Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Podsumowanie

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

Streszczenie

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Wprowadzenie

Epigenetic events are frequent in gliomas and likely play an important role in tumor pathogenesis. Indeed, in pediatric high-grade glioma, mutations in genes encoding histone variants H3.3 and H3.1 occur frequently¹. The mutations affect histone modifications and have major epigenetic consequences^2,3. In the adolescent to adult spectrum, recurrent mutations in isocitrate dehydrogenase gene 1/2 (IDH1/2), a mutation that inhibits α-KG dependent histone and DNA de-methylasaes, and genetic alterations in other chromatin regulators such as ATRX and DAXX occur⁴. Therefore, it is of critical importance to study how mutations that affect epigenetic regulators alter chromatin structure and regulatory histone modifications, which, in turn, have a dramatic impact of the tumor cells' transcriptome.

Chromatin immunoprecipitation (ChIP) is a powerful tool used to evaluate the impact of epigenetic modifications in the genome^5,6,7. In native ChIP, chromatin is digested with micrococcal nuclease (MNase), immunoprecipitated using an antibody raised against the protein of interest, and then DNA is purified from the immunoprecipitated chromatin complex⁶. Cells are not fixed during the procedure so this technique is only applicable for the study of proteins that interact tightly with DNA⁶. The absence of cross-linking aids antibody specificity since antibodies are usually raised against unfixed peptides or proteins⁷. In addition, since there is no cross-linking step, this reduces the chances of fixing transient protein-DNA interactions that are non-specific and not regulatory^7,8. ChIP can be used to identify the enrichment of histone modifications in a specific genomic region. Here, we detail a protocol for performing native ChIP in neurospheres (NS) generated from genetically engineered mouse models of glioma.

Protokół

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan.

1. Generation of brain tumor NS and culturing conditions.

1. Prepare Neural Stem Cell Medium (NSC) with Dulbecco's Modified Eagle Medium/F12B27 supplement (1x), N2 supplement (1x), and an antimicrobial reagent. Supplement NSC medium on the day of use with human recombinant endothelial growth factor (EGF) and basic-fibroblast growth factor (FGF) at a final concentration of 20 ng/mL each.
2. Induce the brain tumor in mouse using the Sleeping Beauty transposon system in which plasmids encoding oncogenes or shRNA targeting tumor suppressors is injected into the lateral ventricles of neonates to generate spontaneous brain tumors⁹. Select a mouse with a large tumor, which is confirmed with bioluminescence imaging.
   NOTE: More detailed information about the Sleeping Beauty Transposon System and plasmid constructs used can be found in Calinescu et al.⁹.
   1. To image the mice, inject 100 µL of 30 mg/mL luciferin solution intraperitoneally using a 26 gauge needle.
   2. 5 min after injection of luciferin, anesthetize the animal using an anesthesia chamber with oxygen/isoflurane flow set to 2% isoflurane.
   3. When the animals are anesthetized (usually 2-3 min), place the animals in the bioluminescence chamber with oxygen isoflurane set to 2% isoflurane. If the animals will be under anesthesia for longer than 5 min, use an ophthalmic ointment to prevent dryness while under anesthesia.
   4. Image the mice using an in vivo optical imaging system with the following parameters: automatic exposure, large binning, and aperture f=1. The parameter to consider it a large tumor is a bioluminescence >10⁶ photons/s/cm²/sr.
3. Euthanize the mouse with an overdose of isoflurane inhalation anesthetic in a closed chamber, check by toe pinch that the animal is anesthetized, and decapitate the mouse.
4. Extract the brain and dissect it as described previously in Calinescu et al.⁹.
5. Place the brain in a 10 cm Petri dish. If the tumor induced expresses a fluorescent protein, use a fluorescent dissecting microscope set to the appropriate fluorescent channel and 0.3X magnification, otherwise identify tumor mass by the differences in tissue color and texture. Use scalpel and forceps to separate the tumor tissue from the normal brain.
6. Mince the tumor into small pieces in the Petri dish. Place the dissected tumor in a 1.5 mL tube with 300 µL of the NSC medium.
7. Use a disposable plastic pellet pestle that fits to the walls of a conical microcentrifuge tube to gently homogenize the tumor. Add 1 mL of cell detachment medium and incubate the sample for 5 min at 37 °C.
   NOTE: The use of a pestle may kill some cells. If the reader wants to maximize the cell viability, the use of a pestle may be omitted.
8. Pass the cell suspension from step 1.7 through a 70 µm cell strainer and wash the strainer with 25 mL of NSC medium.
9. Centrifuge the suspension at 300 x g for 5 min at room temperature, decant the supernatant and re-suspend the pellet into 6 mL of NSC medium.
10. Plate the tumor cell suspension from step 1.9 into a T-25 culture flask and culture it at 37 °C in a tissue culture incubator with an atmosphere of 95% air and 5% CO₂. Usually after 3 days, there will be a mixture of some dead cells, adhered cells and some cells forming NS which float in the medium.
11. Transfer the cell suspension into a 15 mL conical tube, collect only the cells that sink to the bottom using a micropipette and re-plate them onto a T-75 tissue culture flask.
12. Maintain the cells at relatively high density (1-3 X 10⁶ cells in a T-25). Passage the cells when they are confluent.
    NOTE: Confluency is determined by the spheres size. Black centers of large spheres mean that there are dead cells in the center and indicate that the cells should be passaged immediately.
13. Add the growth factors (EGF and basic-FGF; 20 ng/mL each) every three days. Change the medium when the cells are not confluent and the medium turns light orange.
14. To change the medium, collect the old medium and centrifuge it at 300 x g at room temperature for 5 min and re-suspend the cell pellet in NSC medium supplemented with EGF and FGF at the concentration specified in step 1.1.
    NOTE: If the spheres are at mid-to high confluency, but not ready to be passaged (sphere size is small with a diameter < 100 µm), then the pellet can be split into two flasks.
15. To passage the cells, centrifuge the cells at 300 x g for 5 min at room temperature, decant the supernatant and incubate the cells in cell detachment medium for 5 min at 37 °C.
    1. Add 5 mL of balanced salt solution to dilute detachment medium and centrifuge it at 300 x g for 5 min at room temperature.
    2. Decant the supernatant, resuspend the cell pellet in 18 mL of NSC medium and divide the suspension equally into three T-25 flasks.
16. To freeze the aliquots of cells, dissociate the cells as in step 1.15, and freeze the cells in 1 mL aliquots of 90% fetal bovine serum with 10% dimethyl sulfoxide. Slowly cool down the cells by placing the cells on ice for 10 min, then keeping the cells -20 °C for 30 min, and -80 °C for 1 day. The next day, transfer the cells to a liquid nitrogen storage container.

2. Native ChIP

1. Chromatin preparation
   1. After making stock of derived NS, culture NS in a T-75 in 15 mL of NSC medium at 37 °C in a tissue culture incubator with an atmosphere of 95% air and 5% CO₂ until NS become confluent. One confluent T-75 plate will yield 3-5 X 10⁶ cells.
   2. When the cells become confluent, centrifuge NS at 300 x g for 5 min, decant the supernatant and add 1 mL of cell dissociation medium. Incubate the cells at 37 °C for 5 min. Add 5 mL of medium and count the cells using a hemocytometer¹⁰.
      NOTE: 1 x 10⁶ cells are needed per immunoprecipitation (IP) reaction. Note that a pre-immune serum or IgG control must also be included¹¹.
   3. Centrifuge the NS in one 1.5 mL microcentrifuge tube at 300 x g for 5 min, decant the supernatant and re-suspend NS pellet with 1 mL of balanced salt solution and transfer the suspension to low protein binding 1.5 mL microcentrifuge tubes.
   4. Centrifuge NS at 300 x g for 5 min, decant the supernatant and re-suspend the cell pellet in 95 µL of digestion buffer (50 mM Tris-HCl, pH 8.0, 1 mM CaCl₂, 0.2% polyethylene glycol octylphenyl ether) per 1 x 10⁶ cells supplemented with protease inhibitor cocktail at a high concentration (1:100). Immediately pipet the cells up and down to prevent clumping and avoid creating any bubbles.
   5. Resuspend MNase in 50% glycerol to make 1 mg/mL solution (1.15 units/µL, stored at -20 °C) that will be referred to as "1x" stock. Make a "0.1x" stock by doing a second 1:9 dilution with 50% glycerol (e.g., 900 µL of "1x" MNase and 100 µL of 50% glycerol) and store it at -20 °C.
   6. Mix 5 µL of "0.1x" MNase and 145 µL of digestion buffer. Keep the enzyme on ice all the times. Add 5 µL of diluted MNase in digestion buffer per 1 x 10⁶ cells. Flick the tube to mix and place the tube in 37 °C block for exactly 12 min. Process the samples one at a time to ensure an accurate incubation time of 12 min.
   7. Add 10 µL of 10x MNase Stop Buffer (110 mM Tris-HCl, pH 8.0, 55 mM EDTA) per 1 x 10⁶ cells. Samples must be kept on ice from this point onwards.
   8. Add 110 µL "2x RIPA buffer" (280 mM NaCl, 1.8% polyethylene glycol octylphenyl ether, 0.2% sodium dodecyl sulfate (SDS), 0.2% Na-deoxycholate, 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA); stored at 4 °C) supplemented with protease inhibitor cocktail at high concentration (1:100) per 1 x 10⁶ cells. Flick the tube to mix.
   9. Centrifuge the tubes for 15 min in a microfuge at 4 °C and 1700 x g. Transfer the supernatant to a new regular 1.5 mL microcentrifuge tubes (low protein binding microcentrifuge tubes are no longer necessary) and store them on ice. Discard the pellet.
2. Immunoprecipitation (IP)
   1. Mix Protein A and Protein G magnetic beads in a 1:1 ratio. For every IP, prepare 25 µL of beads.
   2. Wash the beads by adding 1 mL of RIPA buffer (10 mM Tris pH 8.0, 1 mM EDTA, 140 mM NaCl, 1% polyethylene glycol octylphenyl ether, 0.1% SDS, 0.1% Na-deoxycholate; stored at 4 °C) supplemented with protease inhibitors at low concentration (1:1000).
   3. Use a magnet to allow beads to separate from the washing solution and decant the washing solution (approximately 1 min). Perform the wash step twice.
   4. Re-suspend the beads to the original volume using RIPA buffer supplemented with protease inhibitors (1:1000).
   5. If necessary, dilute the chromatin using RIPA buffer supplemented with protease inhibitors (1:1000) so that each IP has a volume of 100-200 µL. Reserve 10% of the volume used per IP of chromatin for the input.
      NOTE: For example, if 200 µL are used per IP, 20 µL of diluted chromatin should be reserved for the input. For a total of four IPs, total chromatin volume should be diluted to 820 µL.
   6. Prepare the input. Add 100 µL of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) supplemented with proteinase K (0.5 mg/mL) to the input and incubate the input at 55 °C for 1 h.
      NOTE: Steps 2.2.6-2.2.7 can be performed along with steps 2.3.4 and 2.4.1.
   7. Purify the input using a PCR purification kit following the manufacturer's instructions and elute in 50 µL of elution buffer provided in kit.
   8. Measure the concentration of the input using a microvolume spectrophotometer and record the results in a notebook. Blank with elution buffer from kit.
      NOTE: In mouse neurospheres, we used approximately 6 µg of DNA for each IP.
   9. Run input on a 1% gel or load 1 µL on a DNA Bioanalyzer using standard settings. Store the remainder for use as input in downstream applications.
   10. Add 10 µL of washed protein A & G magnetic beads for every IP and incubate IP for 1 h at 4 °C.
       NOTE: For example, add 30 µL of protein A & G magnetic beads for three IPs.
   11. Place the samples on a magnet and allow beads to separate. Divide chromatin by transferring the previously determined amount into a new 1.5 mL tube.
   12. Add the antibody and wrap the caps with plastic paraffin film to avoid evaporation. Incubate the samples overnight at 4 °C with rotation at 20 rpm.
       NOTE: Concentration should be determined empirically following manufacturer recommendations.
   13. The next day, spin the tubes using a mini centrifuge at room temperature, 2000 x g, for 10 s. Then add 10 µL of beads to each IP and incubate IP for 3 h at 4 °C with rotation at 20 rpm.
3. IP Washes and Protein Digestion
   1. Add 150 µL of RIPA buffer supplemented with protease inhibitors at low concentration (1:1000) to each IP and incubate IP at 4 °C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow the magnetic beads to separate. Use a pipet to remove the supernatant. Repeat this tep five washes.
   2. Add 150 µL of LiCl buffer (250 mM LiCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.5% NP-40, 0.5 % Na-deoxycholate; store at 4 °C) supplemented with protease inhibitors at low concentration (1:1000) to each IP and incubate IP at 4 °C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow magnetic beads to separate. Use a pipet to remove supernatant.
   3. Add 150 µL of cold TE (no protease inhibitors) and incubate the sample at 4 °C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow magnetic beads to separate. Use a pipet to remove the supernatant.
   4. After the final washing step, re-suspend the sample in 100 µL of TE buffer supplemented with proteinase K (0.5 mg/mL) and incubate the sample at 55 °C for 1 h.
4. DNA Purification and ChIP Quantitative Real-Time PCR (qPCR)
   1. Purify immunoprecipitated DNA using a PCR purification kit. After addition of DNA binding buffer from the kit to each sample, place the tube on a magnet and wait until magnetic beads aggregate, then transfer the supernatant to the cleanup column. Follow the manufacturer's instructions and elute in 50 µL of elution buffer provided in kit.
   2. To check if the IP is successful, perform a qPCR. Ideally, primers should be designed for positive and negative control regions.
      NOTE: For example, for an IP performed with H3K4me3 and H3K27me3, the following samples should be run on qPCR: H3K4me3, H3K27me3, and IgG ChIP DNA, input DNA, and a no template control (NTC) using primers for regions to be enriched with H3K4me3 (positive control) and regions to be enriched with H3K4me3 (negative control); likewise, for H3K27me3.
   3. Follow standard protocols to perform qPCR¹². Briefly, perform a qPCR using SYBR green master mix, 0.5 µL of 10 µM working concentration of each forward and reverse primer, and 2 µL of ChIP or input DNA. Read plates using a Real Time PCR system. Primer sequences are provided in Table 2. The Gapdh primer sequences from Hwang et al. were used¹³.
   4. Analyze ChIP data relative to the input, i.e., percent input method (% IP)¹⁴. Calculate the percent input using the following formulas:
      Adjusted input = mean ct(input)-log₂(DF)
      where DF is the dilution factor of the starting input and
      DF = total volume of IP/ volume of input
      % IP = 100×2^(Adjusted input - Ct (IP))
      where Ct is the threshold factor.

Wyniki

A schematic representation of tumor NS generated from a brain tumor where brain tumor cells are Katushka positive is presented in Figure 1. Figure 2 is a schematic representation of the entire ChIP technique. Figure 3 shows the representative results of chromatin from brain tumor NS digested with MNase for 12 min, yielding a majority of mono, di-, and tri- nucleosomes. Following ChIP, a qPCR may be p...

Dyskusje

The protocol presented here will enable the user to perform native ChIP on NS derived from genetically engineered brain tumors. In contrast to cross-linking ChIP, this protocol is limited for the study of proteins that associate tightly with DNA⁶. The number of cells used can be modified as necessary and the protocol can be scaled up. We used 1 X 10⁶ cells per IP, however, native ChIP may also be performed with as little as 4 x 10⁴ cells⁵. In this prot...

Materiały

Name	Company	Catalog Number	Comments
Agilent 2100 Bioanalyzer	Agilent	G2946-90004	bioanalyzer
AccuSpin Micro 17R, refrigerated	Fisher Scientific	13-100-676
C57BL/6	Taconic	B6-f	C57BL/6 mouse
Calcium Chloride	Aldrich	22350-6	buffer reagent
D-Luciferin, Potassium Salt	Goldbio	LuckK-1g
DiaMag1.5 magnetic rack	Diagenode	B04000003	for magnetic bead washes
DiaMag Rotator EU	Diagenode	B05000001	rotator
DMEM/F-12	Gibco	11330-057	NSC component
Dynabeads Protein A	Thermo Fisher Scientific	10001D	protein A magnetic beads
Dynabeads Protein G	Thermo Fisher Scientific	10003D	protein G magnetic beads
EGF	PeproTech	AF-100-15	prepare 20 μg/mL stock in 0.1% BSA and aliquot.
Ethylenediaminetetraacetic acid (EDTA)	Sigma	E-4884	buffer reagent
ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (EGTA)	Sigma	E-4378	buffer reagent
Fast SYBR Green Master Mix	Applied Biosystems	4385612	qPCR reagent
Fetal Bovine Serum	Gibco	10437028	for freezing cells
FGF	PeproTech	100-18b	Prepare 20 μg/mL stock in 0.1% BSA and aliquot.
Forceps	Fine Science Tools	11008-13	for dissection of tumor
Glycerol, MB Grade	EMD- Millipore	356352
H3K4me3	Abcam	Ab8580
H3K27me3	Millipore	07-449
HBSS	Gibco	14175-103	balanced salt solution
Fluriso	VETone	501017	inhalation anesthetic
Ivis Spectrum	Perkin-Elmer	124262	in vivo optical imaging system
Hyqtase	HyClon	SV3003001	cell detachment media
Lithium Chloride	Sigma	L8895	buffer reagent
Low binding microtubes	Corning Costar	CLS3207	low protein binding microcentrifuge tube
Microcentrifuge tube	Fisher	21-402-903	regular microcentrifuge tube
Micrococcal Nuclease	Thermo Fisher Scientific, Affymetrix	70196Y	each batch may differ; purchase sufficient amount for experiments and aliquot.
N2	Gibco	17502-048	NSC component
Normal Rabbit IgG	Millipore	12-372
Normocin	Invivogen	NOL-36-063	anti-microbial agent, use at 0.1 mg/mL.
NP-40 (Igepal CA-630)	Sigma	18896-50ML	buffer reagent
Kimble Kontes Pellet Pestle	Fisher Scientific	K749515-0000
Protease Inhibitor Cocktail	Sigma-Aldrich	P8340	aliquot and store at -20 °C.
Protinase K	Sigma-Aldrich	P2308	make 10 mg/mL stock in water; aliquot and store at -20 °C.
QIAquick PCR Purification Kit	Qiagen	28104	DNA purification kit
Scalpel	Fine Science Tools	10007-16	for dissection of tumor
Sodium Chloride	VWR	0241-5KG	buffer reagent
Sodium Deoxycholate	Sigma-Aldrich	D670-25G	buffer reagent
Sodium Dodecyl sulfate (SDS)	Sigma	L-4390	buffer reagent
Tris Base	Thermo Fisher Scientific	Bp152-1	buffer reagent
Triton X-100	Thermo Fisher Scientific	BP 151-500	polyethylene glycol octylphenyl ether
Standard Mini Centrifuge	Fisherbrand	12-006-901	standard mini centrifuge
SZX16 microscope	Olympus	SZX16	flourescent dissecting microscope
ViiA 7 Real-Time PCR System with Fast 96-Well Block	Applied Biosystems	4453535
Nanodrop One	Thermo-Fisher Scientific	ND-ONEC-W