Name: Capturing Common Fragile Site Breaks by Native γH2A.X ChIP (Video) | JoVE Journal
Uploaded: 2025-01-24T14:20:00.000+00:00
Duration: 9 min 46 s
Description: Learn a rapid native γH2A.X ChIP method for detecting common fragile site breaks in cancer cells. This streamlined approach reduces time and labor while maintaining high reproducibility for genomic instability research.

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5 minutes

This work was supported by University of South China&#39;s startup funding.

1. Cell harvesting
<ol>
	<li>Seed about 5 x 105 HEK 293T cells into each of the four 6 cm dishes, each containing 4 mL of complete DMEM medium.</li>
	<li>After 24 h, treat one dish with 2 &#181;L of 1 mM Aphidicolin (refer to Table of Material) stock solution (final concentration of 0.5 &#181;M) and another dish with 20 &#181;L of 1 M hydroxyurea (refer to Table of Material) stock solution (final concentration of 5 mM) to induce replication stress. Add DMSO to the remaining two dishes to serve as controls.</li>
	<li>After 24 h of treatment, discard the culture media. One 6 cm plate typically yields approximately 2 x 106&#160;cells at 60%-70% confluence.</li>
	<li>Rinse each dish 2x with 5 mL of ice-cold 1x PBS. Use cell scrapers to detach the cells and transfer the cell suspension to four individual 1.5 mL tubes. Gently pipette up and down with a P1000 pipette to dissociate any cell clumps.</li>
	<li>Centrifuge the cells at 500 x g for 5 min at 4 &#176;C, then discard the supernatant. Place the cells on ice.</li>
</ol>
2. Subcellular fractionation
<ol>
	<li>Resuspend the cell pellet in 500 &#181;L of freshly prepared cold Buffer A (refer to Table 1), ensuring the complete dissociation of cell clumps by gentle pipetting.</li>
	<li>Incubate the lysates on ice for 5-10 min. Check lysis progression under a microscope to make sure complete cell lysis.
	<ol>
		<li>Take a small aliquot of the lysate (around 5 - 10 &#181;L) and place it on a clean microscope slide. Cover it with a coverslip to avoid contamination.</li>
		<li>Use a light microscope with an appropriate magnification (e.g., 20x - 40x) to visualize cells or debris. Compare with an un-lysed control sample to differentiate between intact cells and lysed material. 
		NOTE: A properly lysed sample will have no distinct cell outlines, only diffuse chromatin or cellular material. Adjust the focus to clearly observe the lysate. If needed, apply mechanical force during the lysis step, such as using a Dounce homogenizer, when working with certain cell types.</li>
	</ol>
	</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C, once cells are completely lysed. Carefully discard the supernatant. Resuspend the nuclei pellet in 500 &#181;L of cold Buffer A using wide-orifice pipette tips. 
	NOTE: Wide-orifice tips help minimize shear forces and protect delicate samples like chromatin. Make wide-bore tips by cutting the end of standard tips with a sharp blade.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C. Carefully discard the supernatant.</li>
	<li>Prewarm an incubator to 37 &#176;C and prepare a stopping buffer (100 mM EDTA, pH 8.0; Table 1).</li>
	<li>Optimize Micrococcal Nuclease (MNase, refer to Table of Material) concentrations and incubation times in advance.
	<ol>
		<li>Divide 40 &#181;L of testing chromatin sample into several equal aliquots to test different MNase concentrations and incubation times.</li>
		<li>Use a range of MNase concentrations (e.g., 0.0625 U, 0.125 U, 0.25 U, 0.5 U, 1 U, 2 U, 4 U, 8 U per reaction) and test multiple incubation times (e.g., 2, 5, 10, and 15 min).</li>
		<li>Add MNase Buffer (refer to Table 1) containing various concentrations of MNase to the chromatin aliquots and incubate samples at 37 &#176;C for the specified times.</li>
		<li>Terminate the reaction by adding 1/4 volume of stopping buffer (final concentration: 20 mM EDTA) immediately after the desired incubation time.</li>
		<li>Isolate DNA from the digested chromatin samples using a phenol/chloroform/isoamyl alcohol extraction method.</li>
		<li>Run the extracted DNA on a 1.5% agarose gel to visualize digestion patterns: Under-digestion will show high molecular weight bands (Figure 2, lane 1-4); over-digestion will result in a smear or very short fragments (Figure 2, lane 6-8), and optimal digestion will yield a clear nucleosomal ladder pattern (Figure 2, lane 5, e.g., mono-, di-, tri-nucleosomes).</li>
		<li>Identify the conditions that produce the desired nucleosomal resolution without excessive over-digestion. 
		NOTE: CaCl2 acts as a cofactor for MNase activity. Optimize the digestion by adjusting the CaCl2&#160;concentration between 1 mM and 5 mM.</li>
	</ol>
	</li>
	<li>Gently resuspend the intact nuclei with 100 &#956;L of MNase Buffer by pipetting 5 - 10 times with wide-orifice tips. Immediately add the pre-determined amount of MNase to the samples (1.25 U MNase/100 &#181;L MNase Buffer). 
	NOTE: When working with multiple samples, digest each one individually to avoid over-digestion.</li>
	<li>Place the tubes on a rotator and incubate for 5 min at 37 &#176;C. Immediately return the tubes to ice and terminate MNase digestion by adding EDTA to a final concentration of 20 mM and mix by vortexing.</li>
	<li>Add 500 &#181;L of Buffer B (refer to Table 1) to each sample and mix thoroughly by pipetting up and down 5x - 10x. Solubilize proteins by incubating on ice for 5 min. 
	NOTE: The salt and detergent in Buffer B help dissociate weak chromatin-bound proteins and expose the epitopes for immunoprecipitation.</li>
	<li>Pellet the insoluble material by centrifuging at maximum speed for 5 min at 4 &#176;C. Transfer the clear supernatant to new 1.5 mL tubes labeled as the native chromatin fraction. The samples can either be stored at -80 &#176;C or used to validate the efficiency of chromatin fragmentation. 
	NOTE: Avoid frequent freeze-thaw cycles, as they may disrupt protein-DNA interactions of interest. Minimize freeze-thaw cycles whenever possible.</li>
</ol>
3. Verification of chromatin fragmentation
<ol>
	<li>Aliquot 10 &#181;L of the supernatant from each sample into a new 1.5 mL tube. Mix with 20 &#181;L of distilled water and 30 &#181;L of phenol/chloroform/isoamyl alcohol (25:24:1).</li>
	<li>Close the tubes tightly and vortex vigorously for 15-30 s. Centrifuge at 20,000 x g (or the maximum speed of the centrifuge) for 10 min at 4 &#176;C. After centrifugation, three distinct layers will be observed: a clear top layer, a white middle layer, and a yellow bottom layer.</li>
	<li>Carefully transfer 20 &#181;L of the upper aqueous phase (containing DNA) to a fresh tube. Separate the purified DNA in 1.5% agarose gel for 30 min at 100 V and visualize the digestion patterns. Ensure that the size of chromatin fragments is primarily between 200 and 1000 base pairs. 
	NOTE: Proper chromatin fragment sizes are crucial for the success of native ChIP and depend on MNase treatment conditions, including enzyme units, incubation time, and CaCl2&#160;concentration. MNase digestion efficiency can also vary based on cell type and number. The chromatin fragmentation pattern shown in Figure 2 (lane 5) is recommended for this ChIP assay.</li>
</ol>
4. Immunoprecipitation
<ol>
	<li>Aliquot 20 &#181;L of digested chromatin from each sample into a fresh 1.5 mL tube and mix with 180 &#181;L of Elution Buffer (refer to Table 1). Label these tubes as Input samples and store at -20 &#176;C.</li>
	<li>Transfer 400 &#181;L of chromatin sample into another 1.5 mL tube for ChIP.</li>
	<li>Add &#947;H2A.X antibody (refer to Table of Material) to one DMSO-treated, one Aphidicolin-treated sample, and one hydroxyurea-treated sample. Add the same amount of normal IgG (refer to Table of Material) to another DMSO-treated sample as a negative control for the ChIP assay. 
	NOTE: Here, 1 &#956;g of primary antibody is typically used for 400 &#956;L of chromatin (i.e., antibody final concentration is 2.5 &#181;g/mL). However, the optimal amount should be empirically determined for different &#947;H2A.X antibodies.</li>
	<li>Place the ChIP tubes on a rotator at 4 &#176;C and incubate for at least 5 h, or preferably overnight.</li>
	<li>Meanwhile, aliquot 100 &#181;L of ChIP-grade magnetic Protein A/G beads (refer to Table of Material) into a new 1.5 mL tube. Use wide-orifice tips and pipette slowly to ensure accurate measurement of the beads. Place the tube on a magnetic stand for at least 1 min, then carefully discard the liquid.</li>
	<li>Resuspend the beads in 1 mL of 1x PBS containing 0.5% BSA. Rotate at 4 &#176;C for approximately 4 h. Place the tube on a magnetic stand for at least 1 min and discard the supernatant.</li>
	<li>Wash the beads again with 1 mL of 1x PBS containing 0.5% BSA. Place the tube on the magnetic stand for 1 min to pellet the magnetic beads, then discard the supernatant. 
	NOTE: Steps 4.5 to 4.7 are the pre-coating of beads to reduce non-specific binding of antibodies to magnetic beads.</li>
	<li>Resuspend the pre-coated beads in 100 &#181;L of Buffer B using wide-orifice tips. Add 25 &#181;L of the pre-coated magnetic bead suspension to each ChIP sample tube. Rotate at 4 &#176;C for 2 h.</li>
	<li>Place the ChIP tubes on the magnetic stand and wait until the beads are completely attached to the side of the tube and the solution becomes clear.</li>
	<li>Discard the clear supernatant without disturbing the magnetic beads. Resuspend the beads with 1 mL of Wash Buffer (refer to Table 1) and rotate at 4 &#176;C for 10 min.</li>
	<li>Place the tubes back on the magnetic stand and wait until the solution becomes clear. Discard the wash buffer. Repeat washing for a total of four washes.</li>
	<li>Discard the wash buffer after final wash and briefly centrifuge the tubes at 400 x g for 30 s at 4 &#176;C to spin down any residual liquid. Place the tubes back on the magnetic stand and carefully remove any remaining liquid from the bottom of the tube.</li>
</ol>
5. Elution and DNA precipitation
NOTE: Antibody efficiency may vary among different batches. It is important to confirm the binding affinity of a new antibody by checking the immunoprecipitated samples through Western blot analysis.
<ol>
	<li>Verify ChIP antibody pull-down efficiency using Western blot (WB) as described below.
	<ol>
		<li>Take a small aliquot of the ChIP sample for analysis (i.e., usually 10% of the ChIP sample). Include Input chromatin (pre-immunoprecipitation) and negative control (e.g., IgG pull-down) for comparison.</li>
		<li>Elute proteins from the antibody-bound beads by heating in 20 &#181;L of 1x SDS-PAGE loading buffer (refer to Table of Material) at 95 &#176;C for 5 min.</li>
		<li>Load the IP samples, input, and controls onto a 15% SDS-PAGE gel. Run the gel.</li>
		<li>Transfer the proteins to a 0.2 &#181;m nitrocellulose (refer to Table of Material) or PVDF membrane using a wet or semi-dry transfer system.</li>
		<li>Block the membrane with 5% non-fat milk or BSA in TBST (refer to Table 1) for 1 h at room temperature to prevent non-specific binding.</li>
		<li>Incubate the membrane with the primary antibody against the &#947;H2A.X (refer to the Table of Material) diluted in blocking buffer for 1-2 h at room temperature or overnight at 4 &#176;C.</li>
		<li>Wash the membrane 3x with TBST to remove unbound antibodies. Incubate the membrane with an HRP-conjugated secondary antibody (refer to the Table of Material) for 1 h at room temperature. Wash the membrane again to remove excess secondary antibodies.</li>
		<li>Develop the membrane using a chemiluminescent substrate and visualize the signal with an imager. Compare the signal intensity between the IP, input, and control lanes to assess the efficiency and specificity of the pull-down. 
		NOTE: A band corresponding to the target protein in the IP lane confirms successful antibody pull-down. This approach ensures you can evaluate the efficacy of the antibody in capturing the target protein during the ChIP experiment.</li>
	</ol>
	</li>
	<li>Add 50 &#181;L of Elution Buffer (refer to the Table 1) to each of the remaining ChIP samples. Place the tubes on a thermomixer and shake for 15 min at room temperature.</li>
	<li>Place the tubes on the magnetic holder for at least 1 min. Collect the elute into new tubes. Repeat 1x and collect the elute in the same tubes.</li>
	<li>Add additional 100 &#181;L of Elution Buffer to each ChIP elution sample and 180 &#181;L of Elution Buffer to each Input sample.</li>
	<li>Add 200 &#181;L of phenol/chloroform/isoamyl alcohol (25:24:1) to each sample and vortex vigorously. Centrifuge the samples at 20,000 x g (or maximum speed) for 10 min at 4 &#176;C.</li>
	<li>Add 19 &#181;L of 3M sodium acetate (NaOAc, pH 5.2; refer to the Table 1) and 2 &#181;L of glycogen solution (20 mg/mL, refer to the Table of Material) to each new 1.5 mL centrifuge tube.</li>
	<li>After centrifugation, carefully transfer the upper aqueous layer (approximately 190 &#181;L) to the tubes containing NaOAc and glycogen and mix by vortexing.</li>
	<li>Add 500 &#181;L of 100% ethanol and vortex. Precipitate the DNA by incubating the samples at -20 &#176;C for at least 2 h or overnight.</li>
	<li>Centrifuge the tubes at 20,000 x g (or maximum speed) for 10 min at 4 &#176;C. Discard the supernatant, taking care not to disturb the white pellet. Resuspend the pellet in 1 mL of 70% ethanol and vortex thoroughly.</li>
	<li>Centrifuge the tubes at 20,000 x g (or maximum speed) for 5 min at 4 &#176;C. Carefully remove the supernatant. Briefly centrifuge the tubes again to spin down any residual ethanol. Carefully remove the ethanol using a P20 pipette. Air dry the DNA pellets for 2-3 min. 
	NOTE: Avoid over-drying the pellet, as this can make the DNA difficult to re-dissolve.</li>
	<li>For ChIP samples, resuspend the DNA in 400 &#181;L of TE buffer (refer to Table 1). For Input DNA, resuspend in 1000 &#181;L of TE buffer. The eluted samples can now be stored at -20&#176;C.</li>
</ol>
6. qPCR quantification
<ol>
	<li>Perform qPCR using a commercial kit (refer to Table of Material) with technical triplicates for each sample. Confirm the presence of a single specific PCR product by conducting melting curve analysis to ensure the specificity of amplification36.</li>
	<li>Data analysis 
	NOTE: In relative quantification analysis, the test sample is expressed as a fold change relative to a control sample (immunoprecipitated using normal purified IgG or mock IP). DNA loci known to be unoccupied by the immunoprecipitated protein (negative locus) can be used in this manner as a reference gene compared to known, occupied, positive control DNA loci36.
	<ol>
		<li>Calculate the percent of input for each ChIP using the formula below 
		%Input = 2(-&#916;Ct [normalized ChIP])</li>
		<li>Normalize the positive locus &#916;Ct values to negative locus (&#916;&#916;Ct) by subtracting the &#916;Ct value obtained for the positive locus from the &#916;Ct value for the negative locus using the formula below 
		(&#916;&#916;Ct = &#916;Ctpositive - &#916;Ctnegative)</li>
		<li>Calculate the fold enrichment of the positive locus sequence in ChIP DNA over the negative locus using the formula below 
		Fold enrichment =2&#916;&#916;Ct 
		The sequences of the qPCR primers used for analysis are provided in Table 2. The genomic organization of FRA3B and FRA16D37is depicted in Figure 3A,B.</li>
	</ol>
	</li>
	<li>Statistical analysis
	<ol>
		<li>Analyze the results statistically using Student&#39;s paired t-test. A p-value of &#8804;0.05 is considered statistically significant, indicating that the observed differences are unlikely to be due to random variation38.</li>
	</ol>
	</li>
</ol>

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<li>Myers, F. A., Evans, D. R., Clayton, A. L., Thorne, A. W., Crane-Robinson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+and+extended+acetylation+of+histones+H4+and+H3+at+active+and+inactive+genes+in+chicken+embryo+erythrocytes%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Targeted and extended acetylation of histones H4 and H3 at active and inactive genes in chicken embryo erythrocytes.</a> J Biol Chem. 276 (23), 20197-20205 (2001).</li>
<li>Litt, M. D., Simpson, M., Recillas-Targa, F., Prioleau, M. N., Felsenfeld, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transitions+in+histone+acetylation+reveal+boundaries+of+three+separately+regulated+neighboring+loci%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci.</a> EMBO J. 20 (9), 2224-2235 (2001).</li>
<li>Madisen, L., Krumm, A., Hebbes, T. R., Groudine, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+immunoglobulin+heavy+chain+locus+control+region+increases+histone+acetylation+along+linked+c-myc+genes%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">The immunoglobulin heavy chain locus control region increases histone acetylation along linked c-myc genes.</a> Mol Cell Biol. 18 (11), 6281-6292 (1998).</li>
<li>Clayton, A. L., Hebbes, T. R., Thorne, A. W., Crane-Robinson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+acetylation+and+gene+induction+in+human+cells%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">Histone acetylation and gene induction in human cells.</a> FEBS Lett. 336 (1), 23-26 (1993).</li>
<li>Tseng, Z., Wu, T., Liu, Y., Zhong, M., Xiao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+native+chromatin+immunoprecipitation+to+interrogate+histone+variant+protein+deposition+in+embryonic+stem+cells%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Using native chromatin immunoprecipitation to interrogate histone variant protein deposition in embryonic stem cells.</a> Methods Mol Biol. 1176, 11-22 (2014).</li>
<li>Durkin, S. G., Glover, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosome+fragile+sites%5BTitle%5D)+AND+%22Ann+Rev+Genetics%22%5BJournal%5D)">Chromosome fragile sites.</a> Ann Rev Genetics. 41, 169-192 (2007).</li>
<li>Lee, J. B., Keung, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+immunoprecipitation+in+human+and+yeast+cells%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Chromatin immunoprecipitation in human and yeast cells.</a> Methods Mol Biol. 1767, 257-269 (2018).</li>
<li>Miyamoto, R., Yokoyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+for+fractionation-assisted+native+ChIP+%28fanChIP%29+to+capture+protein-protein%2FDNA+interactions+on+chromatin%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Protocol for fractionation-assisted native ChIP (fanChIP) to capture protein-protein/DNA interactions on chromatin.</a> STAR Protoc. 2 (2), 100404(2021).</li>
<li>Mendez, J., Stillman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+association+of+human+origin+recognition+complex%2C+cdc6%2C+and+minichromosome+maintenance+proteins+during+the+cell+cycle%3A+Assembly+of+prereplication+complexes+in+late+mitosis%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: Assembly of prereplication complexes in late mitosis.</a> Mol Cell Biol. 20 (22), 8602-8612 (2000).</li>
<li>Nowak, D. E., Tian, B., Brasier, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-step+cross-linking+method+for+identification+of+NF-%CE%BAB+gene+network+by+chromatin+immunoprecipitation%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">Two-step cross-linking method for identification of NF-κB gene network by chromatin immunoprecipitation.</a> BioTechniques. 39 (5), 715-725 (2005).</li>
<li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analyzing+real-time+PCR+data+by+the+comparative+C%28T%29+method%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Analyzing real-time PCR data by the comparative C(T) method.</a> Nat Protoc. 3 (6), 1101-1108 (2008).</li>
<li>Lu, X., Parvathaneni, S., Hara, T., Lal, A., Sharma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Replication+stress+induces+specific+enrichment+of+RECQ1+at+common+fragile+sites+FRA3B+and+FRA16D%5BTitle%5D)+AND+%22Mol+Cancer%22%5BJournal%5D)">Replication stress induces specific enrichment of RECQ1 at common fragile sites FRA3B and FRA16D.</a> Mol Cancer. 12, 29(2013).</li>
<li>Ruijter, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amplification+efficiency%3A+linking+baseline+and+bias+in+the+analysis+of+quantitative+PCR+data%5BTitle%5D)+AND+%22Nucl+Acids+Res%22%5BJournal%5D)">Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data.</a> Nucl Acids Res. 37 (6), e45(2009).</li>
<li>Lyu, X., Chastain, M., Chai, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+mapping+and+profiling+of+%CE%B3H2AX+binding+hotspots+in+response+to+different+replication+stress+inducers%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Genome-wide mapping and profiling of γH2AX binding hotspots in response to different replication stress inducers.</a> BMC Genomics. 20, 579(2019).</li>
<li>Farmer, P. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+epidemiology+studies+of+carcinogenic+environmental+pollutants.+Effects+of+polycyclic+aromatic+hydrocarbons+%28PAHs%29+in+environmental+pollution+on+exogenous+and+oxidative+DNA+damage%5BTitle%5D)+AND+%22Mutat+Res%22%5BJournal%5D)">Molecular epidemiology studies of carcinogenic environmental pollutants. Effects of polycyclic aromatic hydrocarbons (PAHs) in environmental pollution on exogenous and oxidative DNA damage.</a> Mutat Res. 544 (2-3), 397-402 (2003).</li>
<li>Phillips, D. H., Arlt, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genotoxicity%3A+damage+to+DNA+and+its+consequences%5BTitle%5D)+AND+%22EXS%22%5BJournal%5D)">Genotoxicity: damage to DNA and its consequences.</a> EXS. 99, 87-110 (2009).</li>
<li>Nitsch, S., Schneider, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Native+ChIP%3A+Studying+the+genome-wide+distribution+of+histone+modifications+in+cells+and+tissue%5BTitle%5D)+AND+%22Meth+Mol+Biol%22%5BJournal%5D)">Native ChIP: Studying the genome-wide distribution of histone modifications in cells and tissue.</a> Meth Mol Biol. 2846, (2024).</li>
<li>Nikitina, T., Wang, D., Gomberg, M., Grigoryev, S. A., Zhurkin, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combined+micrococcal+nuclease+and+exonuclease+III+digestion+reveals+precise+positions+of+the+nucleosome+core%2Flinker+junctions%3A+implications+for+high-resolution+nucleosome+mapping%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">Combined micrococcal nuclease and exonuclease III digestion reveals precise positions of the nucleosome core/linker junctions: implications for high-resolution nucleosome mapping.</a> J Mol Biol. 425 (11), 1946-1960 (2013).</li>
<li>Teves, S. S., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Salt+fractionation+of+nucleosomes+for+genome-wide+profiling%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Salt fractionation of nucleosomes for genome-wide profiling.</a> Methods Mol Biol. 833, 421-432 (2012).</li>
</ol>

The authors have no conflicts of interest to disclose.

Environmental pollution is a significant contributor to human cancers. Many pollutants are carcinogenic, meaning they can cause genetic damage that leads to the development of cancer40,41. However, determining whether a particular substance is tumorigenic is a challenging task. A fast, reliable, and cost-efficient method for identifying carcinogenic potential would empower scientists to efficiently screen environmental pollutants and assess their impact on genomi...

Common fragile sites (CFSs) are large chromosomal regions found on every human chromosome prone to breaking during metaphase. Under replication stress, replication at these regions is significantly delayed, preventing their complete duplication before mitotic entry1, which ultimately results in site-specific gaps and breaks. CFSs are hotspots for chromosomal instability and are a major cause of chromosomal rearrangements during early cancer development. Replication stress, which is often present under tumorigenic conditions, can lead to the loss of tumor suppressor genes and amplification of oncogenes-collectively referred to as copy number variation (CNV)2,3,4,5,6. Additionally, CFSs are highly prone to viral integration, further promoting cancer development7,8,9,10. Multiple homozygous deletions of tumor suppressor genes have been detected in CFS regions during pan-cancer analyses of primary tumors. The most commonly affected CFSs in cancer include FRA2F, FRA3B, FRA4F, FRA5H, and FRA16D11. CFSs are particularly vulnerable to breakage in the presence of extrinsic carcinogenic agents12. To assess the detrimental carcinogenic effects of environmental contaminants, a fast and reliable method for quantifying CFS break occurrence is needed.
Phosphorylation of H2A.X at the serine residue 139 (&#947;H2A.X) by Ataxia Telangiectasia and Rad3-Related Protein (ATR) or Ataxia Telangiectasia Mutated (ATM) is a key event in signaling replication fork stalling13. &#947;H2A.X serves as an indicator of stalled replication forks prior to double-strand break (DSB) formation13, creating a favorable chromatin environment to facilitate the efficient recruitment of repair proteins to stalled sites. Additionally, &#947;H2A.X can be recruited to break sites following fork collapse14,15, consistent with its primary role in DSB repair. Since CFS breaks are closely associated with chromosomal aberrations that drive cancer progression, detecting these breaks can be instrumental in understanding the early stages of tumorigenesis. The presence of &#947;H2A.X at CFSs can be used as a biomarker to detect early events of genomic instability. This information can help identify potential carcinogens and evaluate the risk associated with exposure to various extrinsic agents. By measuring DNA breaks at CFSs induced by extrinsic agents, &#947;H2A.X chromatin IP (ChIP) can provide insight into how such agents contribute to the mechanisms underlying tumorigenesis.
In the conventional ChIP (i.e., Cross-linked ChIP, X-ChIP), the association of &#947;H2A.X with its target DNA sequences is stabilized by reversible formaldehyde crosslinking. Chromatin is subsequently sheared to fragments of approximately 500 base pairs (bp) through sonication, and the resulting solution is cleared of debris by sedimentation16,17,18. A ChIP-grade &#947;H2A.X antibody is then added to the cleared chromatin fraction, followed by the addition of Protein A/G agarose beads to enrich for &#947;H2A.X-bound chromatin regions16,17,18. The immune complexes (i.e., beads-antibody-&#947;H2A.X-targeted DNA complex) are washed multiple times with stringent washing buffers to remove nonspecifically bound DNA fragments16,17,18. After washing, the specifically bound DNA is eluted from the immune complexes. The formaldehyde cross-links are then reversed, followed by protein digestion using proteinase K, after which the enriched DNA is purified and concentrated16,17,18. To assess the &#947;H2A.X-associated regions, PCR, quantitative PCR (qPCR), or direct sequencing is used16,17,18. The occupancy of &#947;H2A.X at specific regions, such as CFS, is determined by the intensity of the PCR or qPCR signal, which is proportional to the amount of &#947;H2A.X bound at that location, providing insights into site-specific DNA damage and repair events16,17,18.
Despite being a powerful experimental approach, the X-ChIP has several significant limitations: (i) it requires a large number of cells, typically in the range of 1 x 107&#160;to 5 x 107, due to the inefficiency of antibody precipitation associated with fixation, which increases the overall cost of the experiment19; (ii) the process of reversing formaldehyde cross-links and subsequent DNA purification is time-consuming and labor-intensive, making it challenging to maintain consistency and reliability in results; and (iii) &#947;H2A.X-DNA interactions with minor functional significance may not be distinguished from those with greater significance because the cross-linking step can stabilize transient interactions, leading to the detection of interactions that may not be biologically relevant19.
Native chromatin immunoprecipitation (Native ChIP or N-ChIP) is an essential biochemical technique used to study protein-DNA interactions within their native chromatin context under physiological salt conditions. It has been instrumental in elucidating the spatial and temporal organization of chromatin, transcription factor binding, and histone modifications. Native ChIP has a long-standing role in the broader field of chromatin biology and epigenetics, providing unique advantages and limitations compared to X-ChIP. This method, introduced in the late 1980s20, involves the isolation of chromatin from cells by methods that preserve its native structure, such as digestion with micrococcal nuclease (MNase)21. This preserves the inherent protein-DNA and histone-DNA contacts, which makes Native ChIP particularly well-suited for studying histone modifications and nucleosome positioning in their natural chromatin setting22. High-resolution Native ChIP studies have demonstrated the use of MNase digestion to reduce chromatin to individual nucleosomes, which facilitates the mapping of histone modifications with greater accuracy23. Furthermore, because no chemical cross-linking is involved, the risk of introducing biases or artifacts that might misrepresent the protein-DNA interactions is minimized24.
In contrast to X-ChIP, where formaldehyde or other cross-linking agents are used to fix protein-DNA interactions, Native ChIP provides a more realistic view of chromatin by avoiding potential cross-linking artifacts. However, while X-ChIP is generally better suited for detecting transient or dynamic interactions between DNA and regulatory proteins25, Native ChIP is ideal for stable protein-DNA interactions, such as histones or other chromatin-bound proteins26,27. One of the limitations noted for Native ChIP is the inability to capture low-affinity or transient binding events, which are often stabilized through cross-linking in X-ChIP25.
A significant body of work in epigenetics has leveraged Native ChIP to uncover histone modifications in diverse biological settings28. These efforts have been crucial in defining the histone code - the pattern of histone modifications that regulate gene expression and chromatin dynamics29. Although H2A.X is a less strongly associated linker histone, the native H2A.X ChIP method has been successfully applied in embryonic stem cells30. In this study, we optimized a chromatin extraction procedure to perform Native ChIP of &#947;H2A.X in human 293T cells (Figure 1). Hydroxyurea and aphidicolin are widely used in research to investigate DNA replication stress, damage, and genomic instability31. In this study, these agents were applied to cells to induce replication stress and generate DNA breaks at CFS.
Using starting material of approximately 1 x 106&#160;to 5 x 106&#160;cells, this method can be divided into four main stages: (i) subcellular fractionation to isolate chromatin, (ii) micrococcal nuclease (MNase) digestion to fragment chromatin, (iii) immunoprecipitation and elution, and (iv) DNA analysis by quantitative PCR (qPCR). Conducting ChIP following subcellular fractionation provides several benefits and has been well-documented in numerous studies32,33,34,35. This approach allows for the removal of chromatin-unbound proteins and other cellular debris, resulting in a highly purified chromatin fraction. By isolating chromatin before immunoprecipitation, subcellular fractionation helps maintain native chromatin interactions and reduces background noise from non-chromatin-associated proteins, which leads to more specific and reliable results, as only chromatin-bound complexes are retained for analysis. Moreover, subcellular fractionation enables milder conditions for chromatin digestion, thereby preserving physiological protein-DNA interactions and offering a more accurate representation of chromatin dynamics within the native cellular environment.
Using native ChIP of &#947;H2AX to measure the impact of extrinsic agents on common fragile site breakage holds significant potential for cancer research. This technique enables the detection of DNA damage induced by exposure to environmental carcinogens, providing insights into the molecular mechanisms by which pollutants contribute to genomic instability and cancer development. By preserving the native chromatin context, this method facilitates the accurate assessment of DNA damage patterns associated with carcinogenic exposure, aiding in the evaluation of environmental risks and the study of pollution-driven tumorigenesis.

<table><tbody><tr><td>0.2 &amp;micro;m nitrocellulose membrane</td><td>Amersham</td><td>10600011</td><td></td></tr><tr><td>Actin B</td><td>proteintech</td><td>20536-1-AP</td><td></td></tr><tr><td>Aphidicolin</td><td>MedChemExpress</td><td>HY-N6733</td><td></td></tr><tr><td>ChIP-grade magnetic Protein A/G beads</td><td>ThermoFisher</td><td>26162</td><td></td></tr><tr><td>Clarity Western ECL Substrate</td><td>Bio-Rad</td><td>#1705061</td><td></td></tr><tr><td>Glycogen, molecular biology grade</td><td>ThermoFisher</td><td>Cat. No.&amp;nbsp; R0561</td><td></td></tr><tr><td>HRP-conjugated secondary antibody&amp;nbsp;</td><td>proteintech</td><td>SA00001-2</td><td></td></tr><tr><td>hydroxyurea&amp;nbsp;</td><td>MedChemExpress</td><td>HY-B0313</td><td></td></tr><tr><td>Micrococcal Nuclease&amp;nbsp;</td><td>NEB</td><td>M0247S</td><td></td></tr><tr><td>normal IgG&amp;nbsp;</td><td>Santa Cruz</td><td>sc-2025</td><td></td></tr><tr><td>Taq Universal SYBR Green Supermix&amp;nbsp;</td><td>BioRad</td><td>1725120</td><td></td></tr><tr><td>&amp;gamma;H2A.X antibody&amp;nbsp; (for ChIP)</td><td>Sigma-Aldrich</td><td>05-636</td><td></td></tr><tr><td>&amp;gamma;H2A.X antibody (for WB)</td><td>Cell Signaling</td><td>#25955</td><td></td></tr></tbody></table>

capturing common fragile site breaks by native &#947;h2a.x chip

Capturing Common Fragile Site Breaks by Native &#947;H2A.X ChIP

Replication stress induced by exposure to extrinsic agents can lead to DNA breaks at common fragile sites, which are regions in the genome known to be prone to structural instability. The &#947;H2A.X chromatin immunoprecipitation (ChIP) assay serves as a powerful tool in genotoxicity studies, as &#947;H2A.X phosphorylation is a well-established marker for DNA double-strand breaks. Traditional &#947;H2A.X ChIP assays, however, are often labor-intensive and involve multiple, time-consuming steps. In this study, we present a simplified yet effective method that combines subcellular fractionation with native ChIP to isolate &#947;H2A.X-associated complexes. This approach is particularly suitable for analyzing &#947;H2A.X-chromatin interactions with enhanced specificity and efficiency. Using subcellular fractionation, chromatin-unbound materials are effectively removed, resulting in a purified chromatin fraction. Subsequent micrococcal nuclease (MNase) digestion under mild conditions allows chromatin fragmentation while preserving physiological interactions between &#947;H2A.X and its associated protein complexes. This preservation is essential for studying native interaction partners involved in DNA damage response pathways. This optimized native ChIP protocol substantially reduces the time and labor associated with conventional &#947;H2A.X ChIP assays. The streamlined procedure not only simplifies the workflow but also yields highly reproducible results, making it particularly advantageous in settings where high-throughput processing of multiple samples is required. This method has broad applicability in studies focused on genome stability, DNA repair, and chromatin biology, where accurate and efficient detection of DNA damage sites is critical. By employing optimized protocols and streamlined steps, this method enables the detection of DNA damage at fragile sites with improved sensitivity and minimal sample handling, making it a valuable tool for studies on genome stability and DNA damage response.

The size of chromatin fragments is crucial for the success of Native ChIP, as it directly impacts the accessibility of DNA regions for antibody binding. To determine the optimal MNase concentration for chromatin fragmentation, we prepared a series of microcentrifuge tubes containing varying concentrations of MNase (i.e., 0.0625 U, 0.125 U, 0.25 U, 0.5 U, 1 U, 2 U, 4 U, 8 U per reaction) and 40 µL of isolated nuclei. Each reaction was incubated at 37 °C for 5 min to achieve a range of chromatin fragment sizes. The results...

We present a rapid and efficient method to detect common fragile site breaks through native &#947;H2A.X chromatin immunoprecipitation (ChIP). This approach significantly reduces both the time and labor associated with traditional &#947;H2A.X ChIP assays while maintaining high reproducibility and reliability of results.

Replication stress induced by exposure to extrinsic agents can lead to DNA breaks at common fragile sites, which are regions in the genome known to be ...

Our luxury focuses on in losing dating, the role of common fragile site instability in jive in tumor re-genesis. Try to identify viral mobilities in oncogene gyron cancer cells that can be exploited, therapeutically. Otherwise, imaging and the sequencing technologies, such as single cell sympathy and live cell imaging provide high resolution mapping of genomic instability in mass.The region in vivo models including noise, genetically engineered mouse models, and CRISPR basis systems around us to investigate the mechanisms of oncogene genomic instability in detail. The long-term interest of our lab is in identifying specific weaknesses in cancer cells that can be strategically treated for infected treatments with a focus on minimizing harm to normal cells. This includes exploring genetic and epigenetic vulnerabilities to uncover normal therapeutic opportunities.To begin, treat the HEK 293 T cells with DMSO ethyl decolin and hydroxyurea in three different plates for 24 hours. After washing the cells twice with PBS, centrifuge them at 500 G four, five minutes at four degrees Celsius. Re-suspend the pellet in 500 microliters of freshly prepared cold buffer A, and incubate on ice for five to 10 minutes.Observe the lysis progression under a light microscope to confirm complete cell lysis. Once lysed, centrifuge the cell suspension at 500 G for five minutes at four degrees Celsius, and resuspend the nuclei pellet in 500 microliters of cold buffer A.Centrifuge again, and resuspend the intact nuclei with 100 microliters of micrococcal nuclease buffer by pipetting five to 10 times. Immediately add the predetermined amount of micrococcal nuclease to the samples.Place the tubes on a rotator for five minutes at 37 degrees Celsius. Immediately return the tubes to ice, and add EDTA to terminate the digestion. After vortexing, add 500 microliters of buffer B and pipette up and down for mixing.Incubate the tube on ice for five minutes to solubilize the proteins. To pellet the insoluble material, centrifuge the samples at maximum speed for five minutes at four degrees Celsius. Transfer the clear supernatant to new 1.5 milliliter tubes, labeled as the native chromatin fraction.To verify chromatin fragmentation, aliquot 10 microliters of the supernatant into a new 1.5 milliliter tube. Mix the supernatant with 20 microliters of distilled water and 30 microliters of phenol chloroform isoamyl alcohol. After vigorous vortexing, centrifuge the tubes at 20, 000 G for 10 minutes at four degrees Celsius and observe three distinct layers:a clear top layer, a white middle layer, and a yellow bottom layer.Carefully transfer 20 microliters of the upper aqueous phase, containing DNA to a fresh tube. Separate the purified DNA in a 1.5%agarose gel for 30 minutes at 100 volts. Visualize the digestion patterns, ensuring the size of chromatin fragments is primarily between 200 and 1, 000 base pairs.Transfer 20 microliters of digested chromatin into each tube, containing 180 microliters of elution buffer. Label the tubes as input samples, and store them at minus 20 degrees Celsius. Add 400 microliters of digested chromatin into another 1.5 milliliter tube for chromatin immunoprecipitation, or chip.Transfer gamma H2 AX antibody to DMSO treated, ethyl decolin treated, and hydroxyurea treated sample. Place the tubes on a rotator at four degrees Celsius for five hours, or preferably overnight. Meanwhile, using wide orifice tips, aliquot 100 microliters of chip grade magnetic protein AG beads into a new 1.5 milliliter tube.Place the tube on a magnetic stand for one minute, then carefully discard the liquid. Next, re-suspend the beads in one milliliter of PBS containing 0.5%BSA. Rotate the tube at four degrees Celsius for four hours.Place the tube back on the magnetic stand for one minute and discard the supernatant. After the second wash, resuspend the pre-coded beads in 100 microliters of buffer B.Add 25 microliters of the pre-coded magnetic bead suspension to each chip sample tube. Rotate the tubes at four degrees Celsius for two hours, then place the chip tubes on a magnetic stand and wait until the beads attach to the side of the tube and the solution becomes clear.Discard the clear supernatant without disturbing the magnetic beads. Resuspend the beads in one milliliter of wash buffer and rotate at four degrees Celsius for 10 minutes. Place the tubes back on the magnetic stand and wait until the solution becomes clear before discarding the wash buffer.After the final wash, briefly centrifuge the tubes at 400 G for 30 seconds at four degrees Celsius to remove residual liquid. Place the tubes back on the magnetic stand and carefully remove any remaining liquid from the bottom of the tube. After verifying the chip antibody pull down efficiency, using western blot, add 50 microliters of elution buffer to each of the remaining chip samples.Place the tubes on a Therma-mixer and shake for 15 minutes at room temperature. Place the tubes on a magnetic holder for one minute and collect the eluit into new tubes. Add 100 microliters of elution buffer to each chip elution sample, and 180 microliters of elution buffer to each input sample.Then add 200 microliters of phenol chloroform isoamyl alcohol to each sample and vortex the tubes vigorously to mix. After centrifuging the samples, carefully transfer the upper aqueous layer to the tubes containing 19 microliters of three molar sodium acetate and two microliters of glycogen solution. Mix by vortexing the tubes.Add 500 microliters of 100%ethanol to each sample, and vortex thoroughly. Place the tubes at minus 20 degrees Celsius for two hours, or overnight to precipitate the DNA. Then centrifuge the tubes before discarding the supernatant, and re-suspending the pellet in one milliliter of 70%ethanol.Once residual ethanol is removed by centrifugation, air dry the DNA pellets for two to three minutes before re-suspending them in 400 microliters of TE buffer. Higher concentrations of micrococcal nuclease led to more extensive digestion of chromatin, resulting in a predominance of mono-nucleosome fragments. In contrast, at lower micrococcal nuclease concentrations, the majority of chromatin fragments were larger, often exceeding one kilo base.Gamma H2 AX levels were significantly elevated in replication stressed cells, treated with ethyl decolin, compared to DMSO treated controls as shown in the input samples for both native chip and cross-linked chip.

capturing-common-fragile-site-breaks-by-native-h2ax-chip

Research

JoVE Journal

Biochemistry

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Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

This paper describes a protocol along with a comparative study of two microfluidic fabrication techniques, namely photolithography/wet-etching/thermal-bonding and Selective Laser-induced Etching (SLE), that are suitable for high-pressure conditions. These techniques constitute enabling platforms for direct observation of fluid flow in surrogate permeable media and fractured systems under reservoir conditions.

Microfluidic Fabrication Techniques for High-Pressure Testing of Microscale Supercritical CO2 Foam Transport in Fractured Unconventional Reservoirs

Here, we present a protocol for performing in situ TEM closed-cell gas reaction experiments while detailing several commonly used sample preparation methods.

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

A protocol for the culture and manipulation of mouse embryonic tissue on a microfluidic chip is provided. By applying pulses of pathway modulators, this system can be used to externally control signaling oscillations for the functional investigation of mouse somitogenesis.

A Microfluidics Approach for the Functional Investigation of Signaling Oscillations Governing Somitogenesis

This article traces the life cycle of a silicon nitride membrane resonator, from design through fabrication to characterization.

Fabrication and Characterization of High-Q Silicon Nitride Membrane Resonators

A novel sealing protocol to produce water-tight silicone-based seals for membranes of Polydimethylsiloxane (PDMS) chip devices. Intended for labs setting-up chip models and small-scale chip platform development. We demonstrate the protocol using plastic and native tissue membranes. This procedure only requires PDMS, toluene, mold, membrane, and a vacuum chamber.

Accessible Silicone Chip-to-Membrane Sealing Procedure for Flexible, Reliable Bonding

Learn a rapid native γH2A.X ChIP method for detecting common fragile site breaks in cancer cells. This streamlined approach reduces time and labor while maintaining high reproducibility for genomic instability research.

Capturing Common Fragile Site Breaks by Native γH2A.X ChIP

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