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

Podsumowanie

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

Streszczenie

Multiple 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 DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Wprowadzenie

Epigenetic modifications, including DNA modification, histone modification and RNA modification, have been shown to play essential functions in diverse biological processes and diseases^{1,2,3,4,5,6,7}. 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 TET3^8,9. Further studies show that 5-hmC could serve as a stable marker and play biological roles through regulating gene expression^4,10,11,12.

The present evidences indicate that 5-hmC is highly enriched in neuronal tissues/cells relative to other types of tissues in mammals, and exhibits dynamic features during neuronal development^13,14. In the 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’s disease, Huntington’s disease, etc.^{2,13,15,16,17,18,19,20}.

There are several approaches for detecting 5-hmC in cells and tissues^{14,21,22,23,24}. 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 studies^{25,26,27,28,29,30}. 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 NaOH solution. These two methods together with next-generation sequencing are very useful tools for investigating the function of 5-hmC.

Protokół

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

1. Isolate adult neural stem cells from the forebrain of an adult (8-10 week old) C57/BL6 male mouse as described previously^31,32.
2. 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 °C. Induce the differentiation of adult neural stem cells with 1 µM retinoic acid and 5 µM forskolin for 48 h as described previously^31,32.
3. 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 °C as previously described³³.

2. Transcardial Perfusion of the Mouse

1. Prepare 10% chloral hydrate, 4% paraformaldehyde (PFA) and phosphate buffered saline (PBS) one day before the experiment, and store at 4 °C.
2. 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).
3. 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.
4. Perfuse the mouse with a 10 mL disposable sterilized syringe from the left ventricle with cold PBS (around 30 mL per adult mouse).
5. Perfuse the mouse with 4% PFA (around 30 mL in 10 min per adult mice) until it is stiff.
6. 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 °C.
   NOTE: Clean the surgical area after surgery has finished.
7. At least 24 h later, transfer the brain samples into a 30% sucrose solution for complete dehydration at 4 °C.

3. Brain Sectioning

1. Embed the brain samples in optimal cutting temperature compound (OCT) in a small container, and cool down at -20 °C for at least 1 h.
2. Section brain samples at a thickness of 20-40 μm with a cryostat microtome.
3. Collect sections into PBS and store at 4 °C.

4. Immunofluorescence Staining

1. 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.
2. Wash with PBS on the shaker at room temperature for 10 min. Repeat this twice more.
3. Remove PBS, and treat with preheated 1 M HCl for 30 min at 37 °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 °C.
4. Wash the samples with PBS for 5 min. Repeat this twice more.
5. Block samples with PBS containing 3% normal goat serum and 0.1% Triton X-100 for 1 h on the shaker at room temperature.
6. Incubate sections with specific primary antibodies overnight at 4 °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).
7. Take out the samples, and further incubate at room temperature for 1 h.
8. Wash the samples with PBS for 10 min. Repeat this twice more.
9. 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′-6-diamidino-2-phenylindole (DAPI).
10. Wash the samples with PBS for 10 min on the shaker. Repeat this twice more.
11. Mount brain sections onto the slides, add proper amount of antifade mounting medium (around 100-150 μL), and cover with premium cover glass. Seal with nail polish.
12. Take images with a regular or confocal fluorescence microscope.

5. Genomic DNA Isolation

1. Euthanize an adult C57 BL/6J mouse (male or female) by cervical dislocation and remove the brain.
2. 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 μg of proteinase K per 600 μL 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.
3. The second day, add about 50 μg of RNase A per sample for at least 12 h at 37 °C.
4. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and mix completely.
5. Centrifuge at 20,817 x g for 15 min, and remove the supernatant into a new microcentrifuge tube.
6. Add 600 μL of chloroform to the supernatant to precipitate DNA, and mix thoroughly.
7. Centrifuge at 20,817 x g for 15 min, and remove the supernatant into another new tube.
8. Add 500 μL of isopropanol to the supernatant, and mix thoroughly.
9. Centrifuge at 20,817 x g for 15 min, and remove the supernatant completely.
10. 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.
11. Dry the DNA pellet completely.
12. Dissolve DNA pellet with Tris-HCl buffer (pH 8.5) to the proper concentration.

6. DNA Dot Blot

1. Prepare the solutions: 2 M NaOH, Tris-HCl buffer (pH 8.5), 6x saline sodium citrate (SSC).
2. Make the sample mixture as Table 1.
3. Denature DNA samples at 100 °C for 10 min, and cool down on ice.
4. Cut the proper size of nylon membrane (e.g., Hybond-N+) and rinse with 6x SSC.
5. Put the membrane on dot-blot apparatus and connect to the vacuum pump. Spot 6 μL of mixture per dot onto the membrane.
6. Hybridize for 30 min at 80 °C, and block the sample membrane with fat-free milk in Tris-buffered saline (TBS) for 1 h.
7. Incubate with primary antibody at 4 °C overnight.
   NOTE: the following primary antibody: polyclonal rabbit anti-5-hydroxymethylcytosine (1:5,000).
8. 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.
9. Incubate the membrane with anti-rabbit secondary antibody (1:5,000) for 30 min at room temperature.
10. Wash with TBS for 10 min. Repeat this wash twice more.
11. Visualize the chemiluminescence signals, and quantify signal intensities.

Wyniki

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 differentiat...

Dyskusje

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 demon...

Materiały

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