Name: analysis of the c-kit ligand promoter using chromatin immunoprecipitation
Uploaded: 2017-06-27T15:00:00
Description: Watch this Scientific Journal Video about Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation at JoVE.com

This work was supported by the University of Texas MD Anderson Cancer Center, Houston, TX (startup funds, B.B.).

1. Preparation of Solutions
<ol>
	<li>Prepare the following solutions fresh for each experiment.
	<ol>
		<li>For the fixation solution, prepare 37% formaldehyde and store it at RT. Dilute it in 1x Phosphate-Buffered Saline (PBS) to a final concentration of 1.42%. 
		CAUTION: Formaldehyde is classified as irritant, corrosive, mutagenic, teratogenic, and carcinogenic. Do not ingest. Do not breathe gas/fumes/vapor/spray. In case of insufficient ventilation, wear suitable respiratory ...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+nucleic+acids+by+extraction+with+phenol:chloroform.">Purification of nucleic acids by extraction with phenol:chloroform.</a> CSH Protoc. 2006 (1), (2006).</li><li>Dennler, 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=Direct+binding+of+Smad3+and+Smad4+to+critical+TGF+beta-inducible+elements+in+the+promoter+of+human+plasminogen+activator+inhibitor-type+1+gene.">Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.</a> EMBO J. 17 (11), 3091-3100 (1998).</li><li>Shi, Y., 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=Crystal+structure+of+a+Smad+MH1+domain+bound+to+DNA:+insights+on+DNA+binding+in+TGF-beta+signaling.">Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling.</a> Cell. 94 (5), 585-594 (1998).</li><li>Seidel, H. 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In this report, we demonstrate the TGF-&#946;1-induced binding of SMAD2 to an SBE within the c-KIT ligand promoter and TGF-&#946;1-induced binding of STAT3 to its recognition sequence within the TGF-&#946;1 ligand gene. We demonstrate cytokine-induced binding of both transcription factors using chromatin immunoprecipitation.
Chromatin immunoprecipitation is a powerful tool to demonstrate the direct binding of a protein of interest to DNA, to characterize the stimuli that induce protein binding...

In the nucleus of eukaryotes, DNA interacts with histone proteins and nonhistone proteins and is condensed into chromatin. In the physiological, pathophysiological, and cell biological context, cellular functions are spatially and temporarily controlled by chromatin-coordinated gene expression. DNA-protein interactions have an essential role in the regulation of cellular processes, such as DNA replication, recombination, and repair, as well as protein expression. Therefore, the analysis of DNA-protein interactions is an indispensable tool in the evaluation of gene expression and cell function.
Several techniques exist to assess DNA-protein interactions in vitro, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting1,2. However, these techniques do not analyze the protein-DNA interaction within the chromatin and cellular context. ChIP is a technique that captures proteins bound to their specific DNA binding sites and thereby facilitates the identification of DNA-protein interactions within the chromatin context. The technique was originally developed by Gimour and Lis for the assessment of RNA polymerase II binding to specific genes in Escherichia coli and Drosophila melanogaster3,4. It is done by fixation of the DNA-protein complexes, followed by performing chromatin extraction and shearing the DNA into ~200 base pair (bp) fragments. Subsequently, the DNA-bound protein of interest is isolated by immunoprecipitation. After the reversal of the DNA-protein crosslink, the DNA is purified and analyzed. Several methods can be used for the analysis of the protein binding sites and depend upon the nucleic acid sequence of the protein binding site within the target gene5. In cases where the DNA sequence is known, standard Polymerase Chain Reactions (PCR) can be applied, using specific primer pairs flanking the known binding site. Quantitative Real-Time PCR (qRT-PCR) can also be used6. In cases where the sequence is unknown, ChIP can be combined with DNA microarrays (ChIP-on-chip), DNA sequencing (ChIP-seq), or cloning techniques7,8,9.
The TGF-&#946; pathway has potent tumor suppressing functions and is a key pathway in cell differentiation. It is activated through the binding of the TGF-&#946;1 ligand to its cognate receptor complex, resulting in the serine-phosphorylation of SMAD2/3 transcription factors. Following their association with the common mediator, SMAD4, the SMAD-complex translocates to the nucleus and binds to the SBE within the promoter region of the target genes, where it regulates genes controlling the cell cycle, apoptosis, and cell differentiation. The transcriptional response to TGF-&#946; stimulation is cell type- and context-specific10. Recently, we described a positive feedback loop between TGF-&#946; and the c-KIT pathway11. In this model, TGF-&#946;1-activated SMAD2 binds to the c-KIT ligand promoter and induces its expression and secretion. Subsequently, the c-KIT ligand activates the c-KIT receptor in an auto- and para-crinic&#160;fashion. c-KIT receptor activation results in STAT3 Tyr705-phosphorylation via JAK1/2. Following STAT3-activation and nuclear translocation, STAT3 binds to the TGF-&#946;1 ligand gene and regulates its expression.
Here, we demonstrate the essential role of ChIP analysis for the identification of SMAD2 binding to the c-KIT receptor ligand promoter and for the identification of the Signal Transducer (and) Activator (of) Transcription 3 (STAT3 binding to the TGF-&#946;1-gene).

<table border="0" cellpadding="0" cellspacing="0" width="907">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">HepG2 cells</td>
			<td style="width:193px;">ATCC</td>
			<td style="width:119px;">HB-8065</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Hep3B cells</td>
			<td style="width:193px;">ATCC</td>
			<td>HB-8064</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">TGF-&#946;1</td>
			<td>R&#38;D Systems</td>
			<td>101-B1</td>
			<td>Used at a concentration of 10 ng/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Anti-SMAD2 antibody</td>
			<td style="width:193px;">Cell Signalling Technology</td>
			<td style="width:119px;">5339</td>
			<td>Amount used per IP: 3 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Anti-STAT3 antibody</td>
			<td style="width:193px;">Cell Signalling Technology</td>
			<td style="width:119px;">4904</td>
			<td>Amount used per IP: 3 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">ChIP-IT Protein G Magnetic Beads</td>
			<td style="width:193px;">Active Motif</td>
			<td style="width:119px;">53033</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Protease Inhibitor Cocktail</td>
			<td style="width:193px;">Active Motif</td>
			<td style="width:119px;">37490</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Micrococcal Nuclease</td>
			<td style="width:193px;">Cell Signalling Technology</td>
			<td style="width:119px;">10011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR forward primer: PAI-1</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GGAAGAGGATAAAGGACAAGCTG-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR reverse primer: PAI-1</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-TGCAGCCAGCCACGTGATTGTC-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR forward primer: SCF</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-CACTGATGTTAATGTTCAGC-3&#8217;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR reverse primer: SCF</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GCTCTAATTTAAACCTGGAGC-3&#8217;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR forward primer: TGF-&#946;1 (STB-1)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GAGAGAGACGTGAGTGGCATGTT-3&#8217;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR reverse primer: TGF-&#946;1 (STB-1)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-TAGCTTTCTCTGCCTTGGTCTCCCC-3&#8217;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR forward primer: TGF-&#946;1 (STB-2)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GTACTGGGGGAGGAGCGGCATC-3&#8217;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR reverse primer: TGF-&#946;1 (STB-2)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-TGCCACTGTCTGGAGAGAGGTGTGTC-3&#8217;&#160;&#160;</td>
		</tr>
	</tbody>
</table>

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).

The binding of the TGF-&#946;1 ligand to its cognate receptor complex results in the serine-phosphorylation of SMAD2/3 transcription factors, followed by their association with the common mediator, SMAD4. The SMAD complex translocates to the nucleus. TGF-&#946; can regulate gene transcription, either directly via SMAD binding to SBEs within regulatory regions of the target genes, or indirectly through the SMAD-regulated expression of transcriptional activators or repressors that subsequen...

Watch this Scientific Journal Video about Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation at JoVE.com

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

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. ...

The overall goal of this chromatin immunoprecipitation procedure is to evaluate TGF beta 1-induced binding of the transcription factor SMAD2 to SMAD binding elements in the C kit promoter. This method can answer key questions in the field of cellular biology such as cytokine induced gene transcription. The main advantage of this technique is that it has the potential to demonstrate direct binding of transcription factors and other transcription regulatory proteins to their DNA binding sites in vivo.Grow the cells to between 70 and 80%confluence on ten centimeter tissue culture plates. Stimulate the cells per the experimental aims. Remove the tissue culture medium, and add five milliliters of fixation solution.Then, incubate the cells for 10 minutes at room temperature on a shaking platform. Remove the fixation solution, and wash the cells twice with 10 milliliters of ice cold one X PBS. Remove the one X PBS and add five milliliters of glycine stop fix solution.After incubating the cells for five minutes at room temperature on a shaking platform, remove the glycine stop fix solution and wash the cells twice with 10 milliliters of ice cold one X PBS. After removing the PBS, add two milliliters of cell scraping solution and harvest the cells using a cell scraper. Then, transfer the harvested cells to a conical tube on ice.Centrifuge the samples for 10 minutes at 600 times G, and four degrees celsius. Remove the supernatant, re-suspend the pellet in one milliliter of one X cell lysis buffer, and incubate on ice for 30 minutes. Once the pellet is re-suspended in the cell lysis buffer, transfer the cell suspension to an ice cold Dounce homogenizer, and Dounce for 10 strokes using a type B pestle for cell disruption.Then, transfer the cell suspension to a microcentrifuge tube, and centrifuge the sample for 10 minutes at 2, 400 times G, and four degrees celsius. Discard the supernatant and re-suspend the pellet in 350 microliters of nuclei digestion buffer. After incubating the samples for five minutes at 37 degrees celsius, add micrococcal nuclease and mix by vortexing.Incubate the samples at 37 degrees celsius for five to 20 minutes, mixing the samples every two minutes by inversion. The time in some concentration, if you're between cell lines, it might require optimization in case of suboptimal enzymatic shearing. An alternative shearing procedure using commercially available sonicare can be found in the text protocol.Following the incubation with the micrococcal nuclease, add seven microliters of ice cold 0.5 molar EDTA to stop the reaction. Centrifuge the samples with the sheared DNA for 10 minutes at 16, 200 times G and four degrees celsius. Transfer the sheared DNA containing supernatant to a fresh microcentrifuge tube.Aliquot 50 microliters into a separate microcentrifuge tube, to confirm the successful shearing of the DNA as described in the text protocol. Use the remaining volume immediately for immunoprecipitation or store it at minus 80 degrees celsius. Combine 10 to 25 micrograms of the sheared cross linked chromatin with 25 microliters of chromatin immunoprecipitation grade protein G magnetic beads.Then, add 10 microliters of immunoprecipitation dilution buffer, and one microliter of protease inhibitor cocktail. After four hours of incubation, add one to 10 micrograms of antibody to a 1.5 milliliter microcentrifuge tube. And add distilled water to a final volume of 100 microliters.Incubate the samples on an end-to-end rotator for four to 12 hours at four degrees celsius. Following incubation, place the tubes on a magnetic stand. Once the magnetic beads aggregate to the side of the tube, carefully remove and discard the supernatant.Wash the beads three times with 800 microliters of wash buffer one. For each wash, mix the beads by inverting the tube several times. Then, place the tube on the magnetic stand and carefully remove the wash buffer once the magnetic beads aggregate on the side of the tube.Next, wash the beads once with 800 microliters of wash buffer two using the same procedure. After washes, re-suspend the beads in 50 microliters of elution buffer and incubate the samples on an end-to-end rotator for 15 minutes at room temperature. Then, transfer the supernatant to a fresh tube with the aid of the magnetic stand.Add six microliters of five molar sodium chloride to reverse the cross linking followed by two microliters of proteinase K.After mixing the samples, incubate them for two hours at 65 degrees celsius. At this point, the samples can be directly analyzed for protein binding sites as described in the text protocol, or, can be stored at minus 20 degrees celsius. To confirm the TGF beta 1-induced binding of SMAD2 to the SCF promoter, the chromatin immunoprecipitation assay was performed.The TGF beta 1 treatment resulted in SMAD2 binding to the SCF promoter in human liver cancer lines, HepG2 and Hep3B. In the absence of TGF beta 1 stimulation, no SMAD binding was noted. As a positive control, for TGF beta 1-induced SMAD activation, chromatin immunoprecipitation for PAI-1 was performed.The PAI-1 promoter is a known target of TGF beta and SMAD. Specificity of the SMAD2 immunoprecipitation was confirmed through use of unspecific IgG antibodies. No SMAD2 binding to the SBE was noted after immunoprecipitation with IgG.To confirm the TGF Beta 1-induced binding of STAT3 to the TGF beta ligand gene, chromatin immunoprecipitation assays were performed using TGF beta 1 treated HepG2 and Hep3B cells. The TGF beta 1 treatment resulted in STAT3 binding to the second putative STAT3 binding site of the TGF beta gene, but not to the first one. In this example, the positive STAT3 binding to STB-2 serves as an internal positive control.Similar to the other chromatin immunoprecipitation experiment, STAT3 binding to the ST-2 was not seen after immunoprecipitation using IgG. Once mastered, this technique can be done in one or two days depending on the antibody if it is performed properly. While attempting this procedure, it's important to follow laboratory safety guidelines and be highly accurate at every step of the protocol.Precautions such as checking MSDS and wearing protective gear such as gloves, white coat and safety glasses should be taken while performing this procedure. After watching this video, you should have a good understanding of how to analyze protein binding to the specific DNA binding sequences. Following this protocol, other methods like promoter assays can be performed in order to confirm the functional relevance of protein DNA binding demonstrated by chromatin immunoprecipitation.

analysis-c-kit-ligand-promoter-using-chromatin

Research

JoVE Journal

Genetics

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

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 (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 ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

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 ...