CGmCGCG is a versatile substrate with which to evaluate Tet protein activity

Kizaki, Seiichiro; Sugiyama, Hiroshi

doi:10.1039/c3ob41823e

Cited by 21 publications

(27 citation statements)

References 15 publications

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“…Consistently, it has been reported that TET proteins also act on DNA substrates containing six nucleotides. 21 Using a solid phase DNA synthesis technique, we prepared three palindromic DNA sequences 3 , 4 , and 5 each containing 22, 14 and 8 nucleotides, respectively, based on the original 58-mer DNA substrate (Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

A rapid mass spectrometric method for the measurement of catalytic activity of ten-eleven translocation enzymes

Sudhamalla

Dey

Breski

et al. 2017

Analytical Biochemistry

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Enzymatic methylation at carbon five on cytosine (5mC) in DNA is a hallmark of mammalian epigenetic programming and is critical to gene regulation during early embryonic development. It has recently been shown that dynamic erasure of 5mC by three members of the ten-eleven translocation (TET) family plays a key role in cellular differentiation. TET enzymes belong to Fe (II)- and 2-ketoglutarate (2KG) dependent dioxygenases that successively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5CaC), thus providing a chemical basis for the removal of 5mC which once was thought to be a permanent mark in mammalian genome. Since then a wide range of biochemical assays have been developed to characterize TET activity. Majority of these methods require multi-step processing to detect and quantify the TET-mediated oxidized products. In this study, we have developed a MALDI mass spectrometry based method that directly measures the TET activity with high sensitivity while eliminating the need for any intermediate processing steps. We applied this method to the measurement of enzymatic activity of TET1 and 3, Michaleis-Menten parameters (KM and kcat) of TET-2KG pairs and inhibitory concentration (IC50) of known small-molecule inhibitors of TETs. We further demonstrated the suitability of the assay to analyze chemoenzymatic labeling of 5hmC by β-glucosyltransferase, highlighting the potential for broad application of our method in deconvoluting the functions of novel DNA demethylases.

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Section: Resultsmentioning

confidence: 99%

A rapid mass spectrometric method for the measurement of catalytic activity of ten-eleven translocation enzymes

Sudhamalla

Dey

Breski

et al. 2017

Analytical Biochemistry

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“…A number of assays are available to probe for specific bases qualitatively and quantitatively. As noted earlier, Kizaki and Sugiyama reported using 4–6 nt substrates with direct resolution of the reaction products by HPLC (Kizaki & Sugiyama, 2014). However, longer substrates are needed to understand how strand specificity, sequence context, and other factors impact TET activity.…”

Section: Analysis Of Cytosine Modifications In Vitromentioning

confidence: 80%

“…In general, we use DNA oligonucleotides 12–35 nt in length, containing a single TET substrate (mC, hmC, or fC) in a CpG context (although useful HPLC-based assays for substrates as short as 4–6 nt have also been developed; Kizaki & Sugiyama, 2014). TET enzymes exhibit a strong preference for CpGs (Hu et al, 2013) but are thought to be less sensitive to surrounding sequences (Yu et al, 2012).…”

Section: Analysis Of Cytosine Modifications In Vitromentioning

confidence: 99%

Quantification of Oxidized 5-Methylcytosine Bases and TET Enzyme Activity

Liu

DeNizio

Kohli

2016

Methods in Enzymology

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In eukaryotic DNA, cytosine can be enzymatically modified to yield up to four epigenetic base variants. DNA methyltransferases convert cytosine to 5-methylcytosine (mC), which plays critical roles in gene regulation during development. Ten-eleven translocation (TET) enzymes can sequentially oxidize mC to three products: 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC). These oxidized bases have been found in numerous mammalian cell types, where they potentially carry out independent epigenetic functions and aid in DNA demethylation. To gain insight into the mechanisms and functions of TET family enzymes, rigorous approaches are needed to quantify genomic cytosine modifications in cells and track TET enzyme activity in vitro. Here, we present tools developed by our lab and others to report on each of the five forms of cytosine (unmodified, mC, hmC, fC, and caC) with high specificity and sensitivity. We provide detailed protocols for qualitative and quantitative analysis of cytosine modifications in genomic DNA by dot blotting and LC-MS/MS. We then describe methods for generating synthetic oligonucleotide substrates for biochemical studies, provide optimized reaction conditions, and introduce several chemoenzymatic assays, as well as HPLC, mass spectrometry, and scintillation counting methods to quantify cytosine modifications in vitro. These approaches enable mechanistic studies of TET activity, which are key to understanding the role of these enzymes in epigenetic regulation.

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“…The binding efficiency of the anti‐hmC monoclonal antibody toward the oligomers was assessed by using hexamer DNA containing either mC or hmC (Figure S1 in the Supporting Information) . The results clearly indicate specificity of the antibody toward hmC, even with short oligomers.…”

Section: Resultsmentioning

confidence: 99%

Identification of Sequence Specificity of 5‐Methylcytosine Oxidation by Tet1 Protein with High‐Throughput Sequencing

2016

Self Cite

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Tet (ten-eleven translocation) family proteins have the ability to oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). However, the oxidation reaction of Tet is not understood completely. Evaluation of genomic-level epigenetic changes by Tet protein requires unbiased identification of the highly selective oxidation sites. In this study, we used high-throughput sequencing to investigate the sequence specificity of mC oxidation by Tet1. A 6.6×10(4) -member mC-containing random DNA-sequence library was constructed. The library was subjected to Tet-reactive pulldown followed by high-throughput sequencing. Analysis of the obtained sequence data identified the Tet1-reactive sequences. We identified mCpG as a highly reactive sequence of Tet1 protein.

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CGmCGCG is a versatile substrate with which to evaluate Tet protein activity

Cited by 21 publications

References 15 publications

A rapid mass spectrometric method for the measurement of catalytic activity of ten-eleven translocation enzymes

A rapid mass spectrometric method for the measurement of catalytic activity of ten-eleven translocation enzymes

Quantification of Oxidized 5-Methylcytosine Bases and TET Enzyme Activity

Identification of Sequence Specificity of 5‐Methylcytosine Oxidation by Tet1 Protein with High‐Throughput Sequencing

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