniam et al. 2014). Thus the DNA methylation patterns of the parent cell are propagated with high fidelity and stably maintained through several generations of cell replication.
II. STRUCTURE AND FUNCTION OF DNA METHYLTRANSFERASES DNA methyltransferases, a group of key epigenetic regulators, are highly conserved enzymes present in almost all life forms, both prokaryotes and eukaryotes. In this section we discuss the structural and functional characteristics of two broad categories of DNMTs: the eukaryotic DNMT and the bacterial DNMTs.
A. Eukaryotic DNA Methyltransferase
The most significant component of DNA methylation machinery in mammals appears to be a characteristic methylation pattern of the cytosine residues at the 5-carbon position. This important epigenetic event is globally coordinated by a family of at least four DNMT enzymes encoded independently DNMT1, DNMT3A, DNMT3B and DNMT3L genes (Subramaniam et al. 2014; Leonhardt & Bestor 1993; Rogers 1993;
Jones 2000). Apart from this, a few other methylation patterns are also observed in the cytosine residues of tRNA and in certain adenosine residues of DNA which are executed by other types of methyltransferase enzymes.
Fig. 1.4: Role of CpG methylation in regulating gene expression Source: http://biol10005.tumblr.com/post/31972466118/epigenetics
Characteristic Features of DNMTs
Structural and functional assays performed so far have established a few characteristic features common to all the DNMTs known so far (Meissner 2011;
Subramaniam et al. 2014; Bestor 2000; Robertson 2001; Rondelet & Wouters 2017).
These are summarized below (Fig. 2.1):
All 5CDNMTs are highly conserved enzymes encoded independently
Structurally, the enzymes encoded by DNMT1 and DNMT3 are made up of two distinct domains, the N-terminal regulatory domain and the C-terminal catalytic domain
The conserved catalytic domain includes 10 conserved motifs for DNA targeting and binding, cofactor binding and catalysis
C-terminal catalytic domain further consists of 10 conserved amino acids
N-terminal regulatory domain is variable and may or may not be essential for catalysis
S-adenosyl-L-methionine (SAM), the cofactor of the 5CDNMTs acts as the universal methyl donor during the methylation reactions by all types of DNMTs
i) DNMT1
DNMT1 is the most abundantly expressed of all DNA methyltransferases present in a mature mammalian cell (Ferguson-Smith & Greally 2007). However, in the unicellular zygote DNMT1 is not the first DNMT that comes into play. DNA methyltransferase1 has special affinity for the “hemi-methylated” DNA strands (Subramaniam et al. 2014; Cheng & Blumenthal 2008).
Fig. 2.1: Schematic representation of domain structure of human DNMT isoforms
Source: http://www.frontiersin.org/files/Articles/132809/fgene-06-00090-r2/image_m/fgene-06-00090-g002.jpg
Dnmt1 specifically catalyzes methylation of the cytosine residues in the CpG sites of the non-methylated strand in the newly replicated DNA in which only the parent DNA strand carries the previously methylated cytosine residues.
Thus Dnmt1 is often referred to as the “Maintenance- methyltransferase”
(Subramaniam et al. 2014) as it is involved in faithfully replicating and maintaining the already established
cytosine methylation patterns in the CpG islands for each gene. In mammalian cells, Dnmt1 forms a component of the replication fork itself (Fatemi 2005) so that the newly synthesized DNA strands are simultaneously methylated before release of the replication fork.
DNMT1 gene codes for the largest of all known mammalian DNMTs that consists of a conserved 500 amino acid long C-terminal catalytic domain and a large multi- domain N-terminal regulatory region of variable length (Subramaniam et al.
2014), characterized by a prominent Zinc binding motif, a distinct nuclear localization signal and replication foci targeting motif (Fig. 2.3)
Fig. 2.2: Ribbon structure diagram representing Dnmt1-DNA complex
Fig. 2.3: Multi-domain structure of mouse Dnmt1 http://www.pnas.org/content/108/22/9055/F1.expansion.html
ii) DNMT3:
Dnmt3 family is known to include three related enzymes, Dnmt3A, Dnmt3B and Dnmt3L (Gowher et al. 2005; Chédin 2011). DNMT3A and DNMT3B are activated soon after the embryo implantation and are essential during the early embryonic development in order to initiate and establish the programmed de novo CpG methylation pattern (Xie et al. 1999; Kaneda et al. 2004; Subramaniam et al. 2014).
DNMT3A and DNMT3B genes are coordinately expressed and encode the “de novo methyltransferases” which do not require a hemi-methylated DNA strand (Okano et al. 1999); instead the de novo methyltransferases exhibit similar affinity for the non- methylated and hemi-methylated CpG sites in the DNA (Kato et al. 2007;
Subramaniam et al. 2014). It has been reported that the de novo methyltransferases remain diffuse throughout the nucleoplasm and show selective association with the replication foci during the S-phase also (Bestor et al. 1999; Esteller 2006). It is speculated that these de novo methyltransferases utilize chromatin remodeling complexes and some other auxiliary factors to gain access to their target CpG sequences even in the densely packed chromatin (Di Croce et al. 2002).
DNMT3A and DNMT3B encode corresponding de novo methyltransferases both of which contain a conserved C-terminal catalytic domain similar to that of Dnmt1.
However the N-terminal regulatory domain of these enzymes is significantly different and is characterized by the PWWP and the Cys-rich Plant Homeodomain (PHD) domains (Subramaniam et al. 2014; Gowher et al. 2005). Unlike the N-terminal of Dnmt1, the N-terminal of Dnmt3 is not essential for catalysis rather it is mainly involved in enzyme targeting (Gowher et al. 2005)
Fig. 2.4: Dnmt3-DNA complex
Source: https://www.123rf.com/photo_18482719_dnmt3-is-an-enzyme-from-a-group-of-dna-methlytransferases-which-modify- dna-in-order-to-regulate-gene.html
DNMT3L is a gene of particular interest as it encodes the peculiar Dnmt3L enzyme which itself lacks any direct catalytic activity (Hata et al. 2002; Subramaniam et al.
2014). It functions mainly as an accessory or stimulatory enzyme which promotes and enhances the methyltransferase activity of Dnmt3A
& 3B by many folds by assisting the binding of Dnmt3A & 3B to the methyl donor and cofactor SAM (Suetake et al. 2004; Subramaniam et al. 2014).
Dnmt3L is the smallest of the known enzymes encoded by DNMT3 gene family that has a relatively
smaller C-terminal domain and a much smaller N-terminal domain compared to Dnmt3A & 3B (Fig. 2.6). The C-terminal domain of Dnmt3L shows homology with the conserved C-terminal of other DNMTs but it is totally devoid of the amino acid residues essential to form the catalytic motif, thus lacking any direct catalytic function (Subramaniam et al. 2014; Gowher et al. 2005). The N-terminal domain of Dnmt3L contains the PHD domain but altogether lacks the PWWP domain of the Dnmt3A & 3B (Subramaniam et al. 2014; Robertson 2001; Gowher et al. 2005).
Fig. 2.5: Ribbon structure of Dnmt3a and Dnmt3b
Source: https://www.intechopen.com/books/methylation-from-dna-rna-and-histones-to-diseases-and-treatment/diverse- domains-of-cytosine-5-dna-methyltransferases-structural-and-functional-characterization
Fig. 2.6: Schematic representation of DNA modelling by Dnmt3A and Dnmt3L
Source: Nature Reviews: Molecular Cell Biology
Before we proceed on to the following sections, let us first go through a conceptual summary (Fig. 2.7) of the functional roles of mammalian DNA Mtases where we outline the discrete and vivid roles of different DNA Methyltransferases in eukaryotes. Pre-programmed, de novo methylation pattern is established by Dnmt3A/3B aided by Dnmt3L. During this event, both strands of DNA are methylated at the CpG sites. Due to semiconservative nature of DNA replication, only one of the two strands in the newly synthesized DNA duplex, the parental strand retains previous methylation pattern(s). The newly synthesized DNA strand in the daughter duplex is initially unmethylated, so that, the new DNA duplex exhibits hemi- methylated CpG sites. Eventually, the new DNA strand gets methylated by maintenance methyltransferase Dnmt1 which selectively adds methyl group to the hemimethylated CpG sites immediately after DNA replication.
More recent studies aimed at characterizing DNMTs have established that mammalian DNMTs Dnmt1, Dnmt3a and Dnmt3b also act as cytosine demethylase (Anon 2012; Ji et al. 2014). These have been shown to be involved in Ca2+
dependent demethylation of 5mC converting it back to cytosine during the early zygotic development.
DNMT2: DNMT2 is the smallest of all known eukaryotic methyltransferase enzymes, albeit it is a highly conserved methyltransferase. It is characterized by all the motifs of the conserved C-terminal domain of cytosine-C5 DNMTs but the N- terminal regulatory domain is altogether absent (Subramaniam et al. 2014;
Gowher et al. 2005; Shanmugam et al. 2015). Thus Dnmt2 exhibits a slow methyltransferase activity. Cloning and characterization of DNMT2 has established
Fig 2.7: Conceptual diagram depicting Dnmt family mediated DNA methylation pattern in Eukaryotes Source: Molecular Cell Biology, Aug., 2003
its ability to methylate RNA instead of DNA (Okano-1998). It was later reported that Dnmt2 methylates the 5-carbon of cytosine-38 residue in the tRNA-Asp (Shanmugam et al. 2015; Hermann et al. 2003). Recent studies have established that Dnmt2 is involved in methylation of cytosine residues in several other tRNA in different species, including plants, humans and lower euakryotes and is speculated to have a protective or stabilizing function (Schaefer et al. 2010). Thus Dnmt2 has been aptly renamed as tRNA-aspartic acid methyltransferase 1” (Trdmt1) (Shanmugam et al. 2015).
Apart from cytosine methylation in tRNA, m6A or methylation of Adenosine-N6 in the eukaryotic mRNA converting it into N6-Methyladenosine, is another method of mRNA methylation known to occur in eukaryotes.
B. Prokaryotic DNA Methyltransferases
DNA methyltransferase enzymes in bacteria are known to be associated with several fundamental physiological processes, the important ones being the epigenetic regulation of gene expression, the cell cycle regulatory mechanism, DNA damage repair machinery, virulence and survival mechanisms, establishing bacterial strains and inheritance patterns, and the restriction-modification system (Reisenauer et al.
1999). DNA methylation in bacterial genome represents an epigenetic regulation that occurs as a part of the post-replication modification of nascent DNA by several DNA methyltransferase enzymes which utilize S-adenosyl-methionine as the methyl donor to add a methyl group to a specific target DNA sequence (Casadesús 2016).
The most prevalent patterns of methylation in bacterial genome include formation of C5-methylcytosine, N4-methylcytosine and N6-methyladenosine by the corresponding bacterial DNA methyltransferases (Casadesús 2016). Methylation of nucleotide residues in bacterial DNA is postulated to closely influence survival or defense pathways, DNA stability, DNA topology, gene expression and overall interaction with DNA binding proteins and enzymes. Those DNA methylases in bacteria which are mainly involved in epigenetic regulatory mechanism are denoted as the “orphan” or “solitary” methylases as they are not associated with a respective restriction enzyme while the methylases involved in defense against foreign DNA are closely coupled to a cognate restriction enzyme. It is speculated that the bacterial DNA methylases associated with R-M system may function as the pre-cursors to the orphan methylases (Casadesus & Low 2006).
Several DNA methyltransferases in prokaryotes are involved in the prototypical
“restriction/modificationsystem” (R-M system). Prokaryotes have evolved an elaborate and extensive “restriction-modificationsystem” as a defense mechanism against the bacteriophages and other bacterial pathogens. This method of bacterial defense mechanism involves a wide array of restriction endonuclease enzymes which selectively cleave the genome of the invading species at multiple restriction sites. However these restrictions enzymes do not have the ability to differentiate, by
Here comes the role of the bacterial DNA methyltransferase enzymes which selectively methylate the host genomic DNA while the invading foreign DNA remains unmethylated (Glover 1972; Rao & Srivani 2004; Dryden 2004; Halford 2013). The bacterial endonuclease enzymes can cleave only the unmethylated DNA and while the methylated DNA strands remain protected. Thus, DNA methyltransferase mediated “modification” of bacterial DNA, in the form of methylation, “restricts” the endonuclease enzymatic cleavage of the host DNA while the invading foreign DNA is open to enzymatic cleavage, thus protecting the bacteria from viral invasion.
The bacterial DNA methylation shows certain differences as compared to the eukaryotic DNA methylation. Unlike the eukaryotes, the most obvious function of DNA methylation in bacteria is coordinated action with the R-M system to ensure defense against viral or any foreign DNA through restriction enzyme mediated DNA destruction (Casadesús 2016). Further bacteria use adenine methylation as the preferred DNA methylation pattern in the genome rather than cytosine methylation, which occurs at much less frequency (Seshasayee n.d.). The most well studied bacterial DNA methylases are discussed below:
i) DAM Family:
Dam or DNA adenine methylase (sometimes referred to as deoxyadenosine methylase) belongs to the α-group of bacterial DNA methyltransferases as it contains the ten conserved characteristic motifs forming distinct AdoMet binding site, a target recognition domain and a catalytic domain (Reisenauer et al.
1999; Casadesus & Low 2006). One of the most researched Dam is the E. coli Dam (EcoDam) which is a 32kDa monomeric protein and its homologues have been widely reported in several enteric bacteria
such as Salmonella spp., Serratia marcescens, Yersinia spp., Vibrio cholerae, and also in Haemophilus influenza and some other gram-negative bacteria. Studies have revealed that the binding of Dam to DNA is non-specific in nature; however, the target sequence for methylation is a specific 5’-GATC-3’ sequence in which the deoxyadenine nucleotide is methylated at the 6-nitrogen position. Here again the methyl group is donated by the universal methyl donor S-adenosyl-L-methionine (AdoMet) which is also essential for stabilizing the interaction between the target base and active site of the enzyme (Low et al. 2001; Casadesus & Low 2006).
Fig. 2.8: 3D-crystal structure of EcoDam
Source: http://www.rcsb.org/pdb/explore.do?structureId=2g1p
EcoDam is known to function as a de novo methylase and also a maintenance methylase enzyme. It carries out methylation of the adenine residue in the target sequence on the non-methylated DNA strands as well as hemi-methylated DNA strands. There are mounting evidences that deoxyadenine methylation of bacterial DNA executes epigenetic regulation and also plays a role in virulence in certain bacterial species. The epigenetic regulation is ecxecuted through hemimethylation and other characteristic DNA methylation patterns at the target sites of different genes. Although EcoDam executes epigenetic regulation on the expression of a large group of genes, yet there is no direct evidence that adenine methylation is essential for the growth of E. coli. Albeit, there is reported evidence of direct involvement of Dam in virulence in most of the gram negative bacteria in which it occurs (Casadesus & Low 2006; Low et al. 2001; Reisenauer et al. 1999).
ii) CcrM
Cell cycle-regulated DNA Methyltransferase, a 39 kDa “orphan” methylase reported initially in Caulobacter crescentus belongs to the β-group of methyltransferases based on the organization of its amino acid domains (Fig. 2.10) such that the AdoMet binding site is organized towards the C-terminal and the catalysis site placed at the N-terminal (Reisenauer & Shapiro 2002; Reisenauer et
Fig. 2.9: Methylation pattern in E. coli
Source:
https://www.flickr.com/photos/ag athman/4977090679
Fig. 2.10: Domain organization of Dam and CcrM Source: http://jb.asm.org
CcrM is not coupled to any cognate restriction enzyme thus ruling out its role in protection against phage. CcrM reportedly functions as an adenine methylase which specifically methylates the adenine residue in its target sequence 5’-GANTC- 3’. CcrM appears to preferably bind to hemimethylated DNA strands and catalyzes transference of a methyl group from the universal methyl donor SAM onto the 6-N of adenine in the target sequence. The specific domain essential for adenine-MT activity is the only conserved domain between CcrM and Dam. Unlike Dam, CcrM is reportedly essential for growth and viability of Caulobacter and is one of the first DNA methylase to be reported that is not paired with a cognate restriction enzyme (Reisenauer et al.
1999; Reisenauer & Shapiro 2002; Low et al. 2001;
Casadesús & Low 2013).
Methylation of -GANTC- sites is closely synchronized with cell cycle and genome replication in Caulobacter (Fig. 2.11). DNA replication initiates only after the entire chromosome is fully methylated, which then becomes progressively hemimethylated due to the semi conservative nature of DNA replication as the bidirectional replication proceeds from the origin to the terminus.
Maximal expression and activity of ccrM is limited to and concomitant with the later part of cell division during which ccrM mediated methylation of nearly 30,000 GANTC sites in the two newly replicated chromosomes is achieved (Casadesús & Low 2013; Reisenauer & Shapiro 2002).
iii) Dcm
DNA cytosinemethylase (Dcm)which is another important “orphan” methylase in E. coli is known to methylate the first Cytosine in the “duplex” sequence sequence 5’-CCWGG-3’ (where W represents A or T) (Buryanov et al. 1974; Dar & Bhagwat 1993; Militello et al. 2012). However there are no reports that indicate possible
Fig.2.11: CcrM mediated DNA methylation in Caulobacter during chromosome replication
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC94015/
involvement of Dcm in epigenetic gene regulation in the host organism. As cytosine methylation by Dcm protects the host genome against enzymatic cleavage by EcoRII, it is speculated that Dcm has evolved as an inherent defense mechanism against invading restriction-modification complex (Palmer & Marinus 1994)
iv) R-M system associated DNA Methyltransferase
There has been sufficient evidence which indicate that the restriction-modification systems in prokaryotes have evolved as a mechanism of cellular defense against the invasion of phage and other foreign DNA (Casadesus & Low 2006). As the name suggests, the R-M system includes a paired group of a restriction enzyme coupled with its cognate methyltransferase enzyme. Reports suggest that the spatial and temporal expression profiles of these paired enzymes is strictly coordinated and regulated such that the target sites throughout the entire host genome is methylated completely and thus protected from enzymatic cleavage of DNA by its own restriction enzymes (Bhagwat 1995; Dale & Park 2004). However in case of the invading
foreign DNA, the host restriction enzyme supersedes the host methylase enzyme and executes enzymatic cleavage at multiple target sites on the pathogenic DNA sequence (Fig. 2.12) (Stankevičius et al. 1995; Mruk & Kobayashi 2016).
The mechanisms of action by the R-M systems of prokaryotes may be broadly classified into following types (Chandrashekaran et al. 1999; Goodman et al. 1977):
Fig. 2.12: Step-wise activity of bacterial DNA methyltransferases in mediating bacterial restriction modification system against invading DNA
Source: https://lh3.googleusercontent.com/JWQ4qfg85yjClpb38ICK6ATBB9vSb1Fh- T61eudkHCl3WLyfJZJV1ynxb1B9R3_h0APaKg=s115
(i) Delayed expression: plasmids containing type-II R-M EcoRV type of system have an additional gene (C gene) sandwiched between the R- and M- genes.
M gene (methylase) is reportedly an early expressing geneand R gene (restriction enzyme) is activated late and also requires an optimal level of expression product of C gene for its activation.
(ii) Feedback regulation: CfrBI R-M system of Citrobacter freundii and SsoII R- M system of Shigella sonnei are reported to use feedback mechanism for regulation of R-M activity. (Shiuan & Campbell 1988; Beletskaya 2000).
Methylation of target site 5’-CCATGG-3’ in case of CfrBI R-M system leads to repression of the methylase (CfrBIM) and simultaneous increase in expression of the cognate restriction endonuclease (CfrBIR). Studies on SsoII R-M system have revealed that upon binding of the methylase (SsoIIM) to the target DNA sequence via its N-terminal extension bearing a helix-turn- helix motif, the methylase itself downregulates its expression while simultaneously upregulating expression of its cognate restriction enzyme (SsoIIR). This typical N-terminal extension is observed in several other 5- methylcytosine methyltransferases, important ones being the EcoRII, dcm, MspI, and LlaJI systems.
III. APPLICATIONS
A. Research Applications
Bacterial DNA methylases are known to methylate DNA at adenine as well as cytosine. Further, different DNA methylases in bacteria are either associated with the prototypical Restriction-Modification system or work in close coordination with the epigenetic regulation of various cellular activities (Casadesús 2016). A detailed knowledge of the bacterial DNA methyltransferases and their mode of action give us a leverage to exploit and utilize these enzymes for various research and development studies (Harrison & Parle-McDermott 2011).
A routine protocol in the RDT involves introducing engineered DNA molecules into the E. coli for cloning and expression and other analytical studies. However, the bacteria have an inherent mechanism to destroy foreign DNA by the R-M system.
Hence, if the exogenous DAN is artificially methylated before introducing into the bacteria, that would prevent its endonuclease cleavage (Fig. 3.1).
Further, the orphan DNA methylases in bacteria which are known to be involved in epigenetic regulation of gene expression, DNA stability, DNA topology, cell cycle, virulence and other essential cellular activities can be explored and exploited as potential targets for designing novel anti-microbial therapies other than antibiotics. It has been established that antibiotic resistance and in development of multi-drug resistance in bacteria is mediated by methylation of nucleosides by bacterial DNA Mtases (like Dam) and RNA methylases. Researchers have shown that disruption of
bacterial methylomes can enhance the susceptibility of bacteria to antibiotics.(Rosenthal & Elowitz 2011; Burgess 2014).
B. Clinical Implications
Methylome analysis of pathologically affected tissues, tumor cells and cancer cells from multiple mammalian sources have illustrated with certainty that anomalous methylation patterns and aberrant DNA Mtase expression profiles are predominant in all the cases of tumorigenesis and carcinogenesis. In case of the malignant tumor cells the promoter associated CpG islands have been shown to be mostly hypermethylated for the mammalian tumor suppressor genes while the CpG sites in the promoters of mammalian oncogenes have been shown to be largely hypomethylated compared to the methylation trend in healthy cells (Fig. 3.3) (Carrió
& Suelves 2015; Suelves et al. 2016). Hypomethylation of a promoter, as a rule, causes overexpression of the respective gene while hypermethylation of the promoter invariably leads to suppression or inactivation of the corresponding gene (Fig. 3.2) (Suelves et al. 2016).
Fig. 3.1: Host-mimicking strategies for circumventing restriction–modification (RM) systems in bacteria.
Source: https://www.intechopen.com/books/methylation-from-dna-rna-and-histones-to-diseases-and-treatment/host- mimicking-strategies-in-dna-methylation-for-improved-bacterial-transformation
In most cases of tumorigenesis, enhanced expression of DNMT3A/3B (the de novo methyltransferases) and DNMT3L that stimulates the activity of DNMT3A/3B is concurrent with hypermethylation of the CpG sites present in the promoters regions of the tumor suppressor genes. Variation in expression levels of maintenance methyltransferase DNMT1, which is mainly involved in post replicative methylation, and of DNMT2 which is speculated to participate in DNA damage recognition, DNA recombination and mutational repair has also been correlated with multiple cases of tumorigenesis and malignancy (Fig. 3.4) (Esteller 2006; Subramaniam et al. 2014).
Since the origin and progression of multiple types of human cancer is closely associated with aberrant expression profiles of DNA methyltransferases, hence the mammalian DNMTs are being extensively studied as potential targets for anti-cancer therapy. DNMT inhibitors such as Azacitidine (Čihák 2009; Chekhun et al. 2016) and Decitabine (Scotto et al. 2017; Sheng et al. 2011) are already in advanced stages of clinical trials against leukemia and other types of malignancies. But what have been explored till date is merely the tip of the iceberg and the mammalian methylome and DNA Mtases offer endless scope to be investigated as potential targets for designing novel cancer treatment strategies and anticancer therapies.
Fig. 3.2: Cell type-specific DNA methylation profiles http://journal.frontiersin.org/article/10.3389/fnagi.2015.00019/full
Fig. 3.3: Aberrant DNA methylation profile in cancer cells Source: http://atlasgeneticsoncology.org/Deep/DNAMethylationID20127.html
Fig. 3.4: Induction of apoptosis in tumor cell upon decitabine mediated demethylation of tumor suppressor genes
Source: http://embomolmed.embopress.org/content/8/8/863
In more recent studies several other pathological conditions such as optic atrophy, peripheral neuropathy and other mitochondrial dysfunction disorders (Maresca et al.
2015) and even diseases associated with skeletal muscle development (Carrió &
Suelves 2015) have been correlated with altered dynamics of DNA methylation.
Thus, the methylome analysis in the mammalian cells, in particular the biochemical evidences from the pathologically altered tissues provide us even more convincing reasons to explore the mammalian DNA Methyltransferases as novel targets for biomedical research.
In a nutshell, DNA methyltransferases at present are being explored and exploited for a wide array of applications:
Widely used in recombinant DNA technology as DNA-modifiers, as these can induce target specific in vitro methylation
Used for in vitro methylation of desired DNA in the rDNA so that the restriction sites present in the exogenous DNA are protected from endonuclease cleavage by restriction enzymes of R-M system of the host system being used
Bacterial DNMTs are an integral part of many vital physiological processes and also bacterial R-M defense system. Thus, can be explored as targets for drug-designing and for novel anti-microbial therapeutic strategies.
Mammalian DNMTs are being widely explored as potential targets for
“targeted genome engineering” and molecular therapy against
several human diseases, most important ones being tumorigenesis and carcinogenesis
Fig. 3.5: Establishment and Maintenance of DNA Methylation.
Source: Cancers | Apoptosis and DNA Methylation|2011
IV. SUMMARY
DNA methyltransferases are one of the most highly conserved enzymes across all life forms; DNA Methylation is one of the important epigentic tools
In eukaryotes, the Dnmt3 enzymes initiate de novo methylation and Dnmt1 mediate post-replicative
maintenace-methylation of the CpG sites in the genome DNA Mtases in eukaryotes are closely associated with epigenetic gene regulation, recognition of DNA
damage, recombination and repair
Bacterial DNA Mtases are invovlved in epigenetic gene regulation, cell cycle regulation, virulence mechanism and Restriction-Modification system . Selective targeted DNA methylation leads to silencing or suppression of gene expression in both eukaryotes and prokaryotes
Drastic variation in Dnmt expression levels are associated with many pathological conditions in humans, including carcinogenesis
DNMTs are important tools of RDT and potential targets for
genome engineering and molecular therapy
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