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A Complete Guide to 5-Methylcytosine (5-mC) Derivatives

Comprehensive explanation of 5-mC derivatives, including 5-hmC, 5-fC, and 5-caC and methods for quantification

5-methylcytosine derivatives

The most widely studied epigenetic modification, DNA methylation, involves the addition of a methyl group to position 5 of the cytosine base generating 5-methylcytosine (5-mC). Other modified cytosine nucleotides derived from 5-methylcytosine, namely 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), have been uncovered and are investigated as part of an active DNA demethylation pathway (Figure 1) and as possible epigenetic marks. These modified cytosines are derived from oxidation of 5-mC by members of the Ten-eleven translocation (TET) dioxygenase enzyme family. 5-hydroxymethylcytosine is generated by oxidation of 5-methylcytosine; and 5-formylcytosine and 5-carboxylcytosine are generated subsequently from 5-hmC or can be produced directly from 5-mC. For advancing DNA methylation and demethylation research, EpiGentek offers a variety of products for studying 5-mC and its derived cytosines.

5-methylcytosine (5-mC) generated by addition of a methyl group to position 5 of the cytosine base is the most studied of the epigenetic modifications. DNA methylation represses gene expression and has been the subject of intense investigation. It is catalyzed by DNA methyltransferases (DNMTs) generally at CpG dinucleotide positions, though non-CpG methylation has also been reported at CpA and CpT sites predominantly in plants and in embryonic stem cells. In normal somatic cells, approximately 60-80% of CpG sites are methylated. Some regions of the genome are heavily populated with CpG dinucleotides, particularly gene promoters, and these CpG dense regions are called CpG islands. They are generally unmethylated, which allows for transcriptional activation and gene expression. Aberrant methylation is believed to have a role in the pathogenesis and progression of several diseases, including cancer, which is characterized by global hypomethylation, but hypermethylation at certain gene promoters leading to silencing and repression of a number of tumor suppressor genes.

Researchers often start their DNA methylation studies by measuring the global levels of 5-mC in their samples. For this purpose, EpiGentek offers the MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit, which uses a specific 5-mC antibody to detect 5-mC in fragmented, as well as full-length DNA from a variety of species. This fast and easy one-step ELISA uses a small amount of DNA and results are highly comparable to those obtained by HPLC methods, but with lower cost and higher throughput.

DNA demethylation pathwayDNA Demethylation Pathway.

For gene-specific DNA methylation studies, the gold standard technique is bisulfite sequencing, which involves bisulfite conversion of the DNA combined with sequencing. DNA treatment with sodium bisulfite, converts the unmethylated cytosines to uracil, but the methylated cytosines are resistant to sodium bisulfite treatment and remain as cytosines. This allows for discrimination of the methylation status of the cytosines during sequencing, as the unmethylated cytosines will be read as thymines, while the methylated cytosines will be read as cytosines. For these purposes, EpiGentek offers the Methylamp DNA Modification Kit, which is suitable for bisulfite conversion of very small amounts of DNA, starting as low as 50 picograms. The unique reagents and protocol result in a very high conversion rate and extremely low degradation of DNA, preventing DNA loss. Bisulfite conversion can be combined with other downstream applications, including qPCR (methylation-speciific PCR), for which the Methylamp MS-qPCR Fast Kit is available to perform qPCR with the bisulfite converted DNA.

Other methods used in gene-specific methylation studies, include affinity-enrichment methods to pull-down methylated DNA. For these purposes, EpiGentek offers the EpiQuik MeDIP Ultra Kit for performing methylated DNA immunoprecipitation (meDIP). This kit uses a highly specific antibody to immunoprecipitate 5-mC from single or double-stranded DNA fragments, and assay can be performed with as little as 50 nanograms of DNA per reaction. EpiGentek also offers the EpiQuik Tissue Methylated DNA Immunoprecipitation Kit, which includes all buffers and reagents necessary for performing meDIP directly from mammalian tissues. An alternative approach to immunoprecipitation and enrichment by 5-mC antibodies, is binding of 5-mC by methyl-CpG binding domain proteins (MBDs). MBD proteins bind methylated DNA preferentially at CpG dinucleotides, and EpiGentek offers two kits for chromatin immunoprecipitation (ChIP) of methylated DNA based on MBD pull down using an antibody specific for MBD2. The EpiQuik Methyl-CpG Binding Domain Protein 2 ChIP Kit and the EpiQuik Tissue Methyl-CpG Binding Domain Protein 2 ChIP Kit, are specifically suited for use in cells and tissues, respectively. The immunoprecipitated methylated DNA obtained with the use of these meDIP and MBD2 ChIP kits is suitable for analysis by several downstream applications, including microarrays, PCR and sequencing.

DNMT quantification
High sensitivity and specificity achieved by using (a) recombinant DNMT1 and (b) nuclear extract with the EpiQuik DNMT Activity/Inhibition Assay Ultra Kit (Colorimetric).
EpiGentek also offers several products to researchers interested in studying DNA methyltransferases (DNMTs), the enzymes responsible for adding the methyl group to cytosine. Researchers can perform DNA methyltransferase (DNMT) activity or inhibition studies by using the EpiQuik DNMT Activity/Inhibition Assay Ultra Kit. With this kit, changes in activity of total DNMTs (DNMT1, DNMT3A and DNMT3B) contained in nuclear extracts or purified DNMTs can be quantitatively measured. This can be helpful in measuring activity levels of DNMTs in DNMT inhibitor-treated cells vs untreated cells, as well as measurement of DNMT activity in enzymatic studies. The high-throughput format allows easy and fast measurement in multiple samples. In addition, EpiGentek also offers a series of EpiQuik Dnmt Assay kits for quantification of protein amounts of DNMT1, DNMT3A and DNMT3B in nuclear extracts from cells or tissues, or in purified DNMT preparations.

For use in other DNA methylation studies, EpiGentek offers the 5-Methylcytosine Monoclonal Antibody [33D3], which detects 5-mC from a broad range of species, and is suitable for enrichment of 5-mC during meDIP studies, for staining by IHC, and for use in ELISA assays, among other applications.

Controls for use in DNA methylation studies are also available, including the DNA Methylation/Hydroxymethylation Standard Set, which contains methylated, hydroxymethylated, and unmodified DNA fragments that can be used as positive and negative controls. For instance, they are suitable for use as controls in methylation-specific PCR, or as substrates for studies with 5-mC or 5-hmC interacting proteins. For convenience, this set also includes primers to the unmodified and modified DNA fragments.

5-hydroxymethylcytosine (5-hmC)

5-hmC quantification
Accurate quantification of 5-hmC content of various DNA samples from different species with the MethylFlash™ Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit (Colorimetric). The results are closely correlated with those obtained by MS-LC.
5-hydroxymethylcytosine (5-hmC) is the product of oxidation of 5-mC by TET enzymes, and it’s the first derivative in the active DNA demethylation pathway. In contrast to 5-mC, which is associated with gene repression, 5-hmC is generally associated with gene activation and expression. Its highest levels have been found in adult brain, where it’s enriched in gene coding regions; while in embryonic stem cells, 5-hmC is associated with promoters and other gene regulatory regions. 5-hmC levels are lower in several cancers, hence has been proposed as a possible diagnostic marker.

For measurement of global levels of 5-hmC, EpiGentek offers the MethylFlash Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit. With this one-step ELISA, global levels of 5-hmC can be measured using DNA isolated from cells, tissues, plasma or serum, from a variety of species. It is a highly specific and sensitive assay, which detects as low as 0.01% hydroxymethylated DNA, with no cross-reactivity to 5-mC or unmethylated cytosine.

For gene-specific hydroxymethylated DNA measurement, EpiGentek offers the EpiQuik Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit. This kit allows the enrichment of 5-hmC from DNA of any species using a highly specific 5-hmC antibody, and the immunoprecipitated 5-hmC can be analyzed by several downstream applications, including PCR, microarrays or NGS.

To measure the enzymatic activity of TET enzymes, the enzymes that convert 5-mC to 5-hmC, in DNA demethylation studies, EpiGentek offers the Epigenase 5mC-Hydroxylase TET Activity/Inhibition Assay Kit. With this kit, the activity or inhibition of total TET enzymes can be measured in nuclear extracts, or in purified TET enzyme preparations. For TET enzymatic experiments, EpiGentek also offers TET1 Protein. This active recombinant TET1 protein is suitable for use in TET enzyme kinetic studies, as well as for screening TET inhibitors. In addition, EpiGentek has available a TET1 Polyclonal Antibody, great for performing 5-hmC immunoprecipitation and WB. Antibodies to the other two TET family members, TET2 and TET3, are also available for use in IHC, WB and ELISA.

Additional 5-hmC epigenetic studies can be performed using the 5-Hydroxymethylcytosine (5-hmC) Monoclonal Antibody [HMC/4D9], good to enrich hydroxymethylated DNA by hmeDIP, for IHC staining, and to measure 5-hmC by ELISA, among other applications. This antibody works great in a broad range of species.

For use as controls in 5-hmC studies, the DNA Methylation/Hydroxymethylation Standard Set contains hydroxymethylated, as well as methylated and unmodified DNA fragments, which can be used as positive and negative controls in DNA methylation and demethylation studies.

5-formylcytosine (5-fC)

5-fC quantification
Demonstration of high specificity of 5fC detection achieved by the MethylFlash™ 5-Formylcytosine (5-fC) DNA Quantification Kit (Colorimetric).
5-formylcytosine (5-fC) is derived from the oxidation of 5-mC or 5-hmC by TET family members during DNA demethylation. 5-fC is enriched in the promoters of actively transcribed genes in embryonic stem cells, and in enhancers and other gene regulatory regions. During DNA demethylation, 5-fC can be converted back to unmodified cytosine by a mechanism involving thymine DNA Glycosylase (TDG), followed by base excision repair.

For your 5-fC epigenetic studies, EpiGentek offers the MethylFlash 5-Formylcytosine (5-fC) DNA Quantification Kit for measuring global levels of 5-fC. This kit is suitable for use with DNA from all species and from cells, tissues and body fluids. The fast and easy colorimetric assay just requires a microplate-reader, and can detect as low as 1 picogram of 5-fC. It has been used successfully for quantification of 5-fC in mouse and human brain tissue, as well as cancer tissue. For studies involving thymine DNA Glycosylase, the enzyme that excises 5-fC during conversion of 5-fC to unmodified cytosine, investigators may use the Epigenase Thymine DNA Glycosylase (TDG) Activity/Inhibition Assay Kit. This ELISA-based kit allows direct measurement of TDG activity or inhibition in nuclear extracts or pure TDG enzyme preparations, without the need of time-consuming electrophoresis and chromatography-based methods.

5-carboxylcytosine (5-caC)

5-carboxylcytosine (5-caC) is the last cytosine derivative produced during DNA demethylation. It is generated by subsequent oxidations of 5-hmC, to 5-fC and to 5-caC; or directly from 5-mC. Similar to 5-fC, 5-caC is enriched at the promoters of actively transcribed genes in embryonic stem cells and can also be converted back to unmodified cytosine. Levels of 5-caC are increased in certain types of cancers.

The products highlighted above are just a few of the products available for DNA methylation and demethylation studies. Alternatively, for researchers looking to save time while still achieving excellent results in their epigenetic studies, EpiGentek offers several end-to-end services for DNA methylation and Chromatin analysis, including Bisulfite-Sequencing, meDIP-Seq, hmeDIP-Seq, ChIP-Seq and methylation-specific PCR (MS-PCR).

See Also: DNA Methylation Quantification Kits

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