Name: efficient purification and lc-ms/ms-based assay development for ten-eleven translocation-2 5-methylcytosine dioxygenase
Uploaded: 2018-10-15T13:00:00.000+00:00
Duration: 10 min 33 s
Description: Watch this Scientific Journal Video about Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase at JoVE.com

This research was funded by the US Department of Defense in the form of an Idea Award (W81XWH-13-1-0174), Aplastic Anemia &amp; MDS Foundation Grant, and UMRB grant to M.M. Authors thank Mohit Jaiswal and Subhradeep Bhar for initial cloning of TET2 in pDEST14 vector.

1. Cloning and Purification of Untagged Human TET2 Dioxygenase
<ol>
	<li>Clone human TET2 dioxygenase (TET2 1129-1936, &#916;1481-1843) into the pDEST14 destination vector using the site-specific recombination technique as previously described22. 
	NOTE: Previous studies have demonstrated that the C-terminal TET2 dioxygenase (TET2 1129-1936, &#916;1481-1843) domain is the minimal catalytically active domain21,23. In order to express the untagged TET2 dioxygenase domain using the pDONR221 vector, Shine Dalgarno and Kozak sequences were incorporated in the forward primer during PCR (Table 1).</li>
	<li>For bacterial transformation, add 1 &#181;L of recombinant pDEST14 expression vector containing untagged human TET2 dioxygenase (TET2 1129-1936, &#916;1481-1843) to 100 &#181;L of chemically competent E. coli BL21 (DE3) cells in a 1.7 mL tube. Keep the mixture on ice for at least 15 minutes followed by heat shock at 42 &#176;C for 30 s in a water bath.
	<ol>
		<li>Immediately after heat shock, keep the cells back on ice for minimum of 2 min. Following this, add 250 &#181;L of super optimal broth with catabolite repression (S.O.C media) to cells. Incubate the bacterial cells for 1 h at 37 &#176;C in a shaker.</li>
		<li>After incubation, spin down the cells by centrifuging the tube at 9,000 x g for 1 min. Discard 70% supernatant by pipetting and dissolve the pellet in left over media.</li>
		<li>Spread the cell suspension on a Luria broth (LB) agar plate containing 100 &#181;g/mL ampicillin. Incubate the plate for 16 h at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Select one isolated colony and inoculate it into 10 mL of LB-ampicillin media in a 50 mL tube. Incubate the tube at 37 &#176;C in a shaker for overnight. Following this, use 100 &#181;L&#160; of culture from the 50 mL tube to inoculate 100 mL of LB-ampicillin media. Incubate the flask at 37 &#176;C on a shaker at 180 rpm and use it as primary culture. Next day, use 6 mL each of primary culture to inoculate 15 flasks, each containing 600 mL LB-ampicillin media. Now, incubate 15 flasks at 37 &#176;C on a shaker at 180 rpm. 
	NOTE: For verifying transformed clones, perform DNA sequencing or restriction digestion with the isolated plasmid DNA.</li>
	<li>To check the density of bacterial culture, measure its OD600 using a spectrophotometer. After the culture reaches a density of 0.8 at OD600, induce the expression of TET2 protein with 300 &#181;L of 1 M (final concentration of 0.5 mM in 600 mL) IPTG in each flask and further grow the culture for 16 h at 17 &#176;C.</li>
	<li>After 16 h, transfer the bacterial culture to centrifuge bottles. Centrifuge the bacterial culture expressing the TET2 enzyme at 5,250 x g for 45 min. Use the bacterial pellet for TET2 purification. 
	NOTE: Perform all remaining protein purification steps either on the ice or at 4 &#176;C.</li>
	<li>Resuspend the bacterial pellet in 100 mL of 50 mM MES (2-(N-morpholino)ethanesulfonic acid) buffer, pH 6 and sonicate for 5 &#215; 30 s at power 20 with 60 s cooling intervals.</li>
	<li>Spin the lysate at 5,250 x g for 45 min. Collect the supernatant containing the soluble TET2 enzyme and pass through 0.45-&#181;m filters before loading on an FPLC system. 
	NOTE: In these experiments, the TET2 enzyme was very stable, but if needed a protease inhibitor cocktail containing 1 mM benzamidine-HCl, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 0.5 mM 1,10-o-phenanthroline can be added to the cell lysate to prevent degradation of TET2 enzyme. Avoid using EDTA or EGTA in the inhibitor cocktail as these may interfere with the following cation-exchange chromatography.</li>
	<li>Pack 30 mL of a strong cation exchange resin into a FPLC column. Equilibrate the column with 10 bed volumes of wash buffer (50 mM MES buffer, pH 6) at the constant flow rate of 0.3 mL/min using a FPLC system.</li>
	<li>Load the clarified lysate onto the pre-equilibrated column and wash with &#8764;10 bed volumes of wash buffer until the flow through becomes clear.</li>
	<li>Elute TET2 using a 0-100% gradient from the wash buffer to the elution buffer (50 mM MES buffer, pH 6, 1 M NaCl) in 15 bed volumes followed by holding at 100% elution buffer for two-bed volumes. 
	NOTE: Collect samples (100 &#181;L each) of cell lysate before and after column loading along with all the elution fractions and analyze on 10% resolving SDS-PAGE.</li>
	<li>Pool the fractions containing TET2 protein, freeze dry, dissolve enzyme pallet in 10 mL water and store at -80 &#176;C.</li>
</ol>
2. 5mC Oxidation by TET2 Dioxygenase
<ol>
	<li>Perform all demethylation reactions in triplicate with 3 &#181;g of substrate (25-mer double stranded DNA, Table 2).
	<ol>
		<li>Add 100 &#181;g of purified TET2 enzyme in 50 &#181;L of total reaction buffer containing 50 mM HEPES (pH 8.0), 200 &#181;M FeSO4, 2 mM 2OG (2-oxoglutarate/&#945;-ketoglutarate), and 2 mM ascorbate and incubate at 37 &#176;C for 1 h21.</li>
		<li>Quench TET2 catalyzed oxidation reactions with 5 &#181;L of 500 mM EDTA.</li>
	</ol>
	</li>
	<li>After quenching TET2 reactions, prepare samples for LC-MS/MS analysis by separating the DNA from TET2 reaction mixture using oligo purification columns.
	<ol>
		<li>Add 100 &#181;L of oligo binding buffer to 55 &#181;L of quenched reaction.</li>
		<li>Following this, add 400 &#181;L of 100% ethanol to the mixture. Pass this mixture through an oligo binding column.</li>
		<li>Wash bound DNA with 750 &#181;L wash buffer and elute in 20 &#181;L water.</li>
	</ol>
	</li>
	<li>Digest the isolated DNA with 2 units of DNase I and 60 units of S1 nuclease at 37 &#176;C for 12 h to produce individual nucleoside-monophosphates.</li>
	<li>Following digestion, add 2 units of calf intestinal alkaline phosphatase (CIAP) in the samples and incubate for addition 12 h at 37 &#176;C to remove the terminal phosphate groups from nucleoside-monophosphates to obtain nucleosides.</li>
	<li>Quantify all nucleosides, especially modified cytosines, using the LC-MS/MS method described below.</li>
</ol>
3. Quantitative LC-MS/MS-based Assay Development
<ol>
	<li>Prepare 100 &#181;M stock solution of all modified cytosine nucleosides [5-methyl-2&#8242;-deoxycytidine (5mdC), 5-hydroxymethyl-2&#8242;-deoxycytidine (5hmdC), 5-formyl-2&#8242;-deoxycytidine (5fdC), and 5-carboxy-2&#8242;-deoxycytidine (5cadC)] and normal DNA bases (adenine, thymine, cytosine, and guanine) in HPLC-grade water for the development of the LC-MS/MS method.</li>
	<li>Optimize nucleoside-dependent MS/MS parameters by infusing stock solutions, one at a time, in mass spectrometer at a flow rate of 10 &#181;L/min in EMS scan mode. Optimize following parameters: Declustering Potential (DP), Entrance Potential (EP), Collision Cell Entrance Potential (CEP), Collision Energy (CE), and Collision Cell Exit Potential (CXP) for each DNA nucleoside using automated quantitative optimization feature of the software.</li>
	<li>Optimize source-dependent MS/MS parameters by injecting 10 &#181;L of stock solution using a gradient with 25% solvent B at a flow rate of 0.3 mL/min where solvent A is 10 mM ammonium acetate (pH 4.0) and solvent B is 20% acetonitrile with 10 mM ammonium acetate (pH 4.0). Optimize following parameters: Curtain Gas (CUR): 10-50, Temperature: 0-600 &#176;C, Gas Flow 1 (GS1): 0-50, Gas Flow 2 (GS2): 0-50, Collisionally Activated Dissociation (CAD): Low-Medium-High, Ion spray Voltage (IS): 4000-5500 for each DNA nucleoside using manual quantitative optimization feature of the software in FIA (Flow Injection Analysis) mode.</li>
	<li>To separate all eight DNA nucleosides, perform liquid chromatography using the following gradient: 0% solvent B (0-2 min), 0-20% solvent B (2-5 min), 20-60% solvent B (5-9 min), 60-0% solvent B (9-10 min) and then equilibrate with solvent A for 5 min at a flow rate of 0.3 mL/min on a C18 column (Particle Size: 5 &#181;m, Pore Size: 120 &#197;).</li>
	<li>Using the optimum MS/MS parameters (step 3.2 and 3.3) coupled with the above-mentioned liquid chromatography gradient (step 3.4), determine the response linearity, limit of detection (LOD), and lower limit of quantification (LLOQ) using a two-fold serial dilution of a 100 &#181;M standard mixture containing all eight nucleosides. Draw standard curves for all eight DNA nucleosides.</li>
	<li>Detect and quantify all nucleosides, especially modified cytosines, produced in step 2.4 using the LC-MS/MS method and standard curves.</li>
</ol>

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The authors have no financial interests to declare.

Mutations in TET2 gene are some of the most frequently detected genetic changes in patients with diverse hematopoietic malignancies. To date hundreds of different TET2 mutations, which include nonsense, frame-shift, and missense mutations, have been identified in patients12. Patients with TET2 mutations show low levels of genomic 5hmC in the bone marrow compared to those with wt-TET214. Mutant TET2 knock-in experiments have recapitulated the effects of the...

The C5 position of cytosine bases within CpG dinucleotides is the predominant methylation site (5mCpG) in mammalian genomes1. In addition, a number of recent studies have uncovered extensive C5 cytosine methylation (5mC) in non-CpG sites (5mCpH, where H = A, T, or C)2,3. 5mC modification serves as a transcriptional silencer at endogenous retrotransposons and gene promoters3,4,5. DNA methylation at 5mC also plays important roles in X chromosome inactivation, gene imprinting, nuclear reprogramming and tissue-specific gene expression5,6,7. Methylation of cytosine at the C5 position is carried out by DNA methyltransferases, and mutations in these enzymes cause significant developmental defects8. The removal of 5mC marks are initiated by TET1-3 5mC oxidases9,10. These TET-family dioxygenases convert 5mC into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by sequential oxidation steps11,12,13. Finally, thymine-DNA glycosylase replaces 5fC or 5caC to unmodified cytosine using the base excision repair pathway11.
The human TET2 gene was identified as a frequently mutated gene in diverse hematopoietic malignancies including myelodysplastic syndromes (MDS)14,15,16, MDS-myeloproliferative neoplasms (MDS-MPN), and acute myeloid leukemia (AML) originating from MDS and MDS-MPN16. The levels of 5hmC modification in the bone marrow DNA are lower in patients with TET2 mutations compared to those with wild type (wt)-TET214. A number of groups have developed TET2-knockout mouse models to elucidate its role in normal hematopoiesis and myeloid transformation17,18,19,20. These mice with mutations in the TET2 gene were initially normal and viable, but manifested diverse hematopoietic malignancies as they aged causing their early death. These studies showed the important roles played by the wt-TET2 in normal hematopoietic differentiation. In these mouse models, the heterozygous hematopoietic stem cells (TET2+/- HSCs) and homozygous TET2-/-&#160;HSCs had a competitive advantage over homozygous wt-TET2 HSCs in repopulating hematopoietic lineages as both TET2+/- and TET2-/-&#160;HSCs developed diverse hematopoietic malignancies17,18. These studies demonstrate that haploinsufficiency of TET2 dioxygenase alters the development of HSCs and results in hematopoietic malignancies.
Similar to mice with mutations in the TET2 gene, most leukemia patients manifest haploinsufficiency of TET2 dioxygenase activity. These mostly heterozygous somatic mutations include frame-shift and nonsense mutations dispersed throughout the TET2 gene body while missense mutations that are most clustered in the dioxygenase domain12. To date, little characterization of wt- and mutant-TET2 is reported in the literature mainly due to difficulties with the production of TET2 dioxygenase and its assay21. Here, we report a simple single-step purification of native TET2 dioxygenase using ion exchange chromatography. Further, a quantitative LC-MS/MS assay was optimized and used to measure the enzymatic activity of native TET2 dioxygenase.

<table><tbody><tr><td>HEPES</td><td>Carbosynth</td><td>FH31182</td><td></td></tr><tr><td>Iron(II) sulfate heptahydrate</td><td>Sigma-Aldrich</td><td>F8633</td><td></td></tr><tr><td>&amp;alpha;-Ketoglutaric acid (2-Oxoglutaric acid)</td><td>Sigma-Aldrich</td><td>K1750</td><td></td></tr><tr><td>L-Ascorbic acid</td><td>Sigma-Aldrich</td><td>A4544</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dihydrate</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>Ammonium acetate</td><td>Sigma-Aldrich</td><td>A1542</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher Scientific</td><td>75-05-8</td><td></td></tr><tr><td>HPLC grade water</td><td>Fisher Scientific</td><td>7732-18-5</td><td></td></tr><tr><td>Oligo clean and concentrator</td><td>Zymo Research</td><td>D4061</td><td></td></tr><tr><td>DNAse I</td><td>New England Biolabs</td><td>M0303S</td><td></td></tr><tr><td>S1 Nuclease</td><td>Thermo Scientific</td><td>ENO321</td><td></td></tr><tr><td>CIAP (Calf intestinal alkaline phosphatase)</td><td>New England Biolabs</td><td>M0290S</td><td></td></tr><tr><td>LB Media</td><td>Affymetrix</td><td>J75852</td><td></td></tr><tr><td>IPTG</td><td>Carbosynth</td><td>EI05931</td><td></td></tr><tr><td>MES [2-(N-Morpholino)ethanesulfonic acid monohydrate]</td><td>Carbosynth</td><td>FM37015</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>7647-14-5</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G7893</td><td></td></tr><tr><td>SP Sepharose</td><td>Fisher Scientific</td><td>45-002-934</td><td></td></tr><tr><td>2&#039;-Deoxy-5-methylcytidine</td><td>TCI</td><td>D3610</td><td></td></tr><tr><td>2&#039;-Deoxy-5-hydroxymethyalcytidine</td><td>TCI</td><td>D4220</td><td></td></tr><tr><td>2&#039;-Deoxycytidine-5-carboxylic acid, sodium salt</td><td>Berry &amp;amp; Associates</td><td>PY 7593</td><td></td></tr><tr><td>5-Formyl-2&#039;-deoxycytidine</td><td>Berry &amp;amp; Associates</td><td>PY 7589</td><td></td></tr><tr><td>2&#039;-Deoxycytidine</td><td>Berry &amp;amp; Associates</td><td>PY 7216</td><td></td></tr><tr><td>2&#039;-Deoxyadenosine</td><td>Carbosynth</td><td>ND04011</td><td></td></tr><tr><td>2&#039;-Deoxyguanosine</td><td>Carbosynth</td><td>ND06306</td><td></td></tr><tr><td>2&#039;-Deoxythymidine</td><td>VWR Life Science</td><td>97061-764</td><td></td></tr><tr><td>Gateway technology</td><td>Thermo Fisher</td><td>11801016</td><td></td></tr><tr><td>Beckman Allegra X-15R centrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>392932</td><td></td></tr><tr><td>Sonic Dismembrator 550</td><td>Fisher Scientific</td><td>XL2020</td><td></td></tr><tr><td>&amp;Auml;KTA FPLC system</td><td>Pharmacia (GE Healthcare)</td><td>18116468</td><td></td></tr><tr><td>FreeZone 4.5 freeze dry system</td><td>Labconco</td><td>7750020</td><td></td></tr><tr><td>Zymo Oligo purification columns&amp;nbsp;</td><td>Zymo Research</td><td>D4061</td><td></td></tr><tr><td>BDS Hypersil C18 column</td><td>Keystone Scientific, INC</td><td>105-46-3</td><td></td></tr><tr><td>3200 Q-Trap mass spectrometer</td><td>AB Sciex</td><td></td><td></td></tr><tr><td>HPLC&amp;nbsp;</td><td>Shimadzu HPLC&amp;nbsp;</td><td></td><td></td></tr><tr><td>XK16/20 FPLC column</td><td>Pharmacia (GE Healthcare)</td><td>28988937</td><td></td></tr></tbody></table>

efficient purification and lc-ms/ms-based assay development for ten-eleven translocation-2 5-methylcytosine dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Dynamic modification of 5mC in DNA by TET-family dioxygenases plays important roles in epigenetic transcriptional regulations. TET2 dioxygenase is frequently mutated in diverse hematopoietic malignancies12. To investigate the role of the TET2 enzyme in normal development and disease, we have cloned its minimal catalytically active domain without any affinity tag into the pDEST14 vector22. The untagged TET2 dioxygenase was produced at &#8764;...

Watch this Scientific Journal Video about Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase at JoVE.com

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

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Research

JoVE Journal

Biochemistry

8.1K Views.  University of Missouri-Kansas City. Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

Video: Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many lineages for regenerative medicine. ES cell culture in vitro has long been the subject of widespread concerns. Classically, mouse ES cells are maintained in serum and leukemia inhibitory factor (LIF)-containing medium. However, under serum/LIF conditions, cells show heterogeneity in morphology and the expression profile of pluripotency-related genes, and are mostly in a metastable state. Moreover, cultured ES cells exhibit global hypermethylation, but na&#239;ve ES cells of the inner cell mass (ICM) and primordial germ cells (PGCs) are in a state of global hypomethylation. The hypomethylated state of ICM and PGCs is closely associated with their pluripotency. To improve mouse ES cell culture methods, we have recently developed a new method based on the selectively combined utilization of two small-molecule compounds to maintain the DNA hypomethylated and pluripotent state. Here, we present that the co-treatment of vitamin C (Vc) and PD0325901 can erase about 90% of 5-methylcytosine (5mC) at 5 days in mouse ES cells. The generated 5mC content is comparable to that in PGCs. The mechanistic investigation shows that PD0325901 up-regulates Prdm14 expression to suppress Dnmt3b (de novo DNA methyltransferase) and Dnmt3l (the cofactor of Dnmt3b), by reducing de novo 5mC synthesis. Vc facilitates the conversion of 5mC to 5-hydroxymethylcytosine (5hmC) catalyzed mainly by Tet1 and Tet2, indicating the involvement of both passive and active DNA demethylations. Moreover, under Vc/PD0325901 conditions, mouse ES cells show homogeneous morphology and pluripotent state. Collectively, we propose a novel and chemical-synergy culture method for achieving DNA hypomethylation and maintenance of pluripotency in mouse ES cells. The small-molecule chemical-dependent method overcomes the major shortcomings of serum culture, and holds promise to generate homogeneous ES cells for further clinical applications and researches.

We described in detail two chemical-based protocols for culturing mouse embryonic stem cells. This new method utilizes synergistic mechanisms of promoting Tet-mediated oxidation (by vitamin C) and repressing de novo synthesis of 5-methylcytosine (by PD0325901) to maintain DNA hypomethylation and pluripotency of mouse embryonic stem cells.

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many ...

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

Genetics

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype&#8722;phenotype relations in various biological systems.

Detailed protocols for introducing an engineered split-TET2 enzyme (CiDER) into mammalian cells for chemical inducible DNA hydroxymethylation and epigenetic remodeling are presented.

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control ...

Chemistry

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

Developmental Biology

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of ...

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Histone Proteomic Analysis and Post-Translational Modification Characterization

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...