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Epigenetics & Chromatin

DNA Methylation & Enrichment

 

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What is DNA Methylation?

Methylation of mammalian DNA has long been recognized to play a major role in different cellular functions as development or control of gene expression and is generally associated with transcriptional repression. The DNA methyltransferases (DNMTs) catalyze the transfer of a methyl group from S-adenosyl methionine to the 5'-position of cytosines, mostly within the CpG dinucleotide motifs1. DNA methylation is involved in a lot of cellular functions such as embryonic development, genetic imprinting, X chromosome inactivation and control of gene expression. Aberrant methylation patterns are associated with certain human tumors and developmental abnormalities.

CpG islands are small regions of the DNA in which the CpG dinucleotide frequency is higher than would normally be expected. While CpG islands are only found in approximately 1% of the genome, they coincide with more than 60% of human promoters. CpG islands are normally not methylated, however, if a CpG island within a promoter becomes methylated the gene associated with the promoter is permanently silenced, and this silencing can be transmitted through mitosis. This means that CpG island methylation is an epigenetic means of inheritance.

Three families of DNMTs have been identified: DNMT1, DNMT2 and DNMT3. The DNMT3 family contains two active methyltransferases, DNMT3A, DNMT3B and one DNMT3-Like protein (DNMT3L). DNMT3A and DNMT3B establish the initial CpG methylation pattern de novo and show the same propensity for methylating unmethylated duplex DNA as for hemi-methylated DNA. DNMT3L shows significant sequence homology to the cysteine-rich N-terminal domains of DNMT3A and DNMT3B but has only weak homology to the C-terminal methyltransferase catalytic domain. This enzyme has no DNA methyltransferase activity. However, DNMT3L influence DNA methylation pattern by acting as a co-factor of DNMT3A2. DNMT1 is considered as maintenance DNMT, which ensures the maintenance of methylation marks during DNA replication. Indeed, this enzyme shows a specificity for hemi-methylated DNA and is responsible of the establishment and regulation of tissue-specific patterns of DNA methylation in regulatory sequences3. DNMT2 lacks the large N-terminal regulator domain common to other eukaryotic methyltransferases, but it possesses a catalytic domain. It is involved in the methylation of aspartic acid transfer RNA at position 38, giving the enzyme the alternative name of TRDMT1. Although referred to as a DNA methyltransferase, DNMT2 does not methylate DNA, but instead is the first RNA cytosine methyltransferase to be identified4.

The complex series of events leading to a repressive chromatin state involve the coordinated regulation of DNA methyltransferases, other proteins called Methyl-CpG binding proteins (MBD proteins) and the Kaiso family proteins. The MBD family proteins include MeCP2, MBD1, MBD2, MBD3 and MBD45. Whereas MeCP2, MBD1 and MBD2 have been found to have strong methyl-binding activity and transcriptional repression domains6, MBD3 harbors a critical mutation in the MBD domain and does not bind to methylated DNA. MBD3 regulates transcription by forming a Mi-2/NuRD complex with nucleosome remodeling and histone deacetylase (HDAC) activities in mammalian cells7. MBD4 is a thymine glycosylase that recognizes the product of deamination at methyl-CpG sites, as a part of DNA repair system. MBD4 is able to bind the hemimethylated DNA or methyl-CpG TpG mismatches8.

In mammalian cells, DNA methylation is generally associated with gene silencing, either directly by inhibiting binding of transcription factors to their recognition sequences9, or indirectly by preventing transcription factors from accessing their target sites through attachment of MBD proteins that “read” DNA methylation patterns. These MBDs can recruit histone deacetylases and histone methyltransferases, thereby resulting in formation of a closed repressive chromatin structure.

DNA methylation and chromatin modifications interact intimately to bring about transcriptional silencing. The association of DNMTs with HDACs leads to histone deacetylation and, in some instances at least, to CpG methylation. For example, DNMT1 binds HDAC2 and a co-repressor DMAP1 to form a complex at replication foci during late S-phase10. It was reported that the DNMT-HDAC interaction is mediated by the non-catalytic N-terminal part of DNMTs. DNMT3L can therefore recruit HDAC repressive machinery despite its lack of DNMT activity11. DNMTs appear to associate with histone methyltransferase activities that modify lysine 9 of H3. Interaction with the histone methyltransferase, such as Suv39h may be involved12-13. Interactions between DNMTs and proteins HP1α and HP1β have also been demonstrated. It was also reported that Suv39h mediated H3K9 trimethylation (H3K9me3) can direct DNMT3B to major satellite repeats present in pericentric heterochromatin13. DNMTs bound to an adaptor molecule, such as HP1 would add methyl groups to DNA only on chromatin that is methylated at lysine 9 of histone H3. Association of the DNMTs with an H3K9 methyltransferase (e.g., Suv39h) would have a direct impact of H3K9 methylation states on the DNMTs. These would also make contacts with HDACs. This would lead to partial gene silencing. MBD will also be recruited to methylated DNA. The bound MBDs would in turn interact with H3K9 methyltransferase(s) and facilitate lysine methylation. As deacetylation of acetylated histone H3 at lysine 9 (H3K9ac) is necessary for methylation to take place on this residue14. So deacetylation of histone H3 at lysine 9 would be followed by histone methylation, which in turn might result in the recruitment of proteins such as HP115.

Due to the association of DNA methylation in development and disease, much research depends on the ability to accurately quantify DNA methylation. Active Motif offers products for several techniques that can be employed for DNA methylation analysis. To see a list of available DNA Methylation products, click Available Products.

 

What is DNA Demethylation?

DNA demethylation can be achieved passively by the failure of the maintenance methylation during DNA synthesis. But the paternal pronucleus loses essentially all paternal methylation by the first cleavage division, suggesting an active demethylation process. However, the mechanism of this demethylation and the enzymes responsible are still elusive. An initial report that MBD2b might exhibit demethylating activity has not been verified by other groups. Although active demethylation might be the result of enzymatic activity that removes the methyl group from the cytosine base, this might not be energetically feasible and other mechanisms for active demethylation have been suggested. Mammalian glycosylases seem to be unable to perform demethylation efficiently, like in plants. Alternatively, deaminases could convert 5mC to thymidine. The resulting G:T mismatch would attract a glycosylase to remove the T, which would be replaced by a C by base excision repair enzymes. Candidate glycosylases include MBD4 and TDG. It has recently been proposed that DNMT3A and DNMT3B can function as deaminases, allowing them to catalyze rapid cyclical methylation and demethylation at the promoters of actively transcribed genes1,16.

A second form of DNA CpG methylation has recently been linked to epigenetic events. Several papers have recently been published describing the relative abundance of 5-hydroxymethylcytosine (5-hmC) in specific cell types and the conversion of 5-methylcytosine (5-mC) into 5-hmC. 5-hmC is replaced by cytosine by DNA repair proteins, thus conversion of 5-mC into 5-hmC may represent a pathway by which DNA is demethylated. The methyl group at position 5 of 5-hydroxymethylcytidine is oxidized to 5-hydroxymethylcytidine by the TET family of iron-dependant oxygenases.

 

Bisulfite Conversion

Bisulfite Conversion is a process in which double-stranded genomic DNA is treated with sodium bisulfite, leading to deamination of unmethylated cytosines into uracils, while methylated cytosines remain unchanged. The DNA is then amplified by PCR with primers that differentiate between methylated and unmethylated sequences followed by sequencing analysis. A comparison of the sequences of converted and untreated DNA will reveal the methylation profile of the sample. The results provide single nucleotide resolution information about the methylation status of a particular region of DNA. Alternatively, non-sequencing methods can be used to determine the methylation status at a genome-wide level.

Active Motif's MethylDetector™ Bisulfite Modification Kit simplifies analysis of DNA methylation. It comes complete with optimized reagents for performing DNA conversion with bisulfite, plus time-saving DNA purification columns and positive control PCR primers to validate your results.

In the MethylDetector method, DNA of interest is rapidly heat denatured in a thermocycler in the presence of the bisulfite conversion reagent. The temperature is then lowered and the conversion reaction is performed. Unlike other methods, MethylDetector does not require an initial acid denaturation step as the conversion reagent includes a DNA denaturant, which saves you time and effort. After DNA conversion, the sample is added to the included DNA purification columns, and a simple, on-column desulfonation is performed. Ready-to-use DNA is then eluted from the columns. For your convenience, the included positive control PCR primers can be used to assess the success of the bisulfite conversion before you spend time and money on DNA sequencing. This is because the included primers only anneal to converted human DNA

 

Affinity Enrichment

Affinity enrichment is a technique that is often used to isolate methylated DNA from the rest of the DNA population. This is usually accomplished by antibody immunoprecipitation methods or with methyl-binding domain (MBD) proteins. Methylated DNA Immunoprecipitation (MeDIP) is an antibody immunoprecipitation method that utilizes a 5-methylcytidine antibody to specifically recognize methylated cytosines. The MeDIP method requires the input DNA sample to be single stranded in order for the 5-methylcytidine antibody to bind. Another method for the enrichment of methylated CpG islands uses a recombinant methyl-binding protein MBD2b, or the MBD2b/MBD3L1 complex, as in the MethylCollector™ Ultra Kit. One advantage of a methyl-CpG binding protein enrichment strategy is the input DNA sample does not need to be denatured, the protein can recognize methylated DNA in its native double-strand form. Another advantage is that the MBD method only binds to DNA methylated in a CpG context to ensure the enrichment of methylated-CpG DNA.

MethylCollector™ Ultra is based on the Methylated CpG Island Recovery Assay (MIRA), which utilizes a His-tagged recombinant methyl-binding protein complex, MBD2b/MBD3L1, that specifically binds methylated CpGs of genomic DNA fragments that have been prepared by sonication or enzymatic digestion. Nickel-coated magnetic beads capture the protein-DNA complexes, which are then separated from the rest of the genomic DNA using the included magnet. Optimized buffers ensure that fragments with little or no methylation are removed. The methylated DNA is then eluted from the beads in the presence of Proteinase K. Following clean up, the eluted DNA is ready for use in PCR analysis or other downstream applications. MethylCollector™ Ultra can efficiently enrich for methylated DNA from as few as 170 cells (~1 ng DNA).

Because CpG islands are normally unmethylated, the ability to enrich for unmethylated DNA fragments allows researchers to validate the methylation status of the locus of interest and identify hypomethylated DNA. While other techniques confirm methylation of CpG dinucleotides, a negative result is often designated as unmethylated DNA. With Active Motif's unique UnMethylCollector™ Kit, unmethylated promoters can now be verified using a recombinant His-tagged CXXC protein, from the mouse Mbd1, which specifically binds unmethylated DNA. This DNA can be analyzed by either endpoint or realtime PCR, or used in a variety of other applications.

Enriched DNA can be used in many downstream applications, such as endpoint or realtime PCR analysis of the methylation status of particular loci in normal and diseased samples, bisulfite conversion followed by cloning and sequencing, or amplification and labeling for microarray analysis.

 

DNA Methyltransferases

DNA methylation is catalyzed by DNA methyltransferase enzymes (DNMTs or DNA MTases) and consist in the addition of a methyl group from S-adenosyl-L-methionine (AdoMet) to the fifth carbon position of cytosine (cytosine-5 or C5), mostly within CpG dinucleotides. Three families of DNA methyltransferase enzymes have been identified: DNMT1, DNMT2 and DNMT3. These enzymes can be further classified as de novo methyltransferases (DNMT3A, DNMT3B and DNMT3L), enzymes that are able to methylate previously unmethylated CpG sequences, or maintenance methyltransferases (DNMT1), which copy pre-existing methylation marks onto new DNA strands during replication. DNMT3L is closely related to DNMT3A and DNMT3B structurally, but is catalytically inactive as a DNA methyltransferase. DNMT3L is known to associate with both DNMT3A and DNMT3B and may be responsible for the recruitment of histone deacetylases to direct repression onto newly established imprints. DNMT2 is not a DNA methyltransferase; it methylates cytosine 38 in the anticodon loop of tRNA16 but does not methylate DNA.

Active Motif's DNMT Activity / Inhibition Assay is a time-saving, non-radioactive assay to measure DNA methyltransferase activity and/or inhibition from recombinant DNMT enzymes (DNMT1, DNMT3a & DNMT3b) or nuclear extract samples. This sensitive ELISA-based method uses the ability of methyl CpG binding domain (MBD) proteins to bind methylated DNA with high affinity. In the DNMT assay method, a universal CpG-enriched DNA substrate has been immobilized on a 96-stripwell plate. Purified DNMTs or DNMT activities from nuclear extracts will catalyze the transfer of methyl groups from the provided AdoMet reagent to the coated DNA substrate. The resulting methylated DNA will be recognized by the His-tagged recombinant MBD2b in an amount proportional to the enzyme activity. Addition of a poly-Histidine antibody conjugated to horseradish peroxidase (HRP) provides a sensitive colorimetric readout that is easily quantified by spectrophotometry. The assay includes a sample of purified DNMT1 enzyme as a positive control.

 

Methylated Control DNA

In addition to complete assays, Active Motif also offers fully methylated Jurkat DNA that can be used as a positive control in methylation analysis studies. The Fully Methylated Jurkat DNA is supplied with a BRCA1 PCR primer set. As native Jurkat DNA is non-methylated at the BRCA1 locus, this primer set is ideal for use as a control in methylation specific assays.

Active Motif also offers the Methylated DNA Standard Kit. This kit contains three recombinant DNA standards derived from the APC gene promoter: unmethylated DNA, 5-methylcytosine methylated DNA and 5-hydroxymethylcytosine methylated DNA. This kit (which also includes PCR primers specific to the APC promoter) can be used as part of experiments utilizing our antibodies specific to the two different types of DNA methylation (see below).

 

DNA Methylation Antibodies

Active Motif offers a growing list of DNA methylation-related antibodies. Active Motif is committed to providing the highest quality antibodies for studying the biology of the nucleus. Each antibody we make is rigorously tested. Many of the DNA methylation antibodies have been validated for use in ChIP and immunofluorescence (IF).

A second form of DNA CpG methylation has recently been linked to epigenetic events. Several papers have been published in the past year describing the relative abundance of 5-hydroxymethylcytosine (5-hmC) in specific cell types and the conversion of 5-methylcytosine (5-mC) into 5-hmC. 5-hmC is replaced by cytosine by DNA repair proteins, thus conversion of 5-mC into 5-hmC may represent a pathway by which DNA is demethylated. The methyl group at position 5 of 5-hydroxymethylcytidine is oxidized to 5-hydroxymethylcytidine by the TET family of iron-dependant oxygenases. To facilitate the study of this new and potentially important regulatory event, Active Motif has developed the first antibody that recognizes 5-hmC, our 5-Hydroxymethylcytidine antibody. It specifically recognizes 5-hydroxymethylcytosine but not 5-methylcytosine
 

References

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2. Chen, T. & Li, E. Curr Top Microbiol Immunol 301, 179-201 (2006).
3. Svedruzic, Z.M. Curr Med Chem 15, 92-106 (2008).
4. Goll, M.G., et al. Science 311, 395-398 (2006).
5. Hendrich, B. & Bird, A. Mol Cell Biol 18, 6538-6547 (1998).
6. Nan, X., et al. Nature 393, 386-389 (1998).
7. Hendrich, B. & Tweedie, S. Trends Genet 19, 269-277 (2003).
8. Kondo, E., Gu, Z., Horii, A. & Fukushige, S. Mol Cell Biol 25, 4388-4396 (2005).
9. Zhu, W.G., et al. Mol Cell Biol 23, 4056-4065 (2003).
10. Rountree, M.R., Bachman, K.E. & Baylin, S.B. Nat Genet 25, 269-277 (2000).
11. Deplus, R., et al. Nucleic Acids Res 30, 3831-3838 (2002).
12. Fuks, F., Hurd, P.J., Deplus, R. & Kouzarides, T. Nucleic Acids Res 31, 2305-2312 (2003).
13. Lehnertz, B., et al. Curr Biol 13, 1192-1200 (2003).
14. Rea, S., et al. Nature 406, 593-599 (2000).
15. Brenner, C. & Fuks, F. Curr Top Microbiol Immunol 301, 45-66 (2006).
16. Weaver, J.R., Susiarjo, M. & Bartolomei, M.S. Mamm Genome 20, 532-543 (2009).
  

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