Name: An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling (Video) | JoVE Journal
Uploaded: 2017-12-18T14:00:00.000+00:00
Duration: 8 min 34 s
Description: Discover how to use the engineered split-TET2 enzyme CiDER for chemical-inducible DNA hydroxymethylation in mammalian cells. This method enables precise temporal control of epigenetic remodeling.

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5 minutes

We thank the financial supports from the National Institutes of Health grant (R01GM112003 to YZ), the Welch Foundation (BE-1913 to YZ), the American Cancer Society (RSG-16-215-01 TBE to YZ), the Cancer Prevention and Research Institute of Texas (RR140053 to YH, RP170660 to YZ), the American Heart Association (16IRG27250155 to YH), and the John S. Dunn Foundation Collaborative Research Award Program.

1. Cell Culture, Plasmid Transfection and Chemical Induction
<ol>
	<li>Culture an adherent cell line (e.g., the human embryonic kidney HEK293T cell) in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and streptomycin at 37 &#176;C with 5% CO2.</li>
	<li>Transfect cells with the CiDER plasmid.
	<ol>
		<li>At least 18 h before transfection, seed 0.3 x 106 cells in 2.5 mL of appropriate DMEM per well in a 6-well plate and incubate cells at 37 &#176;C overnight to reach 60-80% confluency.</li>
		<li>Pre-warm the transfection reagent to room temperature before use.</li>
		<li>Place 250 &#181;L of serum-free medium in a sterile tube. 
		NOTE: This amount is tailored for a 6-well plate with 80% confluency. If needed, proportionally adjust the medium amounts according to the sizes of plates or cell numbers.</li>
		<li>Add 2.5 &#181;g of the plasmid encoding CiDER18 or the catalytic domain of TET2 (TET2CD as positive control)12,13 into the serum-free medium and gently mix by pipetting.</li>
		<li>Add 7.5 &#181;L of transfection reagent to the diluted DNA mixture and gently mix by pipetting.</li>
		<li>Incubate the mixture at room temperature for 20-30 min.</li>
		<li>Change pre-warmed media and add the mixture drop-wise to the wells, and gently shake the cell culture plate to evenly distribute the transfection reagent and DNA mixture.</li>
		<li>Incubate cells and mixture overnight, and then replace the transfection reagent containing media with 2ml of fresh complete DMEM.</li>
	</ol>
	</li>
	<li>Chemical induction
	<ol>
		<li>Dilute 2 mM of rapamycin stock (dissolved in DMSO) to a concentration of 20 &#181;M in appropriate complete medium.</li>
		<li>Add 20 &#181;L of diluted rapamycin to transfected cells maintained in 2 mL medium to reach a final concentration of 200 nM. 
		NOTE: After incubation for 48 h, cells are ready for quantification of 5hmC generation or any other downstream applications. If needed, the induction efficiency can be monitored by taking out cells every 3 h for further 5hmC quantification as described below and shown in Figure 1.</li>
		<li>For better quantification of 5hmC induction efficiency, seed separate wells to monitor cells every 3 hours.</li>
	</ol>
	</li>
</ol>
2. Quantification of Rapamycin-induced 5hmC Production by Immunostaining
<ol>
	<li>5hmC immunofluorescence staining 
	NOTE: Add sufficient amounts of the fixation solution, composed of 4% paraformaldehyde, 0.2% surfactant (such as Triton X-100) in PBS with 3 N HCl, to cover all the cells in plate. For example, 500 &#181;L of solution would be enough for a well in a 6-well plate. Perform all steps at room temperature.
	<ol>
		<li>To fix cells, add 4% paraformaldehyde in PBS to rapamycin-treated cells for 15 min at ambient temperature. For the control group, use cells expressing mCherry-CiDER without rapamycin treatment. Do not agitate. 
		Caution: Paraformaldehyde is toxic. Wear appropriate protection.</li>
		<li>Discard fixative solution. Incubate fixed cells with 0.2% surfactant in PBS for 30 min to permeabilize cell membrane.</li>
		<li>Discard permeabilization solution. Add 3 N HCl to the permeabilized cells and incubate for 15 min to denature the DNA.</li>
		<li>Discard HCl. Add 100 mM Tris-HCl buffer (pH 8.0) to the cells for 10 min for neutralization of pH.</li>
		<li>Discard all the solution. Wash cells thoroughly with PBS for 3-5 times. Remove PBS thoroughly. 
		NOTE: Add sufficient amounts of PBS for every wash step. For example, 1 mL of PBS is enough for a 6-well plate.</li>
		<li>Block cells by adding the blocking buffer comprising 1% BSA and 0.05% of surfactant (such as Tween 20) in PBS for 1 h with gentle shaking.</li>
		<li>Discard the blocking solution. Add the diluted 5hmC antibody (1:200) to the cells and incubate for 2 h at room temperature or overnight at 4 &#176;C.</li>
		<li>Wash the cells with PBS three times for 15 min.</li>
		<li>Add a diluted fluorophore (FITC)-conjugated secondary antibody (1:200) to the cells and incubate for 1 h.</li>
		<li>Wash the cells with PBS three times extensively to remove residual antibodies.</li>
		<li>Add 250 ng/mL of 4&#39;,6-diamidino-2- phenylindole (DAPI) to stain the nucleus for 5 min. Remove the staining solution.</li>
		<li>Mount the slide or glass-bottom culture dish with immunostained cells for confocal imaging. A representative result was shown in Figure 1B.</li>
	</ol>
	</li>
	<li>Flow cytometry 
	NOTE: Use a 96-well round bottom plate for the following staining procedure. Usually, 150 &#181;L of reagents is sufficient for each well.
	<ol>
		<li>Resuspend transfected and rapamycin-induced cells in FACS buffer (PBS with 1% BSA, 2 mM EDTA). For the control group, use CiDER-expressing cells without rapamycin treatment.</li>
		<li>Fix and permeabilize the cells as described in Step 2.1.</li>
		<li>Add 2 N HCl to the cells to denature the DNA for 10 min.</li>
		<li>Discard HCl. Neutralize and block the cells as described in Step 2.1.</li>
		<li>Add a diluted anti-5hmC antibody (1:200) to the cells and incubate for 1 h at room temperature or overnight at 4 &#176;C.</li>
		<li>Wash the cells with FACS buffer three times. Remove FACS buffer thoroughly.</li>
		<li>Add a diluted fluorophore (FITC)-conjugated secondary antibody (1:400) for fluorescence-activated cell sorting (FACS) analysis, and incubate for 1 h at room temperature.</li>
		<li>Wash the cells with FACS buffer three times.</li>
		<li>Samples are ready for FACS analysis. A representative result was shown in Figure 1C-D.</li>
	</ol>
	</li>
</ol>
3. Dot-blot Assay to Quantify 5hmC and 5-methylcytosine (5mC) Amounts
<ol>
	<li>Isolate genomic DNA from transfected and rapamycin-induced cells using a commercial DNA extraction kit. For the control group, use CiDER-transfected cells without rapamycin treatment.</li>
	<li>Heat the vacuum oven to 80 &#176;C and pre-soak nitrocellulose membrane in double-distilled H2O and then in 6x saline-sodium citrate (SSC) buffer for 10 min.</li>
	<li>Load 60 &#181;L of equal amounts of DNA (total 2 &#181;g in TE buffer) to a 96-well PCR plate. Add 30 &#181;L of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to the rest wells.</li>
	<li>Make two-fold serial dilutions vertically on the 96-well plate by transferring 30 &#181;L of solution on row 1 to the same column position on row 2. Mix again and transfer 30 &#181;L of solution to the wells in the subsequent row till reaching the boundary. Remove the last 30 &#181;L.</li>
	<li>Add 20 &#181;L of 1 M NaOH and 10 mM EDTA to each well, seal the plate and heat the mixture for 10 min at 95 &#176;C to completely denature the DNA duplex.</li>
	<li>Immediately cool down the samples on ice and add 50 &#181;L of ice-cold 2 M ammonium acetate (pH 7.0) and incubate on ice for 10 min. 
	NOTE: Steps 3.7-3.10 involves the use of vacuum.</li>
	<li>Assemble the dot blot apparatus with pre-soaked membrane and remove residual buffer using full vacuum.</li>
	<li>Wash the membrane by adding 200 &#181;L of TE buffer to each well of the dot blot apparatus. Turn on vacuum to remove TE buffer.</li>
	<li>Load the denatured DNA (prepared in previous steps 3.1-3.6) to each well of the dot blot apparatus.&#160;Turn on vacuum to remove solution.</li>
	<li>Wash the membrane by adding 200 &#181;L of 2x SSC and remove residual solutions completely using vacuum.</li>
	<li>Disassemble the dot blot apparatus, take out the membrane and rinse the whole membrane with 20 mL of 2x SSC.</li>
	<li>Air-dry the membrane for 20 min.</li>
	<li>Bake the membrane at 80 &#176;C under vacuum for 2 h.</li>
	<li>Add the blocking solution (5% skim milk in TBST) to the membrane and incubate for 1 h at room temperature.</li>
	<li>Discard the blocking solution. Add a diluted 5hmC (1:5,000) or 5mC (1:1,000) antibodies to the membrane and incubate overnight at 4 &#176;C.</li>
	<li>Wash the membrane with TBST three times for 10 min to remove residual antibodies. Remove TBST thoroughly.</li>
	<li>Add an HRP-conjugated secondary antibody (1:10,000 for anti-rabbit HRP(5hmC) or 1:3,000 for anti-mouse HRP(5mC)) to the membrane and incubate for 1 h at room temperature.</li>
	<li>Wash the membrane with TBST three times for 10 min.</li>
	<li>Add ECL western blotting substrate to the membrane for visualization of 5hmC signals using a chemiluminescence detector. A representative result was shown in Figure 1E.</li>
</ol>

<ol><li>Li, E., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+methylation+in+mammals%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Perspectv+Biol%22%5BJournal%5D)">DNA methylation in mammals.</a> Cold Spring Harbor Perspectv Biol. 6 (5), a019133(2014).</li>
<li>Tahiliani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conversion+of+5-methylcytosine+to+5-hydroxymethylcytosine+in+mammalian+DNA+by+MLL+partner+TET1%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.</a> Science. 324 (5929), 930-935 (2009).</li>
<li>He, Y. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tet-mediated+formation+of+5-carboxylcytosine+and+its+excision+by+TDG+in+mammalian+DNA%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA.</a> Science. 333 (6047), 1303-1307 (2011).</li>
<li>Ito, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tet+proteins+can+convert+5-methylcytosine+to+5-formylcytosine+and+5-carboxylcytosine%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine.</a> Science. 333 (6047), 1300-1303 (2011).</li>
<li>Spruijt, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+readers+for+5-%28hydroxy%29methylcytosine+and+its+oxidized+derivatives%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives.</a> Cell. 152 (5), 1146-1159 (2013).</li>
<li>Lio, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tet2+and+Tet3+cooperate+with+B-lineage+transcription+factors+to+regulate+DNA+modification+and+chromatin+accessibility%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Tet2 and Tet3 cooperate with B-lineage transcription factors to regulate DNA modification and chromatin accessibility.</a> eLife. 5, e18290(2016).</li>
<li>Kellinger, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((5-formylcytosine+and+5-carboxylcytosine+reduce+the+rate+and+substrate+specificity+of+RNA+polymerase+II+transcription%5BTitle%5D)+AND+%22Nat+Struct+Mol+Biol%22%5BJournal%5D)">5-formylcytosine and 5-carboxylcytosine reduce the rate and substrate specificity of RNA polymerase II transcription.</a> Nat Struct Mol Biol. 19 (8), 831-833 (2012).</li>
<li>Su, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((5-Formylcytosine+Could+Be+a+Semipermanent+Base+in+Specific+Genome+Sites%5BTitle%5D)+AND+%22Angew+Chem+Int+Ed+Engl%22%5BJournal%5D)">5-Formylcytosine Could Be a Semipermanent Base in Specific Genome Sites.</a> Angew Chem Int Ed Engl. 55 (39), 11797-11800 (2016).</li>
<li>Ko, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impaired+hydroxylation+of+5-methylcytosine+in+myeloid+cancers+with+mutant+TET2%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.</a> Nature. 468 (7325), 839-843 (2010).</li>
<li>Huang, Y., Rao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Connections+between+TET+proteins+and+aberrant+DNA+modification+in+cancer%5BTitle%5D)+AND+%22Trends+Genet%22%5BJournal%5D)">Connections between TET proteins and aberrant DNA modification in cancer.</a> Trends Genet. 30 (10), 464-474 (2014).</li>
<li>Lu, X., Zhao, B. S., He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TET+family+proteins%3A+oxidation+activity%2C+interacting+molecules%2C+and+functions+in+diseases%5BTitle%5D)+AND+%22Chem+Rev%22%5BJournal%5D)">TET family proteins: oxidation activity, interacting molecules, and functions in diseases.</a> Chem Rev. 115 (6), 2225-2239 (2015).</li>
<li>Hu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crystal+structure+of+TET2-DNA+complex%3A+insight+into+TET-mediated+5mC+oxidation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Crystal structure of TET2-DNA complex: insight into TET-mediated 5mC oxidation.</a> Cell. 155 (7), 1545-1555 (2013).</li>
<li>Hashimoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+a+Naegleria+Tet-like+dioxygenase+in+complex+with+5-methylcytosine+DNA%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.</a> Nature. 506 (7488), 391-395 (2014).</li>
<li>Banaszynski, L. A., Liu, C. W., Wandless, T. J. J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/15796538">Characterization of the FKBP.rapamycin.FRB ternary complex.</a> J Am Chem Soc. 127 (13), 4715-4721 (2005).</li>
<li>Clackson, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Redesigning+an+FKBP-ligand+interface+to+generate+chemical+dimerizers+with+novel+specificity%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity.</a> Proc Natl Acad Sci U S A. 95 (18), 10437-10442 (1998).</li>
<li>Ibrahimi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+efficient+multicistronic+lentiviral+vectors+with+peptide+2A+sequences%5BTitle%5D)+AND+%22Hum+Gene+Ther%22%5BJournal%5D)">Highly efficient multicistronic lentiviral vectors with peptide 2A sequences.</a> Hum Gene Ther. 20 (8), 845-860 (2009).</li>
<li>Kim, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+cleavage+efficiency+of+a+2A+peptide+derived+from+porcine+teschovirus-1+in+human+cell+lines%2C+zebrafish+and+mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.</a> PLoS One. 6 (4), e18556(2011).</li>
<li>Lee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+Split-TET2+Enzyme+for+Inducible+Epigenetic+Remodeling%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Engineered Split-TET2 Enzyme for Inducible Epigenetic Remodeling.</a> J Am Chem Soc. 139 (13), 4659-4662 (2017).</li>
<li>Huang, Y., Pastor, W. A., Zepeda-Martínez, J. A., Rao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+anti-CMS+technique+for+genome-wide+mapping+of+5-hydroxymethylcytosine%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine.</a> Nat Protoc. 7 (10), 1897-1908 (2012).</li>
<li>Buenrostro, J. D., Wu, B., Chang, H. Y., Greenleaf, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ATAC-seq%3A+A+Method+for+Assaying+Chromatin+Accessibility+Genome-Wide%5BTitle%5D)+AND+%22Curr+Protoc+Mol+Biol%22%5BJournal%5D)">ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide.</a> Curr Protoc Mol Biol. 109 (21.29), 1-9 (2015).</li>
</ol>

The authors declare no competing financial interests.

Here we have illustrated the use of an engineered split-TET2 enzyme to achieve temporal control of DNA hydroxymethylation. Following the discovery of TET family of 5-methycytosine dioxygenase, many studies have been performed to decipher the biological functions of TET proteins and their major catalytic product 5hmC1,2,3,4,5,6</sup...

DNA methylation, mostly refers to the addition of a methyl group to the carbon 5 position of cytosine to form 5-methylcytosine (5mC), is catalyzed by DNA methyl-transferases (DNMTs). 5mC acts as a major epigenetic mark in the mammalian genome that often signals for transcriptional repression, X-chromosome inactivation and transposon silencing1. The reversal of DNA methylation is mediated by the Ten-elven translocation (TET) protein family. TET enzymes belong to the iron (II) and 2-oxoglutarate dependent dioxygenases which catalyze the successive oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). TET-mediated 5mC oxidation imposes an additional layer of epigenetic control over the mammalian genome. The discovery of TET has sparked intense interest in the epigenetic field to unveil the biological functions of TET proteins and their major catalytic product 5hmC. 5hmC is not only an intermediate during TET-mediated active DNA demethylation2,3,4, but also acts as a stable epigenetic mark5,6,7,8. Although DNA hydroxymethylation is highly correlated with gene expression and aberrant changes in DNA hydroxymethylation are associated with some human disorders9,10,11, the causal relations between epigenetic modifications on DNA and the phenotypes often remain challenging to be established, which can be partially ascribed to the lack of reliable tool to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution.
Here we report the use of a chemical-inducible epigenome remodeling tool (CiDER) to overcome the hurdle facing studies of causal relationships between DNA hydroxymethylation and gene transcription. The design is based on the assumption that the catalytic domain of TET2 (TET2CD) can be split into two inactive fragments when expressed in mammalian cells and its enzymatic function can be restored by taking a chemically inducible dimerization approach (Figure 1A). To establish a split-TET2CD system, we selected six sites in TET2CD, composed of a Cys-rich region and a double-stranded &#946;-helix (DSBH) fold, on the basis of a reported crystal structure of TET2-DNA complex that lacks a low complexity region12. A synthetic gene encoding rapamycin-inducible dimerization module FK506 binding protein 12 (FKBP12) and FKBP rapamycin binding domain (FRB) of the mammalian target of rapamycin14,15, along with a self-cleaving peptide T2A polypeptide sequence16,17, was individually inserted into the selected split sites within TET2CD. The selection of TET2 as our engineering target is based on the following considerations. First, somatic mutations in TET2 with concomitant reduction in DNA hydroxymethylation are frequently observed in human disorders including myeloid disorders and cancer10, which provides useful information on sensitive sites to be avoided for insertion. Second, a large fraction of the TET2 catalytic domain, particularly the low complexity region, is dispensable for the enzymatic function12, thus enabling us to craft a minimized split-TET2 for inducible epigenetic modifications. After screening over 15 constructs, a construct that exhibited highest rapamycin-inducible restoration of its enzymatic activity in the mammalian system was chosen and designated as CiDER18. We describe herein the use of mCherry-tagged CiDER to achieve inducible DNA hydroxymethylation and epigenetic remodeling with rapamycin, and present three methods for validating CiDER-mediated 5hmC production in a model cellular system HEK293T.

<table><tbody><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>11668027</td><td></td></tr><tr><td>Rapamycin</td><td>Sigma-Aldrich</td><td>37094</td><td></td></tr><tr><td>Paraformaldehyde, 16% Solution</td><td>VWR</td><td>100503-916</td><td></td></tr><tr><td>Triton X-100</td><td>Amresco</td><td>0694-1L</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Amresco</td><td>0369-500ML</td><td></td></tr><tr><td>Albumin, Bovine</td><td>Amresco</td><td>0332-100G</td><td></td></tr><tr><td>Tween 20</td><td>Amresco</td><td>0777-1L</td><td></td></tr><tr><td>5-Hydroxymethylcytosine (5-hmC) antibody (pAb)</td><td>Active Motif</td><td>39769</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A-11008</td><td></td></tr><tr><td>4,6-Diamidino-2-phenylindolde (DAPI)</td><td>Biotium</td><td>40043</td><td></td></tr><tr><td>SVAC1E SHEL LAB Economy Vacuum Oven, 0.6 Cu.Ft. (17 L)</td><td>SHEL LAB</td><td>SVAC1E</td><td></td></tr><tr><td>UltraPure SSC, 20X</td><td>Thermo Fisher Scientific</td><td>15557036</td><td></td></tr><tr><td>Bio-Dot Apparatus</td><td>BIO-RAD</td><td>1706545</td><td></td></tr><tr><td>Anti-5-methylcytosine (5mC) Antibody, clone 33D3</td><td>Millipore</td><td>MABE146</td><td></td></tr><tr><td>Anti-mouse IgG, HRP-linked Antibody</td><td>Cell Signaling Technology</td><td>7076S</td><td></td></tr><tr><td>Anti-rabbit IgG, HRP-linked Antibody</td><td>Cell Signaling Technology</td><td>7074S</td><td></td></tr><tr><td>West-Q Pico Dura ECL Solution</td><td>Gendepot</td><td>W3653-100</td><td></td></tr></tbody></table>

an engineered split-tet2 enzyme for chemical-inducible dna hydroxymethylation and epigenetic remodeling

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.

Chemical inducible 5mC-to-5hmC conversion can be validated by immunostaining at single-cell level, flow cytometry analysis on transfected (mCherry-positive) cell populations, or a more quantitative dot-blot assay as illustrated in Figure 1. The catalytic domain of TET2, or TET2CD, were used throughout the study as a positive control. See references 18-20 for further details regarding the genome-wide mapping of rapamycin-induced changes in 5hmC and chromatin a...

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 ...

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Research

JoVE Journal

Chemistry

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing β-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

Described is a two-step labeling process using β-glucosyltransferase (β-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

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.

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

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

Restriction endonucleases with new sequence specificity can be developed from enzymes recognizing a partially degenerate sequence. Here we provide a detailed protocol that we successfully used to alter the sequence specificity of NlaIV enzyme. Key ingredients of the protocol are the in vitro compartmentalization of the transcription/translation reaction and selection of variants with new sequence specificities.

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

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...

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

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol to reduce DNA modifications in bacteriophages using the NgTET enzyme, enabling efficient and scarless CRISPR-Cas mutagenesis. This method facilitates the genetic engineering of phages for applications in biotechnology and phage therapy.

Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems

This protocol describes a non-integrative, episomal CRISPR/dCas9-based system for targeted epigenetic editing in K562 cells, combining dCas9-DNMT3A and dCas9-HDAC1 effectors with specific sgRNAs to induce locus-specific DNA methylation with precision and reduced off-target effects.

Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins

Discover how to use the engineered split-TET2 enzyme CiDER for chemical-inducible DNA hydroxymethylation in mammalian cells. This method enables precise temporal control of epigenetic remodeling.

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

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