Name: an engineered split-tet2 enzyme for chemical-inducible dna hydroxymethylation and epigenetic remodeling
Uploaded: 2017-12-18T14:00:00.000+00:00
Duration: 8 min 34 s
Description: Watch this Scientific Journal Video about An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling at JoVE.com

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>

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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=Tet-mediated+formation+of+5-carboxylcytosine+and+its+excision+by+TDG+in+mammalian+DNA.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet+proteins+can+convert+5-methylcytosine+to+5-formylcytosine+and+5-carboxylcytosine.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+readers+for+5-(hydroxy)methylcytosine+and+its+oxidized+derivatives.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet2+and+Tet3+cooperate+with+B-lineage+transcription+factors+to+regulate+DNA+modification+and+chromatin+accessibility.">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. 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S., He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TET+family+proteins:+oxidation+activity,+interacting+molecules,+and+functions+in+diseases.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+TET2-DNA+complex:+insight+into+TET-mediated+5mC+oxidation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+Naegleria+Tet-like+dioxygenase+in+complex+with+5-methylcytosine+DNA.">Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.</a> Nature. 506 (7488), 391-395 (2014).</li><li>Banaszynski, L. 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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

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...

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

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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6.6K Views.  Texas A&M University. Gedetailleerde protocollen voor de invoering van een gemanipuleerde split-TET2 enzym (appelwijn) in zoogdiercellen voor chemische afleidbare DNA-hydroxymethylation en epigenetische remodeling worden gepresenteerd.

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

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

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

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 &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

Neuroscience

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 ...

Biochemistry

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

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

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.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Genetics

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

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase variants that have more stringent specificity than the parental enzyme. The protocol requires the creation of a library of expression selection cassettes (ESCs) for variants of the REase, ideally with variability in positions likely to affect DNA binding. The ESC is flanked on one side by a sequence for the restriction site activity desired and a biotin tag and on the other side by a restriction site for the undesired activity and a primer annealing site. The ESCs are transcribed and translated in a water-in-oil emulsion, in conditions that make the presence of more than one DNA molecule per droplet unlikely. Therefore, the DNA in each cassette molecule is subjected only to the activity of the translated, encoded enzyme. REase variants of the desired specificity remove the biotin tag but not the primer annealing site. After breaking the emulsion, the DNA molecules are subjected to a biotin pulldown, and only those in the supernatant are retained. This step assures that only ESCs for variants that have not lost the desired activity are retained. These DNA molecules are then subjected to a first PCR reaction. Cleavage in the undesired sequence cuts off the primer binding site for one of the primers. Therefore, PCR amplifies only ESCs from droplets without the undesired activity. A second PCR reaction is then carried out to reintroduce the restriction site for the desired specificity and the biotin tag, so that the selection step can be reiterated. Selected open reading frames can be overexpressed in bacterial cells that also express the cognate methyltransferase of the parental REase, because the newly evolved REase targets only a subset of the methyltransferase target sites.

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.

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

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.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Bioengineering

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Biology

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-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.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

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.

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

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

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

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.

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

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

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