Name: continuous fluorescence-based endonuclease-coupled dna methylation assay to screen for dna methyltransferase inhibitors
Uploaded: 2022-08-05T13:00:00.000+00:00
Duration: 6 min 7 s
Description: Watch this Scientific Journal Video about Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors at JoVE.com

The authors thank Bucknell University and the Department of Chemistry for their support of this work.

1. Prepare assay solutions for the screen
NOTE: The concentrations of substrates used in this assay can be adapted. For RFTS-lacking DNMT1, the experimentally determined Km values for the hairpin DNA substrate and SAM are 1&#8211;2 nM and 2 &#181;M, respectively26,40.
<ol>
	<li>Prepare 600 &#181;L of each assay condition being tested in microcentrifuge tubes on ice. 
	NOTE: The total volume of assay solution needed depends on the number of replicates being conducted. Each assay condition was run in triplicate for this protocol.
	<ol>
		<li>Add ddH2O and 5x methylation buffer (250 mM Tris pH 7.5, 5 mM MgCl2, 500 mM potassium glutamate, 5 mM dithiothreitol (DTT), 25% glycerol) to each tube to achieve a final concentration of 1x buffer. For assays containing 20 &#181;M compound, add 472 &#181;L of ddH2O and 120 &#181;L 5x buffer. For assays containing 50 &#181;M compound, add 470 &#181;L of ddH2O and 120 &#181;L of 5x buffer.</li>
		<li>Add 3.15 &#181;L of 20 mg/mL bovine serum albumin (BSA) to each sample.</li>
		<li>Add 2 &#181;L of 3.15 &#181;M hairpin DNA substrate and 1.33 &#181;L of 4.75 mM SAM to each sample. 
		NOTE: The concentration of substrates in the assay solution mixture is 1.05x the desired final concentrations.</li>
		<li>Add inhibitor to a final concentration of 21 &#181;M (1.26 &#181;L of 10 mM) or 52.5 &#181;M (3.15 &#181;L of 10 mM) to the appropriate samples. Add an equivalent amount of dimethylsulfoxide (DMSO) to a control sample. 
		NOTE: The concentration of inhibitor used in the screen can be varied. The maximum final DMSO concentration should be &#8804;5% (v/v). DMSO concentrations up to 5% (v/v) have no effect on the activity observed in the assay26.</li>
	</ol>
	</li>
	<li>Mix the solutions by vortexing (3,000 rpm) for 3 s. Spin the samples for a few seconds in a tabletop mini centrifuge (1,200 &#215; g) to ensure that the solution is collected at the bottom of the tube.</li>
	<li>Aliquot 95 &#181;L of each assay solution into 6 consecutive wells in a black half-area 96-well plate (Figure 2). 
	NOTE: These screening assays were performed in two batches. First, 20 &#181;M inhibitor samples (4 total conditions) were examined, followed by the 50 &#181;M inhibitor samples (4 total conditions).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62949/62949fig02.jpg" /> 
Figure 2: Assay plate setup. Each assay solution is aliquoted into six wells in the black 96-well plate: DMSO control (blue), compound 1 (green), compound 2 (red), and compound 3 (yellow). Both RFTS-lacking DNMT1 and Gla I will be added to three wells. As a control, Gla I alone will be added to the other three wells. Abbreviations: RFTS = Replication Foci Targeting Sequence; DNMT1 = DNA methyltransferase 1; DMSO = dimethylsulfoxide. <a href="https://www.jove.com/files/ftp_upload/62949/62949fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Prepare enzyme solutions for screen
NOTE: RFTS-lacking DNMT1 can be expressed in E. coli and purified to homogeneity. Expression and purification procedures for RFTS-lacking DNMT1 have been described previously41. The volume of enzyme needed depends on the number of assays being conducted. Here, four different assays are being performed in each set; each assay is completed in triplicate.
<ol>
	<li>Prepare 75 &#181;L of each enzyme solution in microcentrifuge tubes on ice. 
	NOTE: Two enzyme solutions will be needed. One solution will contain DNMT1 and Gla I; the other will contain only Gla I.
	<ol>
		<li>Add ddH2O and 5x methylation buffer (250 mM Tris pH 7.5, 5 mM MgCl2, 500 mM potassium glutamate, 5 mM DTT, 25% glycerol) to each tube to achieve a final concentration of 1x buffer. For the Gla I enzyme solution, add 15 &#181;L of 5x buffer and 58.8 &#181;L ddH2O. For the DNMT1+Gla I enzyme solution, add 15 &#181;L of 5x buffer and 58.2 &#181;L ddH2O.</li>
		<li>Add 1.2 &#181;L of 10 U/&#181;L Gla I to each solution for a final concentration of 0.16 U/&#181;L.</li>
		<li>Add 0.6 &#181;L of 5 &#181;M RFTS-lacking DNMT1 for a final concentration of 40 nM to the DNMT1+Gla I solution.</li>
	</ol>
	</li>
	<li>Tap gently to mix the solutions. Spin the samples for a few seconds in a tabletop mini centrifuge (1,200 &#215; g) to ensure that the solution is collected at the bottom of the tube.</li>
	<li>Aliquot 12 &#181;L of each enzyme solution into 6 wells in a conical-bottomed 96-well plate (Figure 3).</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62949/62949fig03.jpg" /> 
Figure 3: Enzyme plate setup. The Gla I (grey) and DNMT1+Gla I (blue) solutions are each aliquoted into six wells in the 96-well plate. Using a multichannel pipet, the enzyme can be added to a row of assay solutions simultaneously. For each assay condition (six wells), three wells will receive DNMT1+Gla I, and three wells will receive Gla I alone. Abbreviation: DNMT1 = DNA methyltransferase 1. <a href="https://www.jove.com/files/ftp_upload/62949/62949fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Run the assay and analyze the data.
<ol>
	<li>Preheat the plate reader to 37 &#176;C. Insert the black plate containing the assay solutions. Shake the plate (double orbital at 425 cpm for 5 s) and read the fluorescence with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Incubate the plate at 37 &#176;C for 5 min. 
	NOTE: Alternatively, filters with proper wavelength cutoffs can be used for the fluorescence readings.</li>
	<li>Remove the assay plate. Add 5 &#181;L of enzyme solution to each well and mix by pipetting up and down. 
	NOTE: Use multichannel pipets so that a row of samples can be initiated simultaneously.</li>
	<li>Insert the plate into the plate reader. Record fluorescence every 53 s for 30 min. Shake the plate (double orbital at 425 cpm for 4 s) before each reading.</li>
	<li>Average triplicate Gla I control assays for each condition. Subtract the average Gla I-containing control reaction trace from the triplicate traces obtained in the presence of RFTS-lacking DNMT1. Determine the average and standard error of the mean for the corrected replicates.</li>
	<li>Plot the average corrected reaction trace and fit the initial linear portion to a line to determine the initial velocity.</li>
	<li>Determine the percent activity by dividing the velocity observed in the presence of a compound by the velocity observed in the DMSO-containing control reaction and multiplying by 100.</li>
</ol>
4. Additional assay control &#8212; sequential addition of enzymes
<ol>
	<li>Prepare 550 &#181;L of assay solution in a microcentrifuge tube on ice. Add 110 &#181;L of 5x methylation buffer, 3.88 &#181;L of 3.15 &#181;M hairpin DNA substrate, 6.43 &#181;L of 47.5 mM SAM, 3.06 &#181;L of 20 mg/mL BSA, and 426.6 &#181;L of ddH2O.</li>
	<li>Mix the solution by vortexing (3,000 rpm) for 3 s. Spin the samples for a few seconds in a tabletop mini centrifuge (1,200 &#215; g) to ensure that the solution is collected at the bottom of the tube.</li>
	<li>Aliquot 90 &#181;L of the assay solution into 6 consecutive wells in a black half-area 96-well plate.</li>
	<li>Prepare the DNMT1 enzyme solutions on ice. Add 4 &#181;L of 5x methylation buffer, 0.76 &#181;L of 5 &#181;M RFTS-lacking DNMT1, and 15.24 &#181;L of ddH2O to one tube. Add 4 &#181;L of 5x methylation buffer and 16 &#181;L of ddH2O to another tube.</li>
	<li>Tap gently to mix the solutions. Spin the samples for a few seconds in a tabletop mini centrifuge (1,200 &#215; g) to ensure that the solution is collected at the bottom of the tube.</li>
	<li>Add 5 &#181;L of the DNMT1-containing solution to three wells in the black half-area 96-well plate. Add 5 &#181;L of the buffer control solution to the other three wells.</li>
	<li>Place the microtiter plate into the plate reader preheated to 37 &#176;C. Shake the plate (double orbital at 425 cpm for 3 s) and read the fluorescence with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Repeat the shake and read every 60 s for 30 min.</li>
	<li>While the assay plate is in the plate reader, prepare the Gla I solution on ice. Add 8 &#181;L of 5x methylation buffer, 0.64 &#181;L of 10 U/&#181;L Gla I, and 31.4 &#181;L of ddH2O to a microcentrifuge tube. Tap gently to mix the solution. Spin the sample for a few seconds in a tabletop mini centrifuge (1,200 &#215; g) to ensure that the solution is collected at the bottom of the tube.</li>
	<li>Aliquot 6 &#181;L of the Gla I solution into 6 wells in a conical-bottomed 96-well plate.</li>
	<li>Following the initial 30 min read, remove the black plate from the plate reader. Add 5 &#181;L of Gla I solution to all 6 wells and mix by pipetting up and down. 
	NOTE: Use a multichannel pipet so that all wells can be initiated simultaneously.</li>
	<li>Place the black plate back into the plate reader. Record fluorescence every 35 s for 35 min. Shake the plate (double orbital at 425 cpm for 3 s) before each reading.</li>
</ol>

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dnmt3b+methylates+DNA+by+a+noncooperative+mechanism,+and+its+activity+is+unaffected+by+manipulations+at+the+predicted+dimer+interface.">Dnmt3b methylates DNA by a noncooperative mechanism, and its activity is unaffected by manipulations at the predicted dimer interface.</a> Biochemistry. 57 (29), 4312-4324 (2018).</li><li>Bashtrykov, P., Ragozin, S., Jeltsch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+details+of+the+DNA+recognition+by+the+Dnmt1+DNA+methyltransferase.">Mechanistic details of the DNA recognition by the Dnmt1 DNA methyltransferase.</a> FEBS Letters. 586 (13), 1821-1823 (2012).</li><li>Bashtrykov, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutagenesis+results+in+an+activation+of+DNA+methyltransferase+1+and+confirms+an+autoinhibitory+role+of+its+RFTS+domain.">Targeted mutagenesis results in an activation of DNA methyltransferase 1 and confirms an autoinhibitory role of its RFTS domain.</a> Chembiochem. 15 (5), 743-748 (2014).</li><li>Berkyurek, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+methyltransferase+Dnmt1+directly+interacts+with+the+SET+and+RING+finger-associated+(SRA)+domain+of+the+multifunctional+protein+Uhrf1+to+facilitate+accession+of+the+catalytic+center+to+hemi-methylated+DNA.">The DNA methyltransferase Dnmt1 directly interacts with the SET and RING finger-associated (SRA) domain of the multifunctional protein Uhrf1 to facilitate accession of the catalytic center to hemi-methylated DNA.</a> Journal of Biological Chemistry. 289 (1), 379-386 (2014).</li><li>Kanada, K., Takeshita, K., Suetake, I., Tajima, S., Nakagawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+threonine+1505+in+the+catalytic+domain+stabilizes+mouse+DNA+methyltransferase+1.">Conserved threonine 1505 in the catalytic domain stabilizes mouse DNA methyltransferase 1.</a> Journal of Biochemistry. 162 (4), 271-278 (2017).</li><li>Bashtrykov, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+Dnmt1+for+methylation+of+hemimethylated+CpG+sites+resides+in+its+catalytic+domain.">Specificity of Dnmt1 for methylation of hemimethylated CpG sites resides in its catalytic domain.</a> Chemistry &amp; Biology. 19 (5), 572-578 (2012).</li><li>Dolen, E. K., McGinnis, J. H., Tavory, R. N., Weiss, J. A., Switzer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-associated+mutations+G589A+and+V590F+relieve+replication+focus+targeting+sequence-mediated+autoinhibition+of+DNA+methyltransferase+1.">Disease-associated mutations G589A and V590F relieve replication focus targeting sequence-mediated autoinhibition of DNA methyltransferase 1.</a> Biochemistry. 58 (51), 5151-5159 (2019).</li><li>Kilgore, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+DNMT1+selective+antagonists+using+a+novel+scintillation+proximity+assay.">Identification of DNMT1 selective antagonists using a novel scintillation proximity assay.</a> Journal of Biological Chemistry. 288 (27), 19673-19684 (2013).</li><li>Ceccaldi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+inhibitors+of+DNA+methylation+by+screening+of+a+chemical+library.">Identification of novel inhibitors of DNA methylation by screening of a chemical library.</a> ACS Chemical Biology. 8 (3), 543-548 (2013).</li><li>Duchin, S., Vershinin, Z., Levy, D., Aharoni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+kinetic+assay+for+protein+and+DNA+methyltransferase+enzymatic+activities.">A continuous kinetic assay for protein and DNA methyltransferase enzymatic activities.</a> Epigenetics &amp; Chromatin. 8, 56 (2015).</li><li>Syeda, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+replication+focus+targeting+sequence+(RFTS)+domain+is+a+DNA-competitive+inhibitor+of+Dnmt1.">The replication focus targeting sequence (RFTS) domain is a DNA-competitive inhibitor of Dnmt1.</a> Journal of Biological Chemistry. 286 (17), 15344-15351 (2011).</li><li>Switzer, R. L., Medrano, J., Reedel, D. A., Weiss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substituted+anthraquinones+represent+a+potential+scaffold+for+DNA+methyltransferase+1-specific+inhibitors.">Substituted anthraquinones represent a potential scaffold for DNA methyltransferase 1-specific inhibitors.</a> PLoS One. 14 (7), 0219830 (2019).</li></ol>

The authors have no conflicts of interest to disclose.

To identify and characterize inhibitors of DNA methyltransferases, the activity of the enzyme must be measured. Several methods for examining DNA methyltransferase activity exist. Activity is commonly monitored using radioactivity; transfer of the labeled methyl group of SAM can be quantified29,30,31,32,33,34. Gel-based assay...

DNA methylation is an important epigenetic mark that regulates gene expression and chromatin structure. Methylation occurs predominately in CpG dinucleotides &#8212; cytosine followed by guanosine; the methyl group is added to the 5-position of cytosine. Correct DNA methylation patterns, and thus proper gene expression, are needed for appropriate cellular development and function. Many disease states have been associated with changes to the normal methylation pattern1,2,3. For example, there is a link between cancer initiation and progression and alterations to the DNA methylation pattern. Typically, cancer cells exhibit lower overall levels of methylcytosine, which contributes to genome instability. At the same time, the methylcytosine that is present in the genome is concentrated in the promoter regions of tumor suppressor genes, which leads to gene silencing of these important proteins. Notably, epigenetic changes are dynamic and reversible, unlike the DNA mutations associated with tumorigenesis. This has made the proteins involved in epigenetic gene regulation interesting drug targets2,4.
DNA methyltransferases (DNMTs) are the proteins responsible for generating and maintaining DNA methylation patterns. Three catalytically active isozymes, DNMT1, DNMT3a, and DNMT3b, exist in humans. During development and differentiation, the de novo methyltransferases, DNMT3a and DNMT3b, establish methylation patterns. Both enzymes can bind the catalytically inactive DNMT3L protein to form complexes that exhibit increased activity1,5. Following cell division, daughter cells contain hemimethylated DNA &#8212; DNA containing methylcytosine in only one strand of the duplex &#8212; because the newly synthesized DNA is devoid of methylation marks. The major function of DNMT1 is to methylate this hemimethylated DNA, thus re-establishing the full methylation pattern1,5.
Links between DNMT activity and cancer are well established. Overexpression of DNMT1, either by transcriptional or post-translational mechanisms, is a consequence of several common oncogenic pathways6,7,8,9. Genetic approaches to lower DNMT1 activity using hypomorphic alleles result in decreased tumor formation in Apc(Min)mice10. Antisense oligonucleotides that knockdown DNMT1 inhibit neoplasia in cell culture and mouse tumor models11,12. Thus, inhibiting DNMT1 activity seems like a promising cancer therapy approach. However, the roles the DNMT3 isozymes play are not so straightforward. DNMT3a mutations are found in acute myeloid leukemia13 and myelodysplastic syndrome14. At least one of the identified mutations has been shown to decrease the DNA methylation activity of the enzyme15. However, DNMT3b is overexpressed in breast cancer16 and colorectal cancer17. With the various DNMT isozymes playing different roles in carcinogenesis, identifying isozyme-specific inhibitors will be critical. Not only will these compounds be useful for the development of therapeutics, but isozyme-specific inhibitors would also be an invaluable tool to dissect the role of each DNMT isozyme in cancer etiology.
Several DNMT inhibitors have been reported in the literature. Known DNMT inhibitors can be divided into two classes: nucleoside and non-nucleoside. Nucleoside inhibitors are typically cytidine analogs. These compounds are incorporated into DNA and covalently trap DNMTs. 5-azacytidine and 5-aza-2&#39;-deoxycytidine have been approved for the treatment of myelodysplastic syndrome and acute myeloid leukemia4,18. The high toxicity, low bioavailability, and chemical instability of these compounds present problems. Ongoing work is examining the efficacy of the next generation of nucleoside inhibitors; SGI-110, derived from 5-aza-2&#39;-deoxycytidine, is one example19,20. Nucleoside inhibitors are not isozyme-specific and will inactivate any DNMT isozyme encountered. Therefore, treatment with a nucleoside-demethylating agent results in the depletion of all DNMT isozymes4,18. Non-nucleoside inhibitors do not need to be incorporated into DNA to exert their inhibitory effects. Instead, these molecules bind directly to DNMTs, introducing the possibility for isozyme-specific inhibition. Several non-nucleoside inhibitors have been discovered to date, including SGI-102721, hydralazine22, procainamide23, RG108 and derivatives24, and natural products, (&#8722;)-epigallocatechin 3-gallate (EGCG)25 and laccaic acid A26,27. Most of the non-nucleoside inhibitors discovered to date are not isozyme-selective or display weak preferences for one DNMT isozyme. In addition, the potency of these molecules needs to be improved, especially in cells4,18. Thus, there is a need to discover or develop more potent, isozyme-selective DNMT inhibitors.
A hurdle to discovering new small molecule inhibitors of DNMTs is the laborious assays traditionally used to examine DNMT activity28. Assays are usually discontinuous with multiple steps. The enzymatic activity of DNMTs is still routinely assayed using radioactive S-adenosyl methionine (SAM)29,30,31,32,33,34. Non-radioactive assays for DNA methylation have been developed as well. For example, assays utilizing methyl-sensitive restriction endonucleases and electrophoresis to separate the digestion products have been described35,36. These types of discontinuous, multistep assays are not readily amenable to drug discovery. Since the mid-2000s, several DNA methylation assays with a higher throughput have been developed28. A scintillation proximity assay was used to screen for DNMT1 inhibitors37. Another assay utilizing a methyl-sensitive restriction endonuclease was used to screen for DNMT3a inhibitors25,38. While both assays allowed for higher throughput than traditional DNA methylation assays, the assays require multiple steps and do not allow the observation of methylation activity in real time. More recently, a continuous kinetics assay has been described that couples the formation of S-adenosylhomocysteine (SAH), one product of the methylation reaction, to the spectroscopic change at 340 nm associated with NADPH oxidation39. This assay utilizes three coupling enzymes to generate a spectroscopic signal.
We developed a fluorescence-based endonuclease-coupled DNA methylation assay that utilizes a single commercially available coupling enzyme and can generate data in real time (Figure 1). A hairpin oligonucleotide containing three methylcytosines is used as a substrate. The substrate DNA contains a fluorophore on the 5&#39; end and a quencher on the 3&#39; end. Methylation of the hemimethylated CpG site generates the cleavage site for the endonuclease Gla I &#8212; fully methylated GCGC. Gla I cleavage of the product oligonucleotide releases the fluorophore from the quencher and generates fluorescence in real time. The assay can be used to examine the activity of any isoform of DNMT; however, higher activity is observed with DNMT1 as this isozyme preferentially methylates hemimethylated DNA1,5. Even more robust activity is observed if the autoinhibitory Replication Foci Targeting Sequence (RFTS) domain is removed from DNMT1. This domain, found in the N-terminal regulatory region, binds to the catalytic site and prevents DNA binding. Removal of the first ~600 amino acids results in a truncated enzyme that is significantly more active than the full-length enzyme (~640-fold increase in kcat/Km)40. This activated form of the enzyme, referred to as RFTS-lacking DNMT1 (amino acids 621&#8211;1616), allows for the easier identification of inhibitors due to its increased catalytic power. This paper presents a protocol to utilize RFTS-lacking DNMT1 in assays to screen for potential small molecule inhibitors. Using the endonuclease-coupled continuous assay, the initial velocity is determined in the presence and absence of a few small molecules. Each potential inhibitor is examined at two concentrations to look for concentration-dependent DNMT1 inhibition. The percent activity observed in the presence of the small molecules was calculated in each case.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62949/62949fig01.jpg" /> 
Figure 1: DNA methylation assay. A hemimethylated hairpin DNA with a fluorophore on the 5&#39; end and a quencher on the 3&#39; end is used as a substrate. DNMT1 catalyzes the transfer of the methyl group from S-adenosylmethionine to the nonmethylated CpG site, generating S-adenosylhomocysteine and fully methylated DNA. The DNA product contains the cleavage site for the endonuclease Gla I, which cleaves fully methylated GCGC sites. Cleavage of the product DNA releases the 5&#39; fluorophore from the 3&#39; quencher, generating fluorescence. Abbreviations: Fl = fluorophore; Q =quencher; DNMT1 = DNA methyltransferase 1; SAM = S-adenosylmethionine; SAH = S-adenosylhomocysteine. <a href="https://www.jove.com/files/ftp_upload/62949/62949fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>96-well Half Area Black Flat Bottom Polystyrene Not Treated Microplate</td><td>Corning</td><td>3694</td><td></td></tr><tr><td>96-Well Polystyrene Conical Bottom Plates</td><td>ThermoFisher</td><td>249570</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>NEB</td><td>B9000S</td><td></td></tr><tr><td>compound 1</td><td>ChemBridge</td><td>5812086</td><td>screening compound; resuspended in DMSO to 10 mM</td></tr><tr><td>compound 2</td><td>ChemBridge</td><td>6722175</td><td>screening compound; resuspended in DMSO to 10 mM</td></tr><tr><td>compound 3</td><td>ChemBridge</td><td>5249376</td><td>screening compound; resuspended in DMSO to 10 mM</td></tr><tr><td>Dithiothreitol</td><td>Sigma</td><td>D0632</td><td></td></tr><tr><td>Gla I</td><td>SibEnzyme</td><td>E494</td><td>methyl-sensitive endonuclease</td></tr><tr><td>Glycerol</td><td>RPI</td><td>G22025</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma</td><td>M0250</td><td></td></tr><tr><td>Oligonucleotide (5&#039;-FAM-CCTATGCGmCATCAGTTTTCTGATGmCGmCATAGG-3&#039;-Iowa Black Quencher)</td><td>IDT</td><td>custom synthesized</td><td>internally quenched hairpin DNA (substrate)</td></tr><tr><td>Potassium Glutamate</td><td>Sigma</td><td>G1501</td><td></td></tr><tr><td>S-adenosylmethionine</td><td>Sigma</td><td>A4377</td><td>methyl-donating co-factor (substrate)</td></tr><tr><td>Tris Base</td><td>RPI</td><td>T60040</td><td></td></tr></tbody></table>

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.

Active DNMT1 is a prerequisite for this analysis. RFTS-lacking DNMT1 was expressed in E.coli and purified to homogeneity following previously published procedures41. To ensure the purified enzyme was active, a discontinuous endonuclease-coupled assay was used to examine DNA methylation activity36. This assay utilizes a 32 base pair duplex DNA with a single hemimethylated CpG positioned in a Sau3A1 cleavage site. Sau3A1 can cleave the hemimethylated substrate DNA; h...

Watch this Scientific Journal Video about Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors at JoVE.com

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

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) ...

continuous-fluorescence-based-endonuclease-coupled-dna-methylation

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Biochemistry

2.5K Views.  Bucknell University. 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. 

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

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Genetics

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.

This work describes an optimized methyl-CpG-binding domain (MBD) sequencing protocol and a computational pipeline to identify differentially methylated CpG-rich regions in chronic lymphocytic leukemia (CLL) patients.

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

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.

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 ...

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Biology

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 ...

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

Cancer Research

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation

Source: Jia, Z. et al., <a href="https://app.jove.com/v/55565">A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro.</a>&#160;J. Vis. Exp.&#160;(2017)
This video describes the technique of dot-blot to determine the level of DNA methylation in human chondrocytes in vitro via the blotting of DNA samples on a nylon membrane and the subsequent visualization using monoclonal antibodies. This helps to determine the chondrocytes&#39; phenotype at various stages in culture.