Name: chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae
Uploaded: 2017-12-29T15:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

The authors would like to thank members of the Green lab for helpful discussions. This work was supported in part by NIH grants R03AG052018 and R01GM124342 to E.M.G.

1. Pre-bind Antibody to Magnetic Beads
<ol>
	<li>Mix the protein A/G magnetic beads well and transfer 20 &#181;L of magnetic beads per one immunoprecipitation (IP) sample to a 1.5 mL tube. 
	NOTE: When pipetting beads, use wide-bore, low-retention tips.</li>
	<li>Place the tube with beads into a magnetic stand and allow beads to collect on side of the tube. Remove the supernatant.</li>
	<li>Wash the beads 3 times with 1 mL of cold Tris-buffered saline (TBS) (50 mM Tris-HCl pH 7.5, 150 mM NaCl).</li>
	<li>Add 200 &...

<ol>
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The procedure described here allows for the efficient recovery of DNA associated with modified histones in yeast cells by immunoprecipitation. This is followed by qPCR using primers which amplify regions of interest to determine local enrichment or depletion of specific histone modifications. Despite being developed as a method almost 20 years ago, chIP remains the defining assay for investigating histone modification status at different genomic regions and under diverse conditions. Although chIP coupled to next generati...

The dynamic post-translational modification (PTM) of histones is a key regulatory mechanism for many DNA-templated processes, including transcription, replication and DNA repair1,2. The ability to determine the abundance and precise localization of modified histones concomitant with these processes is therefore critical to understanding their regulation under different conditions in the cell. The development of chromatin immunoprecipitation (chIP) as a method largely stemmed from biochemical studies of the interactions of proteins with DNA, particularly in vitro methods using chemical crosslinkers, coupled with the need to evaluate the dynamic nature of protein-DNA interactions in vivo and at specific regions of the genome3,4,5. The advancement of quantitative PCR (qPCR) and sequencing technologies has also expanded the ability to perform chIP experiments with quantitative comparisons and across whole genomes, making it a powerful tool for dissecting DNA-protein interactions at multiple levels.
Currently, chIP is a required method for any research group interested in chromatin-mediated regulation of the genome as there are no comparable methods for directly interrogating the physical link between a modified histone and a specific genomic locus in vivo. Although variations of this method using next generation sequencing to map histone modifications throughout the genome6,7 are available, these approaches may address different scientific questions and their scale, cost and technical resources may be limiting for some research groups. Additionally, targeted chIP-qPCR is necessary to complement these approaches by providing methods to both optimize the chIP protocol prior to sequencing and to validate results from the epigenomic datasets. Mass spectrometry based approaches for identifying the full complement of histone marks associated with genomic regions have also emerged8,9,10,11, however, these approaches have some limitations regarding which regions of the genome can be probed and they require technical expertise and instrumentation that will not be available to all research groups. Therefore, chIP remains a foundational method for analyzing the abundance and distribution of histone modifications under diverse conditions for all research groups interested in epigenetics, chromatin and the regulation of genomic functions.
Here, we describe a method for chIP using the budding yeast model Saccharomyces cerevisiae (S. cerevisiae) to investigate the distribution of histone PTMs at chromatin. This approach relies on a number of core components of chIP protocols developed in yeast and also applied to diverse model systems12,13. Interactions between modified histones and DNA in the cell are preserved by crosslinking with formaldehyde. Following lysate preparation, chromatin fragments are solubilized into uniformly-sized fragments by digestion with micrococcal nuclease. Immunoprecipitation of the modified histones is performed with either commercial or lab-generated antibodies and any associated DNA is isolated and analyzed for enrichment at particular genomic regions using qPCR (Figure 1). For many histone modifications, the quantity of DNA obtained from this protocol is sufficient for testing more than 25 different genomic loci by qPCR.
This chIP method is highly versatile for monitoring the distribution of a single histone modification across multiple mutant strains or environmental conditions, or for testing multiple histone modifications in wildtype cells at a number of genomic loci. Furthermore, numerous components of the protocol are easily adjustable to optimize detection of either highly- or lowly-abundant histone marks. Finally, performing chIP of modified histones in budding yeast provides the opportunity to use key controls for antibody specificity that are largely unavailable in other systems. Namely, yeast strains can be generated that carry point mutations in histone residues that are targeted for modification, and, in some cases, there is only a single enzyme that catalyzes modification on a particular histone residue (e.g. histone lysine methyltransferases). Therefore, chIP can be performed in either the histone mutant or enzyme deletion strains to assay the extent to which non-specific binding of the antibody may be occurring and generating false positive results. This control is particularly valuable for newly-developed antibodies, and may even be used to validate antibody specificity for conserved histone modifications prior to their use in other systems. This approach complements other methods to test antibody specificity that distinguish among different modification states (such as mono-, di- and tri-methylation), including probing arrays of modified peptides and performing western blots of histones or nucleosomes with defined modifications. Overall, chIP in budding yeast is a powerful method for assessing the dynamics of histone PTMs throughout the genome and dissecting the mechanisms governing their regulation.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Yeast Extract</td>
			<td style="width:301px;">Research Products International (RPI)</td>
			<td style="width:164px;">Y20025-1000.0</td>
			<td style="width:167px;"></td>
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			<td height="20" style="height:20px;width:244px;">Peptone</td>
			<td style="width:301px;">Research Products International (RPI)</td>
			<td style="width:164px;">P20250-1000.0</td>
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			<td height="20" style="height:20px;width:244px;">Dextrose</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">BP350-1</td>
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			<td height="20" style="height:20px;width:244px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
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			<td height="20" style="height:20px;width:244px;">Glycine</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td>AC12007-0050</td>
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			<td height="20" style="height:20px;width:244px;">Tris</td>
			<td style="width:301px;">Amresco</td>
			<td style="width:164px;">0497-5KG</td>
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			<td height="20" style="height:20px;width:244px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758-500G</td>
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			<td height="20" style="height:20px;width:244px;">NaCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">BP358-10</td>
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			<td height="20" style="height:20px;">4-Nonylphenyl-polyethylene glycol</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">74385</td>
			<td>Equivalent to NP-40</td>
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			<td height="20" style="height:20px;width:244px;">MgCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">S25533</td>
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			<td height="26" style="height:26px;width:244px;">CaCl2</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">20899-25G-F</td>
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			<td height="20" style="height:20px;width:244px;">LiCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">AC413271000</td>
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			<td height="20" style="height:20px;width:244px;">Sodium Dodecyl Sulfate</td>
			<td style="width:301px;">Amresco</td>
			<td style="width:164px;">M107-1KG</td>
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			<td height="20" style="height:20px;width:244px;">Sodium Deoxycholate</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">30970-100G</td>
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			<td height="20" style="height:20px;width:244px;">Sodium Acetate</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">S2889</td>
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			<td height="26" style="height:26px;width:244px;">NaHCO3</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">S25533</td>
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			<td height="20" style="height:20px;width:244px;">PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>P7626-5G</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Yeast protease inhibitor cocktail</td>
			<td>VWR</td>
			<td>10190-076</td>
			<td></td>
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			<td height="40" style="height:40px;width:244px;">25 Phenol:24 Chloroform:1 Isoamyl Alcohol</td>
			<td style="width:301px;">VWR Life Science</td>
			<td>97064-824</td>
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			<td height="20" style="height:20px;width:244px;">Ethanol</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">E7023</td>
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			<td height="20" style="height:20px;width:244px;">Nuclease-Free Water</td>
			<td style="width:301px;">VWR</td>
			<td style="width:164px;">100720-992</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Micrococcal Nuclease</td>
			<td>Worthington Biochemical</td>
			<td>LS004797</td>
			<td></td>
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			<td height="20" style="height:20px;width:244px;">Glycogen</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">R0561</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Proteinase K</td>
			<td>Research Products International (RPI)</td>
			<td>P50220-0.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">RNase A&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>R6513-50MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Bradford Assay Reagent</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">23238</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;BSA Standard 2 mg/mL</td>
			<td style="width:301px;">ThermoScientific</td>
			<td>23210</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H4</td>
			<td>EMD Millipore</td>
			<td>04-858</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H4K16ac</td>
			<td>EMD Millipore</td>
			<td>ABE532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H3</td>
			<td style="width:301px;">Abcam</td>
			<td style="width:164px;">ab1791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H3K4me2</td>
			<td>Active Motif</td>
			<td>39142</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;High Rox qPCR Mix</td>
			<td>Accuris qMax Green, Low Rox qPCR Mix</td>
			<td>ACC-PR2000-L-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Protein A/G Magnetic Beads</td>
			<td style="width:301px;">ThermoScientific</td>
			<td>88803</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">magnetic stand for 1.5mL tubes</td>
			<td>Fisher Scientific</td>
			<td>PI-21359</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Acid-Washed Glass Beads</td>
			<td>Sigma-Aldrich</td>
			<td style="width:164px;">G8772</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Microtube Homogenizer</td>
			<td style="width:301px;">Benchmark</td>
			<td>D1030</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">2.0 mL screw-cap tubes with sealing rings</td>
			<td>Sigma-Aldrich</td>
			<td>Z763837-1000EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Gel loading tips</td>
			<td>Fisher Scientific</td>
			<td>07-200-288</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Cuvettes</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td style="width:164px;">50-476-476</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Parafilm</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">50 mL conical tubes</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td style="width:164px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">384-Well PCR Plate</td>
			<td>Fisher Scientific</td>
			<td>AB-1384W</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Gyratory Floor Shaker</td>
			<td style="width:301px;">New Brunswick Scientific</td>
			<td style="width:164px;">Model G10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Spectrophotometer</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">ND-2000c</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">&#160;Real-Time PCR Detection System</td>
			<td style="width:301px;">Bio-Rad</td>
			<td style="width:164px;">1855485</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

One key component of this protocol is optimizing the concentration of micrococcal nuclease (MNase) used to digest the chromatin into soluble fragments, as outlined in Step 10. This is critical for obtaining high resolution data regarding the distribution of histone modifications at genomic regions of interest. An MNase titration should be performed to determine the most suitable concentration to achieve primarily mono-nucleosomes with a smaller amount of di-nucleosomes in the soluble chro...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

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.

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 ...

The overall goal of this chromatin immunoprecipation protocol is to determine the abundance and localization of histone modifications at defined regions throughout the budding yeast genome. This method can help answer key questions in chromatin regulation, such as how histone modifications are linked to gene expression control and how these modifications change in response to different environmental conditions. An extremely versatile protocol that can be used to investigate many different histone modifications and multiple E strains at one time.Though this method can provide insight into histone modifications, it can also be applied to investigating other chromatin bonding proteins through the use of epitope tags or available antibodies. Start this procedure by growing the yeast cells inoculate 10 milliliters of YPD medium with a single colony of each appropriate strain, and grow it 30 degrees celsius in a shaking incubator at 220 rpm overnight. On the following day after measuring the optical density at 600 nanometers or OD600 of the culture, dilute the culture to an OD600 of 0.2 in 100 mL of YPD in a new flask.Grow the cells at 30 degree celsius in a shaking incubator at 220 rpm until they reach mid log phase. When the cultures have reached mid log phase, add 2.7 milliliters of 37%formaldehyde directly to the medium in each flask for a final concentration of 1%formaldehyde. Transfer the flasks to a shaker at room temperature, and shake at 50 rpm for 15 minutes.Add five milliliters of 2.5 molar glycine to the medium and continue shaking at 50 rpm at room temperature for five minutes to quench the formaldehyde. Subsequently, spin down the cells and wash them as described in the text protocol. Remove the supernatant after the last spin and proceed to making yeast lysates.Re-suspend the cells in one milliliter of cold chromatin amino precipitation or ChIP Lysis buffer with 1 millimolar PMSF and 1:1000 dilution of a yeast protease inhibitor cocktail. Transfer the suspension to a 2.0 milliliter screw cap tube containing 200 microliters of glass beads. Transfer the cells to a bead-beating apparatus for a high speed agitation at four degree celsius.Bead-beat for 30 seconds and ice for one minute. Bead-beat a total of six times, keeping the cells on ice for at least one minute between each 30 second beat-beating. Using a gel loading tip, transfer the lysate to a 1.5 milliliter tube.Centrifuge the lysate at 15, 500 g at four degree celsius for 20 minutes. Discard the supernatant and re-suspend the pellet in 250 microliters of micrococcal nuclease digestion buffer by gently pipetting up and down. Add 2.5 microliters of micrococcal nuclease to each reaction.Mix gently by inverting four to six times and immediately incubate in a 37 degree celsius water bath for 20 minutes. After 20 minutes, stop the reaction by placing the tubes on ice, adding five milliliters of 0.5 molar EDTA for a final concentration of 10 millimolar EDTA and mixing gently by inversion. Centrifuge at 15, 500 g at four degree celsius for 15 minutes.Then, transfer each supernatant to a new 1.5 milliliter tube. For the most consistent results, it is critical to ensure that the same concentration of protein is used in each amino precipitation. Depending on the number of amino precipitation or IP reactions, make a master mix of each micrococcal nuclease released chromatin fraction and ChIP lysis buffer and allocate one milliliter containing 50 micrograms of total protein to individual tubes.Remove 100 microliters from each IP mix to process as the input sample. Freeze the input samples at 20 degree celsius for later use. It is also important to make sure the same amount of beads bound to the antibody is used for each immunoprecipitation.Using a wide board pipette tip, add 20 microliters of magnetic protein AG beads, pre-bound with antibody to each IP sample. Rotate the samples at eight rpm at four degree celsius for three hours. When the IP is done, place the tubes in a magnetic stand and let beads collect on the side of the tube.Use a pipette to remove the supernatant. Add one milliliter of ChIP lysis buffer to the beads and rotate at eight rpm at four degree celsius for five minutes. Then, place the tubes on the magnetic stand and remove the supernatant.In this manner, wash the beads successively with the following buffers for the number of times indicated. ChIP Lysis Buffer, High Salt Wash Buffer, LiCl/detergent Wash Buffer and TE.With the last TE wash, use 0.5 milliliters of TE to transfer most of the beads to a new tube, and then use another 0.5 milliliters of TE to wash the tube and transfer the remaining beads. Add 250 microliters of freshly made ChIP elution buffer to each tube and vortex the solution briefly.Rotate the samples at eight rpm at room temperature for 15 minutes. Place the tubes in the magnetic stand. Carefully transfer the supernatant to a new tube and avoid the beads.Add another 250 microliters of ChIP elution buffer to the beads. Rotate the samples at eight rpm at room temperature for 15 minutes. Carefully and without disturbing the beads, transfer the supernatant to the tube containing the first elution.Thaw the input samples collected earlier and add 400 microliters of ChIP elution buffer to each input sample. To reverse protein DNA cross links, add to both input and IP samples, 20 microliters of five molar sodium chloride, five microliters of glycogen and 12.5 microliters of Proteinase K.Mix well by inverting or flicking the tubes. Incubate the samples in a 65 degree celsius water bath overnight.To begin this procedure, make a QPCR master mix for each set of primers by combining the appropriate volumes of QPCR mix, the forward primer, and the reverse primer. Make DNA master mixes for each DNA sample where one reaction contains 0.5 microliters of DNA and 4.0 microliters of nuclease free water Add 5.5 microliters of the appropriate primer master mix to each well in a 384 well QPCR plate. Spin the plate at 200 g at room temperature for one minute.Then, add 4.5 microliters of the appropriate DNA master mix to each well. Seal the plate, and spin at 200 g at room temperature for one minute. Perform QPCR using a real time QPCR system with the following conditions, 95 degree celsius for three minutes 40 cycles of 95 degree celsius for 10 seconds and 55 degree celsius for 30 seconds and a melting curve of 65 degree celsius to 95 degree celsius with 0.5 degree celsius increments for five seconds.One key component of this protocol is optimizing the concentration of micrococcal nuclease used to digest the chromatin into soluble fragments. This example result from agarose gel electrophoresis of DNA isolated following digestion of the chromatin pellet with varying amounts of micrococcal nuclease shows that 2.5 microliters of enzyme at 20 units per microliter produced predominantly mononucleosomes. This representative ChIP experiment used antibodies against a histone methyl marker H3K4me2 and histone H3 in wild type and set1 knockout cells.Set1 knockout cells lack the methyl transferase that catalyzes the mark The bars indicate the relative enrichment of H3K4me2 at the indicated low sci. The wild type strain showed clear enrichment for H3K4me2 at the five prime end of two genes known to be direct targets of set1, PMA1 and ERG11 whereas no signal was observed in set1 knockout cells. As expected, there was no enrichment of the H3K4me2 mark at TEL07L, the expression of SPR3 has also been reported to be regulated by set1 but these results indicate the regulation is likely to occur by a different mechanism than at PMA1 or ERG11.Once mastered, this technique can be done within four days if it is performed properly. While attempting this procedure, it's important to remember to carefully test the antibody used for specificity with the proper controls. If available, both positive and negative control regions should be probed in the QPCR.Following this procedure, other methods like GP sequencing can also be performed in order to determine the genome by distribution of histone modifications. After watching this video, you should have a good understanding of how to consistently detect the levels of different histone modifications at unique genomic regions with well controlled experiments.

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DNA and Chromosome Structure

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Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

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 ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

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, ...

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 ...

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription factors, histone modifications, and other DNA-associated proteins. ChIP-seq data can also be used to find differential binding of transcription factors in different environmental conditions or cell types. Initially, ChIP was performed through hybridization on a microarray (ChIP-chip); however, ChIP-seq has become the preferred method through technological advancements, decreasing financial barriers to sequencing, and massive amounts of high-quality data output. Techniques of performing ChIP-seq with bacterial biofilms, a major source of persistent and chronic infections, are described in this protocol. ChIP-seq is performed on Salmonella enterica serovar Typhimurium biofilm and planktonic cells, targeting the master biofilm regulator, CsgD, to determine differential binding in the two cell types. Here, we demonstrate the appropriate amount of biofilm to harvest, normalizing to a planktonic control sample, homogenizing biofilm for cross-linker access, and performing routine ChIP-seq steps to obtain high quality sequencing results.

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) is a method used to establish interactions between transcription factors and the genomic sequences they control. This protocol outlines techniques for performing ChIP-seq with bacterial biofilms, using Salmonella enterica serovar Typhimurium bacterial biofilm as an example.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription factors (TFs), chromatin regulators, and histone modifications, as well as detecting entire genomes for uncovering TF binding patterns and histone posttranslational modifications. Chromatin-modifying activities, such as histone methylation, are often recruited to specific gene regulatory sequences, causing localized changes in chromatin structures and resulting in specific transcriptional effects. The rice blast is a devastating fungal disease on rice throughout the world and is a model system for studying fungus-plant interaction. However, the molecular mechanisms in how the histone modifications regulate their virulence genes in Magnaporthe oryzae remain elusive. More researchers need to use ChIP-seq to study how histone epigenetic modification regulates their target genes. ChIP-seq is also widely used to study the interaction between protein and DNA in animals and plants, but it is less used in the field of plant pathology and has not been well developed. In this paper, we describe the experimental process and operation method of ChIP-seq to identify the genome-wide distribution of histone methylation (such as H3K4me3) that binds to the functional target genes in M. oryzae. Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...