What is DNA Methylation?
DNA methylation is a biochemical reaction that adds a methyl group to DNA nucleotides. The methylation of DNA has been found to alter the expression of genes in cells during development.
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The DNA in higher eukaryotic cells is known to contain methylated cytosine residues. Most of these methylation sites are found within CG dinucleotides called CpG motifs or elements. The CpG sequence motif is sometimes also called a HTF island and is a classical genetic feature usually found associated with upstream sequence regions of transcriptionally active genes. HTF stands for “HpaII tiny fragments.”
Overview of how methylation of cytosines influences biological processes.
The methyl group on cytosine can either directly or indirectly change the DNA biochemistry. The biochemical modifications can serve as molecular signals for chromosome functions. The resulting effects determine development, physiology, and pathology of an organism. (Source: Franchini et al. 2012).
A HTF island is a tiny DNA fragment, approximately 1000-2000 base pairs long which is usually found associated with expressed genes and is characterized by the relatively rare CpG dinucleotide that occurs unmethylated. Digesting DNA with the restriction nuclease HpaII generates the fragment. For example, chromosome walking together with pulsed field gel electrophoresis allowed to verify the presence of clusters of CpG dinucleotides within the major histocompatibility complex (MHC) class III genomic region in the past and there characterization. These findings suggested that a number or multiples of HTF-islands can be present in genetic DNA regions. Scientists now know that CpG islands surround the promoters of housekeeping genes which encode enzymes involved in essential metabolic pathways such as glycolysis and others. There is increasing experimental evidence that indicate that the state of methylation of CpG islands affect the expression of genes. The recognition that the enzymatic methylation of the cytosine base plays a crucial role in the regulation of chromatin stability, gene regulation, parental imprinting, and X chromosome inactivation in females has led to the new scientific field of epigenetics.
Furthermore, the findings that erroneous DNA methylation may lead to cancer, other diseases and is involved in the aging process suggests that this type of methylation is a major important regulatory epigenetic event. Therefore we can conclude that the analysis of the cytosine methylation status is of great importance for our understanding of gene expression and regulation mechanisms.
Structures of cytosine and 5-methylcytosine.
The cytosine is methylated at the C5 position: Note that the addition of a methylgroup to the cytosine increases the mass of the base by 14.01565 mass units (Da).
The methylation of the cytosine base on the C5 position was the first identified modification of DNA. This type of methylation has been intensively investigated during the last 40 to 50 years and it has now become clear that the major eukaryotic DNA methyl-transferase, Dnmt1, accurately maintains genome-wide methylation patterns and plays an essential role in the epigenetic network that controls gene expression and genome stability during cellular development. The family of DNA methyltransferases (DNA MTase) catalyzes the transfer of a methyl group to DNA bases such as cytosine. DNA methylation has now been identified in a wide variety of biological functions. The methyl-group donor S-adenosyl methionine (SAM) is the donor for all the known DNA methyltransferases.
The DNA methyltransferase (DNMT) uses S-adenosylmethionine (SAM) as the methyl donor. S-adenosylhomocysteine (SAH) is the leaving molecule which acts as an inhibitor of the transferase. The methylation metabolism converts SAH back to SAM. DNMT can copy methylation patterns from one strand of double stranded DNA (dsDNA) to the complementary strand thereby maintaining methylation pattern. However, DNMT can also create new methylation patterns. In general, methylation patterns are well maintained in stable differentiated cells in vivo.
SAM (S-adenosylmethionine,or AdoMet) is a methyl donor for many methyltransferases that modify DNA, RNA, histones and other substrates. It is an important cofactor involved in the transfer of a methyl group. The methyl group (CH3) attached to the methionine sulfur atom in SAM is chemically reactive which allows the donation of this group to acceptor molecules. The molecule was first discovered by G. L. Cantoni in Italy in 1952. It is synthesized from adenosine triphosphate (ATP) and methionine by the enzyme methionine adenosyltransferase (EC 18.104.22.168). Most SAM is produced and consumed in the liver.
Many metabolic reactions involve the transfer of a methyl group from SAM to various substrates, such as nucleic acids, proteins, lipids and secondary metabolites. The compound S-Adenosyl-L-homocysteine (SAH) is generated by the demethylation of SAM during the methyl group transfer reaction as shown in the figure below.
All these molecules are part of the genetic mechanisms that control replication, transcription and translation fidelity, and are involved in nucleotide pair mismatch repair, chromatin remodeling, epigenetic modifications and imprinting. Furthermore, these are topics of great interest and importance in cancer research and aging.
CpG islands are involved in gene silencing. It has been found that the methylation of CpG islands in the promoter region of a gene inactivates the gene. The de-methylation of CpG islands in the promoter region of a gene activates the gene. Misregulation of this mechanism in tissue and cells has been implicated to initiate cancer in humans.
The crystal structure of HaeIII methyltransferase convalently complexed to DNA was solved by Reinisch et al in 1995. It revealed the location of the extrahelical cytosine and the rearrangement of DNA base pairing within the active site of the protein.
Structure of the HaeIII Methyltransferase, MMDB ID: 71850. PDB ID: 1DCT. This structural information provides detailed insights into the inner workings and possible regulation of this intriguing enzyme. (Left) The DNA double helix with the flipped out cytosine is shown. (Right) The DNA-enzyme complex is shown. The DNA duplex is bound so that the major groove faces the small and the minor groove faces the large enzyme domain. (Source: Reinisch et al. 1995). The following table lists the molecules and their interactions in the complex.
(Source: Pubmed, Structure Database: MMDB ID: 71850. PDB ID: 1DCT)
Many organisms expand the information content of their genome through enzymatic methylation of cytosine residues. Reinisch et al in 1995 reported the 2.8 A crystal structure of a bacterial DNA (cytosine-5)-methyltransferase (DCMtase), M. HaeIII, bound covalently to DNA. The structure shows that in the complex, the substrate cytosine is flipped out from the DNA helix and inserted into the active site of the enzyme.The DNA is bound in a cleft between the two domains of the protein and is distorted from the characteristic B-form conformation at its recognition sequence. During the recognition process the remaining bases in its recognition sequence undergo an extensive rearrangement in their pairing in which the bases are unstacked, and a gap 8 Å long opens in the DNA.
More recently the multiple steps of the methyl transfer reaction have been worked out for the related prokaryotic enzymes in molecular detail. Takeshita et al. in 2011 solved the crystal structure of a mammalian Dnmt1 (mouse) that contained the complete catalytic domain and most of the N-terminal regulatory region. The structure revealed the spatial arrangement and possible functional interactions of Dnmt1 domains. The methylgroup transfer reaction involves the flipping out of the target cytosine of the DNA double helix followed by the formation of a covalent complex with the C6 cytosine position to activate the C5 position for transfer of the methyl group from SAM. After that the enzyme is released by β-elimination and the methylated base is flipped back into the DNA double helix. To do this the enzyme requires the N-terminal region for activation. In vivo, Dnmt1 associates with the replication machinery via a PCNA-binding domain (PBD) and a targeting sequence mediates association with heterochromatin. The N-terminal target sequence (TS) was found to be inserted into the DNA-binding pocket of the catalytic domain. Complementary electrostatic surface potentials appear to anchor the TS domain in the catalytic DNA-binding pocket. This structure is further stabilized by specific hydrogen bonds. Furthermore, it was found that hydrophobic interactions between the peptide stretch, connecting the TS and the zinc finger (CXXC) domains, and the PCQ-loop at the catalytic center appear to stabilize the position of the TS domain by narrowing the entrance of the DNA-binding pocket. The PCQ loop appears to represent a sequence module for protein−DNA interactions found in methyltransferases.
Don-Marc Franchini, Kerstin-Maike Schmitz, and Svend K. Petersen-Mahrt; 5-Methylcytosine DNA Demethylation: More Than Losing a Methyl Group. Annual reviews of genetics. Vol. 46: 419–441.
Carina Frauer and Heinrich Leonhardt; Twists and turns of DNA methylation PNAS 2011 ; published ahead of print May 18, 2011, doi:10.1073/pnas.1105804108.
Jamie A. Hackett, Roopsha Sengupta, Jan J. Zylicz, Kazuhiro Murakami, Caroline Lee, Thomas A. Down, M. Azim Surani; Germline DNA Demethylation Dynamics and Imprint Erasure Through 5-Hydroxymethylcytosine. Science 25 January 2013: Vol. 339 no. 6118 pp. 448-452. DOI: 10.1126/science.1229277
Hussain Z, Khan MI, Shahid M, Almajhdi FN; S-adenosylmethionine, a methyl donor, up regulates tissue inhibitor of metalloproteinase-2 in colorectal cancer. Genet Mol Res. 2013 Apr 10;12(2):1106-18. doi: 10.4238/2013.April.10.6.
Reinisch KM, Chen L, Verdine GL, Lipscomb WN.; The crystal structure of HaeIII methyltransferase convalently complexed to DNA: an extrahelical cytosine and rearranged base pairing. Cell. 1995 Jul 14;82(1):143-53.
C A Sargent, I Dunham, and R D Campbell; Identification of multiple HTF-island associated genes in the human major histocompatibility complex class III region. EMBO J. 1989 August; 8(8): 2305–2312. PMCID: PMC401163.
Kohei Takeshita, Isao Suetake, Eiki Yamashita, Michihiro Suga, Hirotaka Narita, Atsushi Nakagawa, and Shoji Tajima; Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1) PNAS 2011 ; published ahead of print April 25, 2011, doi:10.1073/pnas.1019629108.
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