DNA methylation
(節錄自維基 by陳怡睿)
DNA methylation is a type of chemical modification of DNA that can be inherited and subsequently removed without changing the original DNA sequence. As such, it is part of the epigenetic code and is also the most well characterized epigenetic mechanism.
DNA methylation involves the addition of a methyl group to DNA — for example, to the number 5 carbon of the cytosine pyrimidine ring — in this case with the specific effect of reducing gene expression. DNA methylation at the 5 position of cytosine has been found in every vertebrate examined. In adult somatic tissues, DNA methylation typically occurs in a CpG dinucleotide context; non-CpG methylation is prevalent in embryonic stem cells.
In plants, cytosines are methylated both symmetrically (CpG or CpNpG) and asymmetrically (CpNpNp), where N can be any nucleotide but guanine. Some organisms, such as fruit flies, exhibit virtually no DNA methylation.
Research has suggested that long term memories in humans may be stored via DNA methylation.
In mammals
DNA methylation is essential for normal development and is associated with a number of key processes including imprinting, X-chromosome inactivation, suppression of repetitive elements and carcinogenesis.
Between 60-90% of all CpGs are methylated in mammals. Unmethylated CpGs are grouped in clusters called "CpG islands" that are present in the 5' regulatory regions of many genes. In many disease processes such as cancer, gene promoter CpG islands acquire abnormal hypermethylation, which results in heritable transcriptional silencing. DNA methylation may impact the transcription of genes in two ways. First, the methylation of DNA may itself physically impede the binding of transcriptional proteins to the gene and secondly, and likely more important, methylated DNA may be bound by proteins known as methyl-CpG-binding domain proteins (MBDs). MBD proteins then recruit additional proteins to the locus, such as histone deacetylases and other chromatin remodelling proteins that can modify histones, thereby forming compact, inactive chromatin termed silent chromatin. This link between DNA methylation and chromatin structure is very important. In particular, loss of methyl-CpG-binding protein 2 (MeCP2) has been implicated in Rett syndrome and methyl-CpG binding domain protein 2 (MBD2) mediates the transcriptional silencing of hypermethylated genes in cancer.
DNA Methyltransferases
In mammalian cells, DNA methylation occurs mainly at the C5 position of CpG dinucleodtides and carried out by two general classes of enzymatic activities - maintenance methylation and de novo methylation.
Maintenance methylation activity is necessary to preserve DNA methylation after every cellular DNA replication cycle. Without the DNA methyltransferase, the replication machinery itself would produce daughter strands that are unmethylated and overtime would lead to passive demethylation. DNMT1 is the proposed maintenance methyltransferase that is responsible for copying DNA methylation patterns to the daughter strands during DNA replication. Mouse models with both copies of DNMT1 deleted are embryonic lethal at approximately day 9, due to the requirement of DNMT1 activity for development in mammalian cells.
It is thought that DNMT3a and DNMT3b are the de novo methyltransferases that set up DNA methylation patterns early in development. DNMT3L is a protein that is homologous to the other DNMT3s but has no catalytic activity. Instead, DNMT3L assists the de novo methyltransferases by increasing their ability to bind to DNA and stimulating their activity. Finally, DNMT2 (TRDMT1) has been identified as a DNA methyltransferase homolog, containing all 10 sequence motifs common to all DNA methyltransferases; however, DNMT2 (TRDMT1) does not methylate DNA but instead methylates cytosine-38 in the anticodon loop of aspartic acid transfer RNA.
Since many tumor suppressor genes are silenced by DNA methylation during carcinogenesis, there have been attempts to re-express these genes by inhibiting the DNMTs. 5-aza-2'-deoxycytidine (decitabine) is a nucleoside analog that inhibits DNMTs by trapping them in a covalent complex on DNA by preventing the β-elimination step of catalysis, thus resulting in the enzymes' degradation. However, for decitabine to be active, it must be incorporated into the genome of the cell, but this can cause mutations in the daughter cells if the cell does not die. Additionally, decitabine is toxic to the bone marrow, which limits the size of its therapeutic window. These pitfalls have led to the development of antisense RNA therapies that target the DNMTs by degrading their mRNAs and preventing their translation. However, it is currently unclear if targeting DNMT1 alone is sufficient to reactivate tumor suppressor genes silenced by DNA methylation.
In plants
Significant progress has been made in understanding DNA methylation in plants, specifically in the model plant, Arabidopsis thaliana. Whereas in mammals methylation mainly occurs on the cytosine in a CpG context, in plants the cytosine can be methylated in the CpG, CpNpG, and CpNpN context, where N represents any nucleotide but guanine.
The principal Arabidopsis DNA methyltransferase enzymes, which transfer and covalently attach methyl groups onto DNA, are DRM2, MET1, and CMT3. Both the DRM2 and MET1 proteins share significant homology to the mammalian methyltransferases DNMT3 and DNMT1, respectively, whereas the CMT3 protein is unique to the plant kingdom. There are currently two classes of DNA methyltransferases: 1) the de-novo class, or enzymes that create new methylation marks on the DNA, and 2) a maintenance class that recognizes the methylation marks on the parental strand of DNA and transfers new methylation to the daughters strands after DNA replication. DRM2 is the only enzyme that has been implicated as a de-novo DNA methyltransferase. DRM2 has also been shown, along with MET1 and CMT3 to be involved in maintaining methylation marks through DNA replication. Currently, it is not clear how the cell determines the locations of de-novo DNA methylation, but evidence suggests that for many, though not all locations, RNA-directed DNA methylation (RdDM) is involved. In RdDM, specific RNA transcripts are produced from a genomic DNA template, and this RNA forms secondary structures called double-stranded RNA molecules. The double-stranded RNAs, through either the small interfering RNA (siRNA) or microRNA (miRNA) pathways, direct de-novo DNA methylation of the original genomic location that produced the RNA. This sort of mechanism is thought to be important in cellular defense against RNA viruses and/or transposons both of which often form a double-stranded RNA that can be mutagenic to the host genome. By methylating their genomic locations, through a still-poorly-understood mechanism, they are shut off and are no longer active in the cell, protecting the genome from their mutagenic effect.
In fungi
Many fungi apparently have low levels (0.1 to 0.5%) of cytosine methylation while other fungi have as much as 5% of the genome methylated. This value seems to vary both among species and among isolates of the same species. There is also evidence that DNA methylation may be involved in state-specific control of gene expression in fungi.
Although brewers yeast (Saccharomyces) and fission yeast (Schizosaccharomyces) have very little DNA methylation, the model filamentous fungus Neurospora crassa has a well characterized methylation system. Several genes control methylation in Neurospora and mutation of the DNA methyl transferase, dim-2, eliminates all DNA methylation in Neurospora but does not affect growth or sexual reproduction. While the Neurospora genome has very little repeated DNA, half of the methylation occurs in repeated DNA including transposon relics and centromeric DNA.The ability to evaluate other important phenomena in a DNA methylase deficient genetic background makes Neurospora and important system in which to study DNA methylation.
In bacteria
Adenine or cytosine methylation is part of the restriction modification system of many bacteria, in which DNAs are methylated periodically throughout the genome. A methylase is the enzyme that recognizes a specific sequence and methylates one of the bases in or near that sequence. Foreign DNAs (which are not methylated in this manner) that are introduced into the cell are degraded by sequence-specific restriction enzymes. Bacterial genomic DNA is not recognized by these restriction enzymes. The methylation of native DNA acts as a sort of primitive immune system, allowing the bacteria to protect themselves from infection by bacteriophage. These restriction enzymes are the basis of restriction fragment length polymorphism (RFLP) testing, used to detect DNA polymorphisms.
Methylation-Specific PCR（MSP） (甲基化PCR)
(Protocol written by James Herman*)
Methylation-specific PCR (MSP) is a simple rapid and inexpensive method to determine the methylation status of CpG islands. This approach allows the determination of methylation patterns from very small samples of DNA, including those obtained from paraffin-embedded samples, and can be used in the study of abnormally methylated CpG islands in neoplasia, in studies of imprinted genes, and in studies of human tumors for clonality by studying genes inactivated on the X chromosome.
MSP utilizes the sequence differences between methylated alleles and unmethylated alleles which occur after sodium bisulfite treatment. The frequency of CpG sites in CpG facilitate this sequence difference. Primers for a given locus are designed which distinguish methylated from unmethylated DNA in bisulfite-modified DNA. Since the distinction is part of the PCR amplification, extraordinary sensitivity, typically to the detection of 0.1% of alleles can be achieved, while maintaining specificity. Results are obtained immediately following PCR amplification and gel electrophoresis, without the need for further restriction or sequencing analysis. MSP also allows the analysis of very small samples, including paraffin-embedded and microdissected samples.
Basic Protocol
DNA is modified by sodium bisulfite treatment converting unmethylated, but not methylated, cytosines to uracil. Following removal of bisulfite and completion of the chemical conversion, this modified DNA is used as a template for PCR . Two PCR reactions are performed for each DNA sample, one specific for DNA originally methylated for the gene of interest, and one specific for DNA originally unmethylated. PCR products are separated on 6-8% non-denaturing polyacrylamide gels and the bands are visualized by staining with ethidium bromide. The presence of a band of the appropriate molecular weight indicates the presence of unmethylated, and/or methylated alleles, in the original sample.
Prepare the mixes. Thaw 10x , NTP’s and primers. Determine the number of samples to be analyzed, including a positive control for both the unmethylated and methylated reactions, and a water control. Make a master mix for each PCR reaction (methylated and unmethylated). For each 50 µl reaction, the following amounts should be used:
PCR buffer
10x PCR buffer: 5 µL
25mM 4 NTP mix 2.5 µL
Sense primer (300ng/µL) 1 µL
Antisense primer (300ng/µL) 1 µL
Distilled, sterile water 28.5 µL
The 10x PCR buffer we use provides specific and high-efficiency amplification, but it has relative high magnesium and nucleotide concentrations. Other PCR buffers have been used with success as well.
Aliquot 38 µl of this PCR mix into separate PCR tubes (0.5 mL tubes or strips) labeled for each sample. Assure that the components are well mixed prior to aliquoting.
Add 2 µl of bisulfite modified DNA template to each tube. Be sure to have an unmethylated and methylated reaction for each sample, and to have the positive controls, as well as a no DNA control.
Add one to two drops of mineral oil (~25-50 µl) to each tube, and place in thermocycler. Be sure the mineral oil completely covers the surface of the reaction mixture to prevent evaporation. If the thermal cycler has a heated lid to prevent condensation, mineral oil may not be necessary, but longer run times are typical.
Amplify the PCR products in the thermal cycler. Initiate the PCR with a five-minute denaturation at 95°C. Add Taq polymerase after initial denaturation: 1.25 units of Taq polymerase, diluted into 10 µl of sterile distilled water. Mix this 10 µl into the 40 µl through the oil by gently by pipetting up and down. Other methods of hot-start PCR may be more convenient. Continue amplification with the following parameters (35 cycles are usually enough):
30 sec 95°C (denaturation)
30 sec specific for primer (annealing)
30 sec 72°C (elongation)
Final step: 4 min 72°C (elongation)
Store at 40 C until analysis.
Analyze the PCR products by gel electrophoresis.
Prepare 6-8% non-denaturing polyacrylamide gels. 1X TBE provides better buffering capacity and sharper bands for resolving these products. The size of the products typically generated by MSP analysis is in the 80-200 bp range, making acrylamide gels optimal for resolution of size. High-percentage horizontal agarose gels can be used as an alternative.
Run reactions from each sample together to allow for direct comparison between unmethylated and methylated alleles. Include positive and negative controls. Vertical gels can be run at 10 V/cm for 1-2 hours. Stain the gel in ethidium bromide, and visualize under UV illumination.
Critical Parameters
The most critical parameter affecting the specificity of methylation-specific PCR is determined by primer design. Because of the modification of DNA by bisulfite, the two daughter strands of any given gene are no longer complementary after treatment. Either strand can serve as the template for subsequent PCR amplification, and the methylation pattern of each strand could then be determined. In practice, it is often easiest to deal with only one strand, most commonly the sense strand.
Primers should be designed to amplify a region that is 80-250 bp in length, and should incorporate enough cytosines in the original sequence to assure that unmodified DNA will not serve as a template for the primers. In addition, the number and position of cytosines within the CpG dinucleotide determines the specificity of the primers for methylated or unmethylated templates. Typically, 1-3 CpG sites are included in each primer, and concentrated in the 3’ region of each primer. This provides optimal specificity and minimizes false positives due to mispriming. To facilitate simultaneous analysis of the U and M reactions of a given gene in the same thermocycler, we adjust the length of the primers to give nearly equal melting/annealing temperatures. This usually results in the U product being a few base pairs larger than the M product, which provides a convenient way to recognize each lane after electrophoresis.
Since methylation-specific PCR utilizes specific primer recognition to discriminate between methylated and unmethylated alleles, stringent conditions must be maintained for amplification. This means that annealing temperatures should be at the maximum temperature which allows annealing and subsequent amplification. In practice, we typically test newly designed primers with an initial annealing temperature 5-8 degrees below the calculated melting temperature. Non-specificity can be remedied by slight increases in annealing temp, while lack or weak PCR products may be improved by a drop in temperature of 1-3 degrees Celsius. As with all PCR protocols, great care must be taken to ensure that the template DNAs and reagents do not become contaminated with exogenous DNAs or PCR products.
* Herman JG and Baylin SB, Methylation Specific PCR , in Current Protocols in Human Genetics, 1998.
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