XL was supported in part by the National Key R&#38;D Program of China (2017YFE0196600), and the National Natural Science Foundation of China (Grant Nos. 31771395, 31571518). Q.S. was supported by the National Key Research and Development Program of China (2017YFC1001703) and the Key Research and Development Program of Zhejiang Province (2017C03009). W.X. was supported by the Natural Science Foundation of Zhejiang province (LY18H020002) and Science Technology Department of Zhejiang Province (2017C37057).

All the animal procedures have been approved by the Animal Ethics Committee of Zhejiang University.
1. The Culture of Adult Neural Stem Cells and Neurons
<ol>
	<li>Isolate adult neural stem cells from the forebrain of an adult (8-10 week old) C57/BL6 male mouse as described previously31,32.</li>
	<li>Culture adult neural stem cells in DMEM/F-12 medium containing 20 ng/mL FGF-2, 20 ng/mL EGF, 2% B27 supplement, 1% antibiotic-antimycotic, and 2 mM L-Glutamine in a 5% CO2 incubator at 37 &#176;C. Induce the differentiation of adult neural stem cells with 1 &#181;M retinoic acid and 5 &#181;M forskolin for 48 h as described previously31,32.</li>
	<li>Isolate neurons from the embryonic day 17 (E17) hippocampi of the mouse and culture with neurobasal medium containing of 0.25% L-Glutamine, 0.125% GlutaMax and 2% B27 supplement in a 5% CO2 incubator at 37 &#176;C as previously described33.</li>
</ol>
2. Transcardial Perfusion of the Mouse
<ol>
	<li>Prepare 10% chloral hydrate, 4% paraformaldehyde (PFA) and phosphate buffered saline (PBS) one day before the experiment, and store at 4 &#176;C.</li>
	<li>Anesthetize an adult male or female C57 BL/6J mouse with 10% chloral hydrate (50 mg/kg, i.p.), and ensure that the animal is deeply anesthetized by checking the body reaction. Fix each limb with sticky tape on a plastic board (in a face-up position).</li>
	<li>Cut the skin and then the muscle with surgery scissors. Open the thoracic cavity with a surgical scissor. Expose the heart and cut off a little part of the right atrium with a fine surgical scissor.</li>
	<li>Perfuse the mouse with a 10 mL disposable sterilized syringe from the left ventricle with cold PBS (around 30 mL per adult mouse).</li>
	<li>Perfuse the mouse with 4% PFA (around 30 mL in 10 min per adult mice) until it is stiff.</li>
	<li>Open the skull of the mouse with bone forceps. Remove the brain and put into 5 mL of 4% PFA in a 15 mL centrifuge tube for post-fixation at 4 &#176;C. 
	NOTE: Clean the surgical area after surgery has finished.</li>
	<li>At least 24 h later, transfer the brain samples into a 30% sucrose solution for complete dehydration at 4 &#176;C.</li>
</ol>
3. Brain Sectioning
<ol>
	<li>Embed the brain samples in optimal cutting temperature compound (OCT) in a small container, and cool down at -20 &#176;C for at least 1 h.</li>
	<li>Section brain samples at a thickness of 20-40 &#956;m with a cryostat microtome.</li>
	<li>Collect sections into PBS and store at 4 &#176;C.</li>
</ol>
4. Immunofluorescence Staining
<ol>
	<li>Pick up the sections with the targeted brain regions and put them into a 24-well plate with PBS. For cultured cells on a coverslip, go directly to step 4.2.</li>
	<li>Wash with PBS on the shaker at room temperature for 10 min. Repeat this twice more.</li>
	<li>Remove PBS, and treat with preheated 1 M HCl for 30 min at 37 &#176;C. 
	NOTE: Prepare 1 M HCl by adding 1 mL of hydrochloric acid (36-38%) into 10 mL of water in a chemical hood. Preheat 1 M HCl in the incubator at 37 &#176;C.</li>
	<li>Wash the samples with PBS for 5 min. Repeat this twice more.</li>
	<li>Block samples with PBS containing 3% normal goat serum and 0.1% Triton X-100 for 1 h on the shaker at room temperature.</li>
	<li>Incubate sections with specific primary antibodies overnight at 4 &#176;C on the shaker. 
	NOTE: Use the following primary antibodies: polyclonal rabbit antibodies anti-5-hydroxymethylcytosine (1:5,000), mouse monoclonal antibody anti-NeuN (1:500).</li>
	<li>Take out the samples, and further incubate at room temperature for 1 h.</li>
	<li>Wash the samples with PBS for 10 min. Repeat this twice more.</li>
	<li>Incubate with secondary antibodies corresponding to the primary antibodies at room temperature for 1 h on the shaker. Cover the plate with aluminum. 
	NOTE: Use the following secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG (1:500), Alexa Fluor 568 goat anti-mouse IgG (1:500). Counterstain nuclei with 4&#8242;-6-diamidino-2-phenylindole (DAPI).</li>
	<li>Wash the samples with PBS for 10 min on the shaker. Repeat this twice more.</li>
	<li>Mount brain sections onto the slides, add proper amount of antifade mounting medium (around 100-150 &#956;L), and cover with premium cover glass. Seal with nail polish.</li>
	<li>Take images with a regular or confocal fluorescence microscope.</li>
</ol>
5. Genomic DNA Isolation
<ol>
	<li>Euthanize an adult C57 BL/6J mouse (male or female) by cervical dislocation and remove the brain.</li>
	<li>Dissect the hippocampus, cortex and cerebellum tissues on an ice-cooled dish. Grind tissues with a tissue grinder in 1 mL of DNA lysis buffer and transfer into clean microcentrifuge tube. Add 250 &#956;g of proteinase K per 600 &#956;L of lysis buffer. For cell pellets, directly add lysis buffer, proteinase K, and mix thoroughly. 
	NOTE: Prepare DNA lysis buffer: 5 mM EDTA, 0.2% SDS, 200 mM NaCl in 100 mM Tris-HCl, pH 8.5. Wash and autoclave the tissue grinder before the experiment.</li>
	<li>The second day, add about 50 &#956;g of RNase A per sample for at least 12 h at 37 &#176;C.</li>
	<li>Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and mix completely.</li>
	<li>Centrifuge at 20,817 x g for 15 min, and remove the supernatant into a new microcentrifuge tube.</li>
	<li>Add 600 &#956;L of chloroform to the supernatant to precipitate DNA, and mix thoroughly.</li>
	<li>Centrifuge at 20,817 x g for 15 min, and remove the supernatant into another new tube.</li>
	<li>Add 500 &#956;L of isopropanol to the supernatant, and mix thoroughly.</li>
	<li>Centrifuge at 20,817 x g for 15 min, and remove the supernatant completely.</li>
	<li>Wash the precipitation with 1 mL of 70% ethanol, centrifuge at 20,817 x g for 1 min, and remove the supernatant completely. Repeat once.</li>
	<li>Dry the DNA pellet completely.</li>
	<li>Dissolve DNA pellet with Tris-HCl buffer (pH 8.5) to the proper concentration.</li>
</ol>
6. DNA Dot Blot
<ol>
	<li>Prepare the solutions: 2 M NaOH, Tris-HCl buffer (pH 8.5), 6x saline sodium citrate (SSC).</li>
	<li>Make the sample mixture as Table 1.</li>
	<li>Denature DNA samples at 100 &#176;C for 10 min, and cool down on ice.</li>
	<li>Cut the proper size of nylon membrane (e.g., Hybond-N+) and rinse with 6x SSC.</li>
	<li>Put the membrane on dot-blot apparatus and connect to the vacuum pump. Spot 6 &#956;L of mixture per dot onto the membrane.</li>
	<li>Hybridize for 30 min at 80 &#176;C, and block the sample membrane with fat-free milk in Tris-buffered saline (TBS) for 1 h.</li>
	<li>Incubate with primary antibody at 4 &#176;C overnight. 
	NOTE: the following primary antibody: polyclonal rabbit anti-5-hydroxymethylcytosine (1:5,000).</li>
	<li>On the second day, incubate the sample membranes at room temperature for 1 h. Wash with TBS for 10 min. Repeat this wash twice more.</li>
	<li>Incubate the membrane with anti-rabbit secondary antibody (1:5,000) for 30 min at room temperature.</li>
	<li>Wash with TBS for 10 min. Repeat this wash twice more.</li>
	<li>Visualize the chemiluminescence signals, and quantify signal intensities.</li>
</ol>

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No competing financial interests exist.

Epigenetic modifications play essential roles during brain development, maturation, and function. As a stable marker for DNA modification, dynamic 5-hmC responds to behavioral adaptation, neuronal activity, and is positively correlated with gene expression; thus, it is involved in the normal function of the brain and neurological disorder4. To explore its function in cells and tissues, it is necessary to detect the existence of 5-hmC and compare the level before and after treatment. Here, we demonstrated two convenient methods to detect 5-hmC in cells and tissues, which could be performed with common equipment in the lab.
The key reagent of detecting 5-hmC with immunofluorescence staining and DNA dot-blot is the 5-hmC antibody. The 5-hmC antibody used in the method has been proven to have high sensitivity and is very specific. For 5-hmC staining, it requires DNA denaturation with HCl. The proper treatment of tissues and cells with HCl is critical for complete DNA denaturation and affects the results. The DNA dot-blot is a sensitive method to quantify the amount of 5-hmC, and is much more convenient than mass spectroscopy. For a successful dot-blot, precise spreading of DNA samples onto the membrane is required. Further, methylene blue staining helps determine whether DNA samples were equally loaded. Of note, the methods described here detect the global level of 5-hmC in multiple types of cells and tissues. To measure the amount of 5-hmC relative to other bases and distinguish its distribution feature in genome, it requires LC-MS/MS and next-generation sequencing.

Epigenetic modifications, including DNA modification, histone modification and RNA modification, have been shown to play essential functions in diverse biological processes and diseases1,2,3,4,5,6,7. For a long time, DNA methylation (i.e., 5-methylcytosine (5-mC)) has been viewed as a highly stable epigenetic marker and can not be further modified in the genome. Recently, it has been found that 5-mC could be oxidized to 5-hydroxymethylcytosine(5-hmC) by TET (Ten-eleven translocations) family proteins including TET1, TET2, and TET38,9. Further studies show that 5-hmC could serve as a stable marker and play biological roles through regulating gene expression4,10,11,12.
The present evidences indicate&#160;that 5-hmC is highly enriched in neuronal tissues/cells relative to other types of tissues in mammals, and exhibits dynamic features during neuronal development13,14. In the&#160;neuronal system, 5-hmC mediated epigenetic modifications play an important role in regulating neural stem cells, neuronal activity, learning and memory, and is involved in multiple neurological disorders including Rett syndrome, autism, Alzheimer&#8217;s disease, Huntington&#8217;s disease, etc.2,13,15,16,17,18,19,20.
There are several approaches for detecting 5-hmC in cells and tissues14,21,22,23,24. Here, we describe two methods to detect the existence of 5-hmC and quantify the global level of 5-hmC: immunofluorescence staining and DNA dot-blot. These two methods are convenient and sensitive, and have been successfully used in previous studies25,26,27,28,29,30. The key steps of these two methods are DNA denaturation. For immunofluorescence staining of 5-hmC, pre-treatment of samples with 1 M HCl is required. For 5-hmC dot-blot, DNA denaturation is performed with&#160;NaOH solution. These two methods together with next-generation sequencing are very useful tools for investigating the function of 5-hmC.

<table><tbody><tr><td>4&#039;-6-diamidino-2-phenylindole (DAPI )</td><td>Sigma-Aldrich</td><td>D8417</td><td></td></tr><tr><td>Adobe Photoshop software</td><td>Adobe Inc.</td><td>/</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-rabbit IgG</td><td>Thermo Fisher</td><td>A11008</td><td></td></tr><tr><td>Alexa Fluor 568 goat anti-mouse IgG</td><td>Thermo Fisher</td><td>A11001</td><td></td></tr><tr><td>anti-5-hydroxymethylcytosine</td><td>Active Motif</td><td>39769</td><td></td></tr><tr><td>anti-NeuN</td><td>Millipore</td><td>MAB377</td><td></td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>12587-010</td><td></td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>12580-010</td><td></td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>Cryostat microtome</td><td>Leica</td><td>CM1950</td><td></td></tr><tr><td>DMEM/F-12 medium</td><td>OmegaScientific</td><td>DM25</td><td></td></tr><tr><td>Epidermal growth factor</td><td>PeproTech</td><td>100-15</td><td></td></tr><tr><td>Fibroblast growth factor-basic</td><td>PeproTech</td><td>100-18B</td><td></td></tr><tr><td>Forskolin</td><td>Sigma-Aldrich</td><td>F6886</td><td></td></tr><tr><td>GlutaMax</td><td>Thermo</td><td>35050061</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030-149</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>Normal goat serum</td><td>Vector Laboratories</td><td>Z0325</td><td></td></tr><tr><td>Nylon membrane (Hybond&amp;trade;-N+ )</td><td>Amersham Biosciences</td><td>RPN303B</td><td></td></tr><tr><td>OCT</td><td>Leica</td><td>14020108926</td><td></td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Phenol: chloroform: isoamyl alcohol (25: 24:1 )</td><td>Sigma-Aldrich</td><td>516726</td><td></td></tr><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>P0899-10</td><td></td></tr><tr><td>Proteinase K</td><td>VVR</td><td>39450-01-6</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma-Aldrich</td><td>R2625</td><td></td></tr><tr><td>Triton X-100</td><td>Solarbio</td><td>T8210</td><td></td></tr></tbody></table>

the detection of 5-hydroxymethylcytosine in neural stem cells and brains of mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

To reveal the distribution of 5-hmC in the hippocampus of adult mice, we performed immunofluorescence with antibodies against neuronal cells (NeuN) and 5-hmC. In the hippocampus, 5-hmC co-localized well with neuronal cell marker NeuN (Figure 1A-H), suggesting an enrichment of 5-hmC in neurons.
To determine the dynamics of 5-hmC during neuronal development, a dot-blot was first performed with DNA samples isolated from proliferating and differentiated adult neural stem cells (NSCs). Dot-blot results showed that the global level of 5-hmC significantly increased during the differentiation of NSC (Figure 2A-B). Further, dot-blot results showed that the level of 5-hmC in neurons was significantly higher than that of NSCs (Figure 2C-D), suggesting a dynamic 5-hmC modification during neuronal development.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59950/59950fig1.jpg" /> 
Figure 1: Immunofluorescence staining of 5-hmC in hippocampus of adult mice. 5-hmC co-localized well with neuronal cell marker NeuN. Scale bar = 100 &#956;m (A-D); 50 &#956;m (E-F). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59950/59950fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59950/59950fig2.jpg" /> 
Figure 2: DNA dot-blot detection of 5-hmC in adult neural stem cells and neurons. (A) 5-hmC dot-blot of NSCs under proliferation (Proli) and differentiation (Diffe) conditions. (C) 5-hmC dot-blot of NSCs and primary neurons. Methylene blue staining (B, D) indicating an equal loading of genomic DNA at each concentration in (A) and (C), respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59950/59950fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>200 ng/dot</td>
			<td>400 ng/dot</td>
			<td>1,000 ng/dot</td>
		</tr>
		<tr>
			<td>DNA</td>
			<td>470 ng</td>
			<td>940 ng</td>
			<td>2,350 ng</td>
		</tr>
		<tr>
			<td>2 M NaOH</td>
			<td>2.81 &#956;L</td>
			<td>2.81 &#956;L</td>
			<td>2.81 &#956;L</td>
		</tr>
		<tr>
			<td>Tris-HCl buffer, pH 7.5</td>
			<td>Make the volume up to 14.06 &#956;L</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: The preparation of samples for dot-blot.

Watch this Scientific Journal Video about The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice at JoVE.com

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

the-detection-5-hydroxymethylcytosine-neural-stem-cells-brains

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.4K Views.  Zhejiang University. Hier presenteren we een protocol om 5-hydroxymethylcytosine in cellen en hersenweefsels te detecteren met behulp van immunofluorescentie, de nieuwe methode en de DNA dot-vlek. 5-hydroxymethylcytosine, d.w.z. 5hmC, verzacht epigenetische modificatie, speelt belangrijke functie bij het reguleren van genexpressie en betrokken bij meerdere neurologische aandoeningen.Eigenlijk kunnen onze methoden worden gebruikt om 5hmC in meerdere cellen en weefsels te detecteren. Het is handig en kan wor...

Video: The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Genetics

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 ...

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 ...

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

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

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

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

Developmental Biology

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos.

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

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 ...

Biology

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

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