Name: improved protocol for chromatin immunoprecipitation from mouse skeletal muscle
Uploaded: 2017-11-06T17:00:00.000+00:00
Duration: 9 min 30 s
Description: Watch this Scientific Journal Video about Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle at JoVE.com

We thank all the staff of the IGBMC high throughput sequencing facility, a member of &#34;France G&#233;nomique&#34; consortium (ANR10-INBS-09-08) and all IGBMC general services in particular the staff of the IGBMC animal facility. This work was supported by grants from the CNRS, the INSERM, the AFM, the Ligue Nationale contre le Cancer, the French state fund through the ANR under the programme Investissements d&#39;Avenir labelled ANR-10-IDEX-0002-02, the Labex INRT ANR-10-IDEX-0001-02 and the ANR-AR2GR-16-CE11-009-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. S.J was supported by the Ligue Nationale contre le Cancer and the ANR-AR2GR-16-CE11-009-01, and V.U by the Minist&#232;re de l&#39;Enseignement et de la Recherche. ID is an &#39;&#233;quipe labellis&#233;e&#39; of the Ligue Nationale contre le Cancer.

Mice were kept in accordance with the institutional guidelines regarding the care and use of laboratory animals and in accordance with National Animal Care Guidelines (European Commission directive 86/609/CEE; French decree no.87-848). All procedures were approved by the French national ethics committee.
1. Isolation of Muscle Tissue
<ol>
	<li>Sacrifice one adult 6 to 8-week-old mouse by cervical dislocation. Sterlise the limb by rinsing it with 70% ethanol and dissect the hind limb muscles (Gastrocnemius, Tibialis Anterior, Quadriceps) using fine point scissors and forceps. 
	NOTE: One mouse should yield around 500 mg of tissue.</li>
	<li>Mince the muscles in a 2 mL test tube containing 1 mL of ice-cold hypotonic buffer to a homogenous preparation of small (&#60;2 - 3 mm3) pieces using fine scissors and leave the tube shaking on a bench top agitator at 4 &#176;C for 5 - 10 min.</li>
</ol>
2. Tissue Lysis
NOTE: Please refer to Table 1 for all buffer compositions.
<ol>
	<li>Transfer the homogenate to a 14 mL round-bottom tube and resuspend in 5 mL cold hypotonic buffer (EDTA-free protease inhibitor cocktail and PMSF).
	<ol>
		<li>Homogenise the minced muscle tissue using a loose dounce (20 - 30 strokes within 3 min) or a mechanical tissue homogenizer for 15 - 30 s in round-bottom 14 mL tubes. 
		NOTE: The efficiency of lysis can be assessed at this stage by light microscopy, and if required, additional disruption can be performed.</li>
	</ol>
	</li>
	<li>Transfer the homogenate to 15 mL tubes and fill volume to 10 mL using cold hypotonic buffer. Fix the homogenate as described below before proceeding with the next mouse.</li>
</ol>
3. Fixation
<ol>
	<li>Add formaldehyde to 1% final concentration and shake for 10 min at room temperature.</li>
	<li>Add glycine to a final concentration of 0.125 M in each tube to stop fixation and shake for&#160;5 - 10 min at room temperature.</li>
</ol>
4. Nuclei Preparation
NOTE: Please refer to Table 1 for all buffer compositions.
<ol>
	<li>Homogenise the fixed lysate using a loose dounce (5 - 10 strokes).
	<ol>
		<li>Transfer to a 15 mL tube and centrifuge at 1,000 x g for 5 min at 4 &#176;C to obtain nuclei and cellular debris.</li>
	</ol>
	</li>
	<li>Remove the supernatant and resuspend the pellet in fresh 5 mL hypotonic buffer. Filter the lysate through a 70 &#181;m cell strainer into a 50 mL tube. Re-filter the filtrate through a 40 &#181;m cell strainer.</li>
	<li>Transfer the filtrate to a 15 mL tube and centrifuge at 1,000 x g for 5 minutes at 4 &#176;C to obtain the nuclear pellet. 
	NOTE: At this stage, the nuclei can be immediately sonicated, or snap frozen as a dry pellet in liquid nitrogen and stored at -80 &#176;C.</li>
</ol>
5. Sonication
<ol>
	<li>Assess the volume of the nuclear pellet (around 50 &#181;L for both mechanical and dounce homogenisation) and resuspend it in sonication buffer up to 3 to 4 times of packed nuclear volume and sonicate for 10 - 15 min at 4 &#176;C using a sonicator. 
	NOTE: The chromatin can be analysed immediately as described below (6) or frozen and stored at -80 &#176;C.</li>
</ol>
6. Assessing the Chromatin Fragment Size following Sonication
NOTE: Please refer to Table 1 for all buffer compositions.
<ol>
	<li>To de-crosslink, take 30 &#181;L of chromatin in a 1.5 mL test tube and add 20 &#181;L 5 M NaCl. Complete the volume to 500 &#181;L and incubate at 65 &#176;C overnight.
	<ol>
		<li>The next day, perform a treatment with 1 &#181;L proteinase K (20 mg/mL stock), 10 &#181;L 2 M Tris pH 6.8, and 10 &#181;L of 0.5 M EDTA for 1 h at 42 &#176;C. Perform a phenol-chloroform/chloroform extraction and precipitate the DNA with 1 volume of sodium acetate (3 M) and 2 volumes of 100% ethanol for one h at -80 &#176;C or overnight at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Pellet the DNA by centrifugation at 13,500 x g for 15 min. Decant the supernatant and wash the pellet thoroughly with cold 70% ethanol and centrifuge again for 5 min. Decant the supernatant and air-dry the pellet.</li>
	<li>Resuspend the pellet in the original volume (30 &#181;L) of TE buffer. Measure the DNA concentration in a spectrophotometer by absorbance at 260 nm/280 nm. Pipette 500 - 1,000 ng of DNA together with a DNA size ladder on separate lanes of a 1.5% agarose gel and perform electrophoresis to assess the fragment size of chromatin. 
	NOTE: Fragment size should be between 200 - 500 base pairs, and there should be no detectable high molecular weight fragments corresponding to non- or poorly-fragmented DNA. If required, the chromatin solution (step 5) can be re-sonicated for a longer time and then re-analysed as described above until the optimum size is obtained.</li>
</ol>
7. Chromatin Immunoprecipitation (ChIP)
NOTE: Please refer to Table 1 for all buffer compositions.
<ol>
	<li>Block the Protein G sepharose beads by aliquoting 1 mL of the 50% slurry in ethanol. Pellet the beads by centrifugation at 400 x g for 1 min in a benchtop centrifuge and wash with 1 mL of TE buffer centrifuge at 400 x g and repeat the wash.
	<ol>
		<li>After the second centrifugation, re-suspend in 1 mL of ChIP dilution buffer with 25 &#181;L of BSA (stock 20 mg/mL) and 20 &#181;L yeast tRNA (stock 10 mg/mL). Block the beads for at least 2 h prior to use by rotation (40 - 60 rpm) at 4 &#176;C.</li>
		<li>Dilute 50 &#181;g of chromatin (step 5) 8 to 10 times using ChIP dilution buffer. Add 50 &#181;L of blocked bead slurry and incubate for 2 h rotating (40-60 rpm) at 4&#176;C. Centrifuge at 400 x g at 4 &#176;C to obtain pre-cleared chromatin and transfer it to a fresh tube.</li>
	</ol>
	</li>
	<li>For immunoprecipitation, add the appropriate amount of primary antibody (e.g., 1-2 &#181;g per 10 &#181;g of chromatin) and incubate overnight with rotation at 4 &#176;C. 
	NOTE: The optimal amount of antibody may be recommended by the supplier or can be determined empirically by performing ChIP-qPCR with different quantities of antibody until optimum enrichment is observed. Also Protein G sepharose may be replaced by Protein A sepharose depending on the subtype of antibody used.</li>
	<li>Add the blocked beads the next day and incubate for 1 h with rotation (40 - 60 rpm) at 4 &#176;C. Centrifuge at 400 x g for 20 - 30 s to pellet the beads, remove the supernatant, and start the ChIP washes.</li>
	<li>ChIP washes: Perform the following washes with the buffers: once with Low Salt Buffer, and then twice with High Salt Buffer, twice with LiCl Buffer, and finally twice with TE Buffer.
	<ol>
		<li>Perform each wash for 10 min with rotation (40 - 60 rpm) at 4 &#176;C and pellet the beads between each wash by centrifugation at 400 x g for 20 - 30 s in a bench-top centrifuge.</li>
	</ol>
	</li>
	<li>For elution, remove the last wash and resuspend the beads in 250 &#181;L elution buffer (freshly prepared) for 15 min at room temperature while shaking.</li>
	<li>Centrifuge at 400 x g and collect the eluate in a fresh tube. Perform this step twice and then de-crosslink the eluate overnight with 20 &#181;L NaCl 5 M and 1 &#181;L RNase A (10 mg/mL), treat with proteinase K, and phenol-chloroform/chloroform extract as in Step 6.</li>
	<li>Resuspend in 50 &#181;L of TE buffer and use aliquots for ChIP-qPCR as per standard protocols15 to check the quality of the ChIP.</li>
</ol>

<ol>
	<li>Johnson, D. S., Mortazavi, A., Myers, R. M., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mapping+of+in+vivo+protein-DNA+interactions.">Genome-wide mapping of in vivo protein-DNA interactions.</a> Science. 316 (5830), 1497-1502 (2007).</li><li>Furey, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq+and+beyond:+new+and+improved+methodologies+to+detect+and+characterize+protein-DNA+interactions.">ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions.</a> Nat Rev Genet. 13 (12), 840-852 (2012).</li><li>Wade, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+Transcription+Regulatory+Networks+with+ChIP-seq+and+RNA-seq.">Mapping Transcription Regulatory Networks with ChIP-seq and RNA-seq.</a> Adv Exp Med Biol. 883, 119-134 (2015).</li><li>Alpern, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAF4,+a+subunit+of+transcription+factor+II+D,+directs+promoter+occupancy+of+nuclear+receptor+HNF4A+during+post-natal+hepatocyte+differentiation.">TAF4, a subunit of transcription factor II D, directs promoter occupancy of nuclear receptor HNF4A during post-natal hepatocyte differentiation.</a> Elife. 3, e03613 (2014).</li><li>Martianov, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+occupancy+of+an+extended+repertoire+of+CREM+and+CREB+binding+loci+in+male+germ+cells.">Cell-specific occupancy of an extended repertoire of CREM and CREB binding loci in male germ cells.</a> BMC Genomics. 11, 530 (2010).</li><li>Achour, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+identity+genes+regulated+by+super-enhancers+are+preferentially+down-regulated+in+the+striatum+of+Huntington's+disease+mice.">Neuronal identity genes regulated by super-enhancers are preferentially down-regulated in the striatum of Huntington's disease mice.</a> Hum Mol Genet. 24 (12), 3481-3496 (2015).</li><li>Joshi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TEAD+transcription+factors+are+required+for+normal+primary+myoblast+differentiation+in+vitro+and+muscle+regeneration+in+vivo.">TEAD transcription factors are required for normal primary myoblast differentiation in vitro and muscle regeneration in vivo.</a> PLoS Genet. 13 (2), e1006600 (2017).</li><li>Ohkawa, Y., Mallappa, C., Dacwag-Vallaster, C. S., Imbalzano, A. N., DiMario, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Myogenesis: Methods and protocols. 798, 517-531 (2012).</li><li>Dimauro, I., Pearson, T., Caporossi, D., Jackson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+protocol+for+the+subcellular+fractionation+of+skeletal+muscle+cells+and+tissue.">A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue.</a> BMC Res Notes. 5, 513 (2012).</li><li>Thomas, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAK1+and+CtBP1+Regulate+the+Coupling+of+Neuronal+Activity+to+Muscle+Chromatin+and+Gene+Expression.">PAK1 and CtBP1 Regulate the Coupling of Neuronal Activity to Muscle Chromatin and Gene Expression.</a> Mol Cell Biol. 35 (24), 4110-4120 (2015).</li><li>Kuo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+glucocorticoid+receptor-binding+sites+in+myotubes+identifies+gene+networks+modulating+insulin+signaling.">Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling.</a> Proc Natl Acad Sci U S A. 109 (28), 11160-11165 (2012).</li><li>Whyte, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Master+transcription+factors+and+mediator+establish+super-enhancers+at+key+cell+identity+genes.">Master transcription factors and mediator establish super-enhancers at key cell identity genes.</a> Cell. 153 (2), 307-319 (2013).</li><li>Benhaddou, A., Keime, C., Ye, T., Morlon, A., Michel, I., Jost, B., Mengus, G., Davidson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+TEAD4+regulates+expression+of+myogenin+and+the+unfolded+protein+response+genes+during+C2C12+cell+differentiation.">Transcription factor TEAD4 regulates expression of myogenin and the unfolded protein response genes during C2C12 cell differentiation.</a> Cell Death and Differentiation. 19 (2), 220-231 (2011).</li><li>Cowling, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+dynamin+2+expression+rescues+X-linked+centronuclear+myopathy.">Reducing dynamin 2 expression rescues X-linked centronuclear myopathy.</a> J Clin Invest. 124 (3), 1350-1363 (2014).</li><li>. ChIP Analysis Available from: <a target="_blank" href="https://www.thermofisher.com/fr/fr/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html">https://www.thermofisher.com/fr/fr/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html</a> (2017)</li></ol>

The authors declare that they have no competing financial interests.

Here we describe a novel protocol for preparing chromatin from adult mouse skeletal muscles and show that this chromatin is suitable for ChIP experiments that detect transcription factor binding and covalent histone modifications in the muscle fiber nuclei. This protocol involves several critical steps. The first is tissue disruption that can be performed either by dounce or by mechanical shearing. Mechanical shearing is faster and more reproducible, and is therefore the method of choice. Nevertheless, if no suitable apparatus is available, disruption can also be performed by douncing, where assessment of the efficiency of lysis under the microscope is recommended before proceeding to the fixation step. Fixation of whole-tissue lysates, rather than fixation of isolated nuclei, allowed more reproducible sonication in smaller volumes and for short durations. We also chose to fix the lysates after disruption rather than fix the tissue before disruption, as described by Thomas et al.10 Pre-fixing the tissue resulted in reduced efficiency of homogenisation. This protocol based on mechanical shearing is also faster than other proposed solutions, such as collagenase digestion, which involve prolonged incubation of the dissected tissue at 37 &#176;C8. During this period, changes in gene expression and transcription factor binding can occur. Fixing the nuclei as fast as possible is therefore essential to more faithfully capture the normal physiological state.
A second critical step is the purification of nuclei from the fixed lysate. For this, sequential filtering of the fixed lysate solution, first through a 70 &#181;m cell strainer to eliminate the bigger debris and then through a 40 &#181;m cell strainer to remove the fine debris, avoids clogging the strainers and maximizes the yields of nuclei obtained. Once purified by filtration, nuclei are sonicated and the DNA fragment size determined after de-crosslinking. A typical preparation from the two hind limbs of one mouse yields 100 &#181;g of chromatin. Pooling the lysates from up to 3 mice may improve the yield by reducing the losses during preparation. This procedure is efficient as it allows the recovery of up to 75% of the genomic DNA as chromatin (data not shown).
ChIP experiments designed to assess transcription factor binding and epigenetic modifications demonstrated the suitability of this protocol for studying gene regulation in the muscle fiber nuclei. Strong and robust ChIP-seq signals for Pol II and H3K27ac were generated from the chromatin, allowing identification of muscle fiber identity genes by their distinctive H3K27ac and Pol II profiles7. The much higher levels of H3K27ac and Pol II on genes strongly expressed in fiber nuclei, compared to genes expressed in satellite or endothelial cells, showed that the signal came predominantly from the fiber nuclei. This was also confirmed by the loss of Tead4 ChIP signal using chromatin from mice where it was specifically inactivated in muscle fibers. Nevertheless, weaker, but clearly above background signals, for satellite cell markers such as Vcam1, Pax7 (data not shown) can be detected showing that chromatin from these cells is also present.&#160;We anticipate that our protocol should be suitable for other techniques that use formaldehyde-fixed chromatin such as 3C/4C and HiC chromatin conformation capture techniques. Use of our protocol for combining ChIP-seq for transcription factors and chromatin modifications with chromatin conformation capture should in the future allow a detailed understanding of gene regulatory mechanisms in muscle fiber and how these may be regulated or upset by physiological stimuli, such as exercise, fasting, a high fat diet, denervation, or in disease, by using chromatin prepared from genetically modified mice reproducing diseases like X-linked centronuclear myopathy14.
In summary, this low-cost and time-efficient protocol allows the isolation of muscle fiber nuclei that can be sonicated immediately or frozen at -80 &#176;C for ulterior use. Use of chromatin prepared by this protocol has provided the first genome wide ChIP-seq data from muscle fiber7 and will facilitate future studies on gene regulation in this tissue.

Chromatin immunoprecipitation (ChIP) coupled to quantitative polymerase chain reaction (qPCR) and high throughput sequencing (ChIP-seq) have become the methods of choice to study transcription and epigenetic regulation of gene expression in various tissues and cell-types1. This technique allows genome-wide profiling of covalent chromatin modifications, histone variant occupancy, and transcription factor binding2,3.
While performing ChIP from cultured cells is well established, ChIP from mammalian tissues remains more challenging. Preparing chromatin for ChIP involves several critical steps that need to be optimized for every tissue and cell type. Chromatin can be prepared from the cellular lysates of cultured cells or following purification of their nuclei. In the case of mammalian tissues, efficient lysis, formaldehyde fixation, and purification of nuclei are critical in order to ensure optimal recovery of the chromatin. Moreover, the choice of whether to fix the nuclei before or after their purification has to be experimentally determined. Despite these hurdles, ChIP has been successfully performed from tissues such as the liver, testis, or brain4,5,6. In the case of skeletal muscle, disruption of such a physically resistant tissue, which exhibits a high content of structural proteins, and isolation of its nuclei is particularly challenging. Given this specificity, protocols optimized for other tissues do not give satisfactory results for skeletal muscles.
Here we describe a protocol to isolate ChIP-grade chromatin from mouse skeletal muscle tissue that involves physically disrupting the tissue, formaldehyde fixation, and then the isolation of nuclei and sonication. The efficiency of this method to prepare chromatin suitable for ChIP from this tissue was demonstrated by performing ChIP-qPCR and ChIP-seq for various transcription factors, RNA polymerase II, and covalent histone modifications7.
This new technique is much faster than a previously reported protocol8 comprising long collagenase digestion steps during which time, changes in genomic localisation of transcription factors and alterations in gene expression may take place. The rapidity with which the nuclei are isolated and fixed makes the method described here particularly attractive to prepare chromatin that more faithfully captures the native genomic occupancy state. Moreover, although other methods for chromatin isolation or protein extraction from muscle tissue have have been described8,9,10 and used for ChIP-qPCR experiments on selected genes, no ChIP-seq data has been reported using them. The method reported here is suitable for transcription factor and histone modification ChIP-seq, and hence should be also suitable for 3/4C or HiC chromatin conformation capture applications.

<table><tbody><tr><td>T 25 digital ULTRA-TURRAX</td><td>T 18 ULTRA-TURRAX</td><td>0009022800</td><td></td></tr><tr><td>PMSF</td><td>SIGMA-ALDRICH CHIMIE SARL</td><td>P7626-25G</td><td></td></tr><tr><td>Protease Inhibitor Cocktail-EDTA Free</td><td>Roche Diagnostics</td><td>11873580001</td><td></td></tr><tr><td>Protein G Sepharose beads</td><td>SIGMA-ALDRICH CHIMIE</td><td>P-3296</td><td></td></tr><tr><td>Proteinase K</td><td>SIGMA-ALDRICH CHIMIE</td><td>P-2308</td><td></td></tr><tr><td>Cell Strainers 70um</td><td>Corning BV</td><td>431751</td><td></td></tr><tr><td>Cell Strainers 40um</td><td>Corning BV</td><td>352340</td><td></td></tr><tr><td>Formaldehyde EM grade</td><td>Euromedex</td><td>15710-S</td><td></td></tr><tr><td>Rnase A</td><td>Fischer Scientific</td><td>12091039</td><td></td></tr><tr><td>Phenol:Chloroform:Isoamyl Alcohol 25:24:1</td><td>SIGMA-ALDRICH CHIMIE</td><td>P3803</td><td></td></tr><tr><td>BSA</td><td>SIGMA-ALDRICH CHIMIE</td><td>B4287-25G</td><td></td></tr><tr><td>yeast tRNA</td><td>SIGMA-ALDRICH CHIMIE</td><td>R5636</td><td></td></tr><tr><td>Glycogen Blue</td><td>AMIBION</td><td>AM9516</td><td></td></tr><tr><td>Pol II ChIP Antibody</td><td>Santa Cruz</td><td>SC-9001</td><td></td></tr><tr><td>H3K27ac ChIP Antibody</td><td>Active Motif</td><td>39133</td><td></td></tr><tr><td>Tead4 ChIP Antibody</td><td>Aviva Systems Biology</td><td>(ARP38276_P050)</td><td></td></tr><tr><td>IGEPAL</td><td>SIGMA-ALDRICH CHIMIE</td><td>I-3021</td><td></td></tr><tr><td>E220 Focused Ultrasonicator</td><td>Covaris E220</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Additional items&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Loose Dounce</td><td></td><td></td><td></td></tr><tr><td>15 ml and 50 ml falcon tubes</td><td></td><td></td><td></td></tr><tr><td>14 ml round bottom tubes</td><td></td><td></td><td></td></tr><tr><td>1.5 ml and 2 ml Eppendorf tubes</td><td></td><td></td><td></td></tr><tr><td>70 &amp;mu;m and 40 &amp;mu;m cell strainers (Corning)</td><td></td><td></td><td></td></tr></tbody></table>

improved protocol for chromatin immunoprecipitation from mouse skeletal muscle

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

To isolate nuclei, we performed mechanical homogenization of the dissected and minced muscle tissue for either 15 or 45 s, at 18,000 and 22,000 rpm (see Table of Materials). In all conditions, nuclei could be separated from the tissue debris, but the yield was optimal using the lower speed (Figure 1A-B). Nuclei could also be prepared by douncing for 3 - 5 min (Figure 1A, and data not shown), but mechanical homogenization is the method of choice, as optimal yields of nuclei can be achieved in as little as 15 s, allowing an important gain of time especially if multiple samples have to be processed (Figure 1B). Using this protocol, up to 2.7 x 107 nuclei from 500 mg of tissue (equivalent to 1 mouse&#39;s tissue) can be isolated to generate 100 &#181;g of chromatin.
To optimize chromatin fragmentation from fixed nuclei, we sonicated for 5 to 20 min. Under the settings (see Table of Materials), a 10 min sonication was optimal to generate chromatin with an average fragment size of 250 bp (Figure 2). This step should be optimized experimentally taking into account the type of sonicator used.
To assess the quality of the muscle chromatin prepared by the protocol described above, ChIP-qPCR and ChIP-seq experiments were performed against transcription factors, a histone modification or RNA polymerase II (Pol II). ChIP-qPCR against the glucocorticoid receptor (GR) in the presence or absence of dexamethasone (Dex) treatment revealed strong Dex-dependent GR enrichment at regulatory elements of the Redd1 and Murf1 genes that are known to be Dex and GR regulated in muscle fibers11, while no signal was seen at the testis specific Prm1 promoter used as a negative control (Figure 3A).
ChIP-qPCR against acetylated lysine 27 of histone H3 (H3K27ac), a covalent modification found at active enhancers and promoters, showed strong enrichment at the Desmin (Des) promoter that is active in muscle fibers, compared to a negative intergenic control region (Figure 3B). H3K27ac ChIP-seq revealed a high level of this mark throughout the Des locus, typical of a &#39;super-enhancer&#39; regulated tissue identity gene12 (Figure 4A). Similarly, Pol II ChIP-seq showed high levels of transcribing Pol II at this locus.
ChIP-qPCR for transcription factor Tead4, which plays an important role in myogenic differentiation13,7, revealed its enrichment on previously described binding sites at the Ccnd1 and Ifrd1 genes (Figure 3C). Importantly, enrichment was reduced using chromatin prepared from mice where Tead4 was selectively inactivated in muscle fibers (Tead4skm-/-)7 (Figure 3C). Analyses of ChIP-seq data for both Tead1 and Tead4 showed their binding to a site in the promoter of the Kdm5a gene using chromatin from wild-type muscle, whereas the Tead4 signal, but not that of Tead1, H3K27ac, or Pol II, was lost using the chromatin from muscles of the Tead4skm-/- mice (Figure 4B). Similarly, Tead1 and Tead4 binding to a regulatory element downstream of the Adssl1 gene is seen with chromatin from wild-type muscle, whereas Tead4 binding was selectively lost using chromatin from the Tead4skm-/- muscle (Figure 4C). These observations demonstrate that the Tead4 signal seen in the ChIP-seq data came from binding in the muscle fiber.
To determine the contribution of other cell types associated with the muscle fiber to the ChIP-seq results, we assessed the H3K27ac and Pol II signals at the Vcam1 gene that is active in muscle satellite cells and Pecam1, a marker of endothelial cells of blood vessel that irrigate the muscles. Compared to the high levels seen at the Des or Adssl1 genes, the levels of H3K27ac and Pol II seen at both the Vcam1 and Pecam1 genes were much lower (Figure 4A and Figure 4C). The large differences in signal for muscle fiber expressed genes compared to those expressed in associated tissues indicated that the signal seen in ChIP from the chromatin prepared using the protocol described above comes predominantly from binding events in the muscle fiber.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56504/56504fig1.jpg" /> 
Figure 1: Purification of nuclei from adult mouse muscle. (A) Tissue lysates obtained by mechanical homogenisation (18,000 rpm, 45 s) and dounce homogenization (30 strokes/3 min) were stained with trypan blue and observed under a digital microscope and images were captured at 20X&#160;magnification. Scale bar = 100 &#181;m. (B) Number of nuclei obtained from 500 mg of tissue (equivalent of 1 mouse) after preparation under the indicated conditions, 18,000 and 22,000 rpm for 15 and 45 s or 3 min of douncing. Error bars represent mean &#177; S.E.M. for n = 3 mice for mechanical homogenization, and n = 3 mice for douncing. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig1large.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/56504/56504fig2.jpg" /> 
Figure 2: Verification of chromatin fragment size. Isolated nuclei were sonicated for 5, 10, 15 or 20 min as indicated. After de-crosslinking and purification, the DNA fragment size was assessed by agarose gel electrophoresis in the presence of ethidium bromide. M is DNA size marker. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56504/56504fig3.jpg" /> 
Figure 3: Use of chromatin for ChIP-qPCR. (A) Mice were subjected to intra-peritoneal injections of vehicle or dexamethasone (10 mg/kg) and after 1.5 h, chromatin was prepared from hind limb muscles. The enrichment in ChIP at GR target genes Redd1 and Murf1, compared to the negative control protamine promoter (Prm1), is indicated as a % of input precipitated. (B) ChIP was performed for H3K27ac and IgG control antibodies and analysed at the Desmin promoter and compared to an intergenic negative control region. (C) Tead4 ChIP was performed on muscle chromatin obtained from wild type and muscle-specific Tead4 knockout mice (Tead4skm-/-) and analysed at Tead4 binding sites in the Ccnd1 and Ifrd1 promoters, compared to the control intergenic region. In all experiments, chromatin from 3 mice was pooled and error bars represent mean &#177; S.E.M in a Student&#39;s T-test for three technical replicates. P value &#60;0.001**, &#60;0.0001 ***; NS, non-significant. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56504/56504fig4.jpg" /> 
Figure 4: Genomic profiles of muscle fiber chromatin. (A) Screenshots from the University of California at Santa Cruz genome browser of the indicated loci illustrating the much higher H3K27ac and Pol II levels at the Desmin gene, highly expressed in muscle fiber, compared to the Vcam1 and Pecam1 genes expressed in satellite cells and endothelial cells, respectively. (B) UCSC screenshots of the indicated loci illustrating binding of Tead1 and Tead4, as well as H3K27ac and Pol II, in chromatin from wild-type (WT) muscle compared to that from Tead4skm-/- (MT) muscle, where selective loss of Tead4 binding is observed. The ChIP-seq data illustrated is available as GSE82193 in the GEO database. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig4large.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>Buffer Name</td>
			<td colspan="3">Composition</td>
		</tr>
		<tr>
			<td rowspan="6">Hypotonic buffer</td>
			<td colspan="3">10 mM HEPES-KOH (pH 7.3)</td>
		</tr>
		<tr>
			<td colspan="3">10 mM KCl</td>
		</tr>
		<tr>
			<td colspan="3">5 mM MgCl2</td>
		</tr>
		<tr>
			<td colspan="3">0.1% NP-40</td>
		</tr>
		<tr>
			<td colspan="3">0.1 mM PMSF (freshly added)</td>
		</tr>
		<tr>
			<td colspan="3">Protease Inhibitor cocktail (PIC) 1x (freshly added)</td>
		</tr>
		<tr>
			<td rowspan="8">Sonication buffer</td>
			<td colspan="3">1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">10 mM EDTA</td>
		</tr>
		<tr>
			<td colspan="3">20 mM Tris-HCl pH 8</td>
		</tr>
		<tr>
			<td colspan="3">150 mM NaCl</td>
		</tr>
		<tr>
			<td colspan="3">0.1% Sodium Deoxycholate</td>
		</tr>
		<tr>
			<td colspan="3">1% Triton X-100</td>
		</tr>
		<tr>
			<td colspan="3">Freshly added 0.1 mM PMSF</td>
		</tr>
		<tr>
			<td colspan="3">PIC 1x</td>
		</tr>
		<tr>
			<td rowspan="7">ChIP Dilution buffer</td>
			<td colspan="3">0.01% SDS</td>
		</tr>
		<tr>
			<td colspan="3">1.1% Triton X-100</td>
		</tr>
		<tr>
			<td colspan="3">1.2 mM EDTA</td>
		</tr>
		<tr>
			<td colspan="3">16.7 mM Tris HCl pH8</td>
		</tr>
		<tr>
			<td colspan="3">167 mM NaCl</td>
		</tr>
		<tr>
			<td colspan="3">Freshly added 0.1 mM PMSF</td>
		</tr>
		<tr>
			<td colspan="3">PIC 1x</td>
		</tr>
		<tr>
			<td rowspan="5">Low Salt buffer</td>
			<td colspan="3">0.1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">1% Triton X-100</td>
		</tr>
		<tr>
			<td colspan="3">500 mM EDTA</td>
		</tr>
		<tr>
			<td colspan="3">20 mM Tris HCl pH8</td>
		</tr>
		<tr>
			<td colspan="3">150 mM NaCl</td>
		</tr>
		<tr>
			<td rowspan="5">High Salt buffer</td>
			<td colspan="3">0.1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">1% Triton X-100</td>
		</tr>
		<tr>
			<td colspan="3">500 mM EDTA</td>
		</tr>
		<tr>
			<td colspan="3">20 mM Tris HCl pH8</td>
		</tr>
		<tr>
			<td colspan="3">500 mM NaCl</td>
		</tr>
		<tr>
			<td rowspan="4">LiCl Buffer</td>
			<td colspan="3">250 mM LiCl</td>
		</tr>
		<tr>
			<td colspan="3">1% NP-40</td>
		</tr>
		<tr>
			<td colspan="3">1 mM EDTA</td>
		</tr>
		<tr>
			<td colspan="3">10 mM Tris HCl pH 8</td>
		</tr>
		<tr>
			<td rowspan="2">Elution buffer</td>
			<td colspan="3">1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">100 mM NaHCO3</td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE) buffer</td>
			<td colspan="3">50 mM Tris HCl pH 8</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">1 mM EDTA</td>
		</tr>
	</tbody>
</table>
Table 1: Buffer compositions.

Watch this Scientific Journal Video about Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle at JoVE.com

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

improved-protocol-for-chromatin-immunoprecipitation-from-mouse

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.5K Views.  Institut de Génétique et de Biologie Moléculaire et Cellulaire. A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

Video: Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Genetics

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Satellite Cell Isolation from Mouse Limb Muscles and Flow Cytometry Analysis

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion,&#160;the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.

Author Spotlight: Cistrome Analysis in Mouse Muscle Stem Cells 

Presented here is an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells adapted to the study of transcription regulation in muscle fibers by cleavage under targets and release using nuclease (CUT&amp;RUN).

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...

Developmental Biology

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Biology

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. ...

Neuroscience

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...