Chapter Five - Enzymatic Analysis of Tet Proteins: Key Enzymes in the Metabolism of DNA Methylation
Introduction
One of the recent advances in the epigenetic filed is the discovery that Tet family proteins are capable of catalyzing the oxidation of 5-methylcytosine (5mC), a well-characterized epigenetic mark, into 5-hydroxymethylcytosine (5hmC) in mammalian DNA (Ito et al., 2010, Tahiliani et al., 2009). Remarkably, more recent studies have shown that 5hmC can be further oxidized by Tet proteins to generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (He et al., 2011, Ito et al., 2011), which have also been detected in the mouse genome (Ito et al., 2011, Pfaffeneder et al., 2011). These new findings suggest that Tet protein-catalyzed iterative oxidation of 5mC could be the initial steps in DNA demethylation pathways (Fig. 5.1). Indeed, immunostaining of the zygotic DNA have shown that loss of 5mC in the male pronucleus correlates with the appearance of 5hmC, 5fC, and 5caC (Gu et al., 2011, Inoue et al., 2011, Inoue and Zhang, 2011, Iqbal et al., 2011, Wossidlo et al., 2011), which are gradually diluted in a replication-dependent manner during mouse preimplantation development (Inoue et al., 2011, Inoue and Zhang, 2011). In addition to this passive demethylation process, both 5fC and 5caC, but not 5mC and 5hmC, can be actively removed from the genome by thymine-DNA glycosylase (TDG) through a base excision repair pathway (He et al., 2011, Maiti and Drohat, 2011). Thus, Tet-mediated iterative oxidation of 5mC plays an important role in regulating DNA methylation dynamics.
However, conventional approaches for DNA methylation studies, including bisulfite genomic sequencing and methylation-sensitive restriction enzyme digestion, cannot discriminate 5mC from other 5mC oxidation products such as 5hmC (Huang et al., 2010, Jin et al., 2010). It was also reported that 5fC and 5caC are interpreted as unmodified C in bisulfite genomic sequencing (Booth et al., 2012, He et al., 2011). To overcome these technical difficulties and to study Tet-mediated iterative oxidation, several techniques have been developed which allow quantification and genome-wide mapping of the cytosine derivatives. These techniques include (1) thin-layer chromatography (TLC) analysis of modified nucleotides (Ito et al., 2010, Kriaucionis and Heintz, 2009, Tahiliani et al., 2009), (2) liquid chromatography and mass spectrometry (LC-MS) analysis (Globisch et al., 2010, Ito et al., 2011, Munzel et al., 2010), (3) cytosine modification-specific antibodies (Ficz et al., 2011, Williams et al., 2011, Wu et al., 2011), (4) glucosylation of 5hmC (Kinney et al., 2011, Szwagierczak et al., 2010), (5) chemical/enzymatic labeling or conversion of modified cytosine (Booth et al., 2012, Pastor et al., 2011, Song, Szulwach, et al., 2011, Yu et al., 2012), and (6) single-molecule, real-time sequencing (Flusberg et al., 2010, Song, Clark, et al., 2011).
Here, we describe two methods that we have been using in studying the Tet enzymatic activity. The 2D-TLC method is easy to follow and does not require additional instruments, while the mass spectrometry method is more accurate and sensitive and can be used to quantify the levels of cytosine derivatives in genomic DNA.
Section snippets
Expression and Purification of Tet Proteins
For production of recombinant proteins, cDNAs encoding the catalytic domains of mouse Tet1 (aa1367–2039), Tet2 (aa916–1921), Tet3 (aa697–1668), and their corresponding catalytic mutants (Ito et al., 2010) are cloned into a modified pFastBac-HTb (Invitrogen) insect cell expression vector inframe with a FLAG tag at the N-terminus. Constructs are transformed into DH10Bac Escherichia coli (Invitrogen) following manufacturer's instructions to generate bacmid DNA. Since recombinant bacmid DNA is ≥ 135
Preparation of the substrates
To set up the in vitro enzymatic assays, proper DNA substrates are required. If the reaction products are to be analyzed by TLC assay, the modified cytosine should be placed in the context of a restriction site that can be digested regardless of the modification. Since TaqI (with a recognition site of TCGA) is insensitive to all cytosine modifications (Ito et al., 2011), we have been using double-stranded 20-mer DNA containing a TaqI site as our substrate with the following sequences:
Taq20-F:
Analysis of the Cytosine Derivatives by 2D-TLC
TLC is a classic method that separates different nucleotides based on their differential migration rates on TLC plates. TLC assays have been used successfully for analyzing 5hmC in previous studies (Koh et al., 2011, Kriaucionis and Heintz, 2009, Tahiliani et al., 2009). However, under previous TLC conditions, 5hmC and 5fC have almost identical migration patterns, and 5caC fails to migrate (Ito et al., 2011). To overcome this technical problem, we developed a modified 2D-TLC assay using a more
Analysis of the Cytosine Derivatives by Mass Spectrometry
For quantitative analysis of the cytosine derivatives, we developed a sensitive and specific method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reactions monitoring (MRM) to simultaneously detect and quantitate cytosine derivatives (Ito et al., 2011). The instrument we have been using is an ultra-performance liquid chromatography system (Waters) coupled to a TSQ-Quantum Ultra triple-quadrupole mass analyzer (ThermoFinnigan) with a heat-assisted electrospray
Analysis of the Endogenous Level of Cytosine Derivatives by Mass Spectrometry
For endogenous 5fC and 5caC in genomic DNA, they are first enriched by HPLC fractionation before being analyzed by mass spectrometry (Fig. 5.3B and C) (Ito et al., 2011). Before analyzing genomic DNA, the retention time for each of the cytosine derivatives needs to be determined using standard nucleosides (Fig. 5.3C). We have noticed that the retention time of 5caC is sensitive to pH of the digestion buffer and the mobile phase, thus the standard nucleosides should be prepared in the sample
Acknowledgments
We thank Dr. James A. Swenberg and Leonard B. Collins (UNC) for their help in the development of the mass spectrometry method. Y. Z. is a HHMI Investigator. This work was supported by NIH (GM68804 and U01DK089565) and the HHMI.
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