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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 (DNMT3L) protein. 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 5-mC 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 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-hmC17,18. The methyl group at position 5 of 5-methylcytosine is oxidized to 5-hydroxymethylcytosine by the TET family of iron-dependent oxygenases. Further oxidation of 5-hydroxymethylcytosine by the Tet1 protein generates 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) respectively22,23. It has been proposed that the 5-caC modification can be removed from genomic DNA by thymine DNA glycosylase (TDG), thus illustrating a mechanism of DNA demethylation23.

Bisulfite Conversion Kit

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. Active Motif's Bisulfite Conversion Kit simplifies bisulfite conversion for easy 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. For complete details about bisulfite conversion, follow our link to Bisulfite Conversion.

In addition to our Bisulfite Conversion Kit, Active Motif also offers bisulfite conversion as a service through our Epigenetic Services.

Methylated DNA Enrichment Kits

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 (also referred to a Methylated DNA Immunoprecipitation or MeDIP) or with methyl-CpG binding domain (MBD) proteins. Active Motif offers assay kits for both methods of enrichment. Our MeDIP Kit and hMeDIP Kit can be used to specifically enrich for DNA fragments containing 5-methylcytosine and 5-hydroxymethylcytosine respectively, while our MethylCollector™ Ultra Kit specifically enriches for DNA fragments containing 5-methylcytosine methylation and our HypoMethylCollector™ Kit enriches for unmethylated DNA fragments. To learn more about the advantages of each affinity enrichment technique, follow our link to Methylated DNA Enrichment.

Global DNA Methylation Assay

As global hypomethylation has become a hallmark of most human cancers, the need to measure global DNA methylation has become more essential to enable the ability to associate 5-mC (5-methylcytosine) levels with correlative factors such as patient history or clinical outcome. Active Motif's Global DNA Methylation – LINE-1 Kit is designed to quantify methylation in Long Interspersed Nucleotide Element 1 (LINE-1) repeat elements of human genomic DNA. LINE-1 methylation serves as a surrogate for global DNA methylation levels and can be utilized to compare relative changes in 5-mC levels across different sample types, treatment conditions, clinical outcomes, dietary history or environmental backgrounds. The assay is formatted as an easy-to-use ELISA-based assay and is highly sensitive, enabling quantitation of as low as 0.5% methylation and can detect methylation from as little as 10 ng of input DNA. The assay also includes controls derived from human Jurkat genomic DNA as biologically relevant standards for quantitation of 5-mC.

DNA Methyltransferase (DNMT) Activity Assay

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. 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. For complete details on DNMTs, please follow the link to DNA Methyltransferases.

5-Hydroxymethylcytosine (5-hmC) Reagents

In mammalian genomes, DNA methylation usually occurs at the fifth carbon of cytosine residues. The recent discovery that the TET family of iron-dependent deoxygenases can convert 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) has raised questions about the functional relevance of 5-hmC in mammalian genomes. Active Motif offers several reagents to help elucidate the function of 5-hmC.

Our hMeDIP Kit can be used to selectively enrich double-stranded DNA fragments containing 5-hydroxymethylcytosine from the rest of the genomic DNA population. The purified 5-hydroxymethylcytosine antibody used in the hMeDIP Kit is also available separately, as are a mouse monoclonal 5-hydroxymethylcytosine antibody and rabbit polyclonal serum. In addition to our assay kits and antibodies, Active Motif also offers hMeDIP-Seq and hMeDIP-chip services.

DNA containing 5-hydroxymethylcytosine is able to undergo further modification when a β-Glucosyltransferase enzyme transfers the glucose moiety from UDP-Glucose (uridine diphosphoglucose) to 5-hydroxymethylcytosine residues in double-stranded DNA to generate glucosyl-5-hmC DNA. Glucosylated DNA can be quantified20 or used to differentiate 5-hmC DNA from 5-mC DNA using glucosyl-sensitive restriction enzymes. Alternatively, 5-hmC and 5-mC DNA can be directly differentiated with the PvuRts1 I restriction enzyme. The PvuRts1I enzyme cleaves 5-hmC DNA without digesting unmethylated or 5-mC DNA. Additionally, PvuRts1I can cleave both glucosylated and non-glucosylated 5-hydroxymethylcytosine DNA21.

Active Motif's Hydroxymethyl Collector™ Kit is designed for the detection and enrichment of DNA fragments containing 5-hydroxymethylcytosine using a biotin-streptavidin capture method. The Hydroxymethyl Collector Kit utilizes the modification properties of β-Glucosyltransferase to specifically alter 5-hmC residues with a modified glucose. A biotin conjugate is chemcially introduced and streptavidin magnetic beads are used to enrich for DNA fragments containing 5-hydroxymethylcytosine. Enriched DNA can be used for individual gene analysis by PCR or for whole-genome analysis with microarrays or sequencing.

For more information on 5-hmC, please follow the link to 5-Hydroxymethylcytosine.

For more information on 5-hmC, please follow the link to 5-Hydroxymethylcytosine.

DNA Methylation Variants (5-caC & 5-fC)

In addition to 5-hydroxymethylcytosine, other DNA variants have also been identified and characterized in mouse embryonic stem (ES) cells. Researchers have shown that the TET family of cytosine oxygenase enzymes, which convert 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC), can further oxidize hydroxymethylcytosine into 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC)22,23. Both of these DNA modifications have been shown to exist in mouse ES cells, and 5-formylcytosine has also been identified in major mouse organs. Both 5-fC and 5-caC exist in the paternal pronucleus, concomitant with the disappearance of 5-methylcytosine, suggesting that these DNA variants may be involved in DNA demethylation24. One hypothesis for the mechanism of DNA methylation suggests that 5-caC is excised from genomic DNA by thymine DNA glycosylae (TDG), thereby returning DNA to an unmethylated state23. While other research suggests that replication-dependent dilution accounts for paternal DNA demethylation during preimplantation development24.

To better understand the roles that 5-formylcytosine and 5-carboxylcytosine might play in DNA demethylation, Active Motif offers antibodies to each of these DNA modifications. Additionally, positive and negative control DNA for the analysis of 5-carboxylcytosine is available in the 5-Carboxylcytosine DNA Standard Kit.

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 controls for experiments studying 5-methylcytosine and 5-hydroxymethylcytosine methylation. The DNA standards have been validated using Active Motif's DNA methylation antibodies.

To study the newest DNA modifications, Active Motif offers the 5-Carboxylcytosine DNA Standard Kit. This kit contains a double-stranded DNA oligonucleotide with carboxylcytosine modifications and an equivalent unmodified DNA oligonucleotide. The 5-Carboxylcytosine DNA Standard Kit is designed to be used as positive and negative controls for the analysis of 5-carboxylcytosine residues in conjunction with Active Motif's 5-carboxylcytosine antibody.

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). Affinity enrichment antibodies for 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) are also available as stand alone antibodies.

DNA Methylation Services

With the acquisition of Genpathway, Active Motif has expanded its product offering to now include Epigenetic Services. The DNA methylation services include MethylPath™, 5-hmC MeDIP-seq, 5-mC MeDIP-seq and bisulfite sequencing.


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