Name: chromatin immunoprecipitation assay for the identification of arabidopsis protein-dna interactions in vivo
Uploaded: 2016-01-14T16:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo at JoVE.com

The authors would like to acknowledge The Plant Cell for allowing the use of some data published in this journal to elaborate the representative results described here in Figure 4. This work was supported by the EU 7FP Marie Curie-Initial Training Network EpiTRAITS (Grant Agreement 316965), and by the Spanish Ministerio de Econom&#236;a y Competitividad (grants BIO2010-15589 and BIO2013-43098-R).

1. Crosslinking of the Plant Material (1 hr)
<ol>
	<li>Grow the Arabidopsis lines used in the experiment (wild type &#8212; WT&#160;&#8212; versus mutants, and/or lines expressing the tagged version of your protein of interest versus lines expressing the tag not fused to any protein) for 12-18 days on large Petri dishes (150 mm) with MS-agar medium (1 L: 1x Murashige &#38; Skoog salts, 10 g sucrose, 0.5 g MES, pH 5.7 (KOH), 1% agar). Alternatively, grow plants on pots containing 3:1 mix of soil and vermiculite. 
	...

<ol>
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The ChIP protocol described here is a reproducible and powerful technique to analyze interactions between proteins and specific DNA sequences in vivo in Arabidopsis plants. A successful identification of binding sites for the proteins of interest requires an adequate selection of plant organs or developmental stages where the relevant interactions are actually taking place. In addition, it is critical to obtain an appropriate fixation of the plant material and an optimal shearing of the chromatin by sonication. ...

During recent years a wide range of genetic, molecular and genomic tools have been developed in the model species Arabidopsis thaliana. This technology has facilitated enormously the progress in understanding how plant development is regulated. Among the developmental processes studied using Arabidopsis as a model, the genetic control of flowering time has been extensively analyzed. These studies have shown that plants modulate very precisely the time of flowering in response to endogenous cues such as hormones and the age of the plant, and also to environmental signals such as photoperiod and temperature that synchronize flowering time with the natural cycle of seasons1,2. The isolation and characterization of Arabidopsis mutants with alterations in the time of flowering has been determinant in unveiling a complex network of genes that regulate the flowering time in response to endogenous and environmental factors. These genetic circuits are integrated at the level of a few master genes that act as switches of flowering, and the exact timing of the floral initiation depends on the balance of flowering promoting and repressing activities that work upstream of the floral integrator genes1,3.
The functional characterization of genes identified for their role in the control of flowering initiation, aided by the recent use of genomic approaches, have revealed the central role of transcriptional regulation in the modulation of flowering time. In fact, many of the master genes of flowering encode transcription factors4. In addition, a number of chromatin remodeling protein complexes influence the expression of master genes of flowering. A number of the Arabidopsis mutants isolated for their altered flowering time turned out to carry mutations in genes encoding a variety of chromatin modifiers. Different chromatin remodelers that introduce posttranslational modifications in histone tails, reposition nucleosomes relative to the DNA or exchange canonical histones by histone variants are necessary for the proper regulation of flowering in Arabidopsis5,6. Some of these chromatin remodeling activities catalyze the deposition or removal of covalent modifications such as acetylation or methylation in specific histone residues. These histone marks are specifically recognized by specialized effectors that recruit other chromatin remodeling complexes, transcription factors or components of the transcriptional machinery to regulate the transcriptional activity of flowering genes.
Chromatin ImmunoPrecipitation (ChIP) allows the identification of in vivo DNA-binding sites for proteins of interest (Figure 1). This procedure takes advantage of the ability of certain chemicals to cross-link the proteins to the DNA. The resulting DNA-protein complexes can be then immunoprecipitated by using specific antibodies against transcription factors, chromatin-binding proteins, or particular modifications and heterologous epitopes (commonly referred to as &#8220;tags&#8221;) attached to the protein of choice. The DNA purified from these immunoprecipitates can be used as a template in quantitative PCR (qPCR) reactions to assess for enrichment of particular sequences of interest. In this way, the binding sites of transcription factors or the distribution of histone marks in particular genes can be established7,8. In addition, combined with Next Generation Sequencing (NGS) that enables massive parallel sequencing, ChIP technology has made possible the genome-wide identification of the binding sites of transcription factors as well as unveiling epigenomic landscapes of histone modifications. Furthermore, the simultaneous analysis of gene expression allows monitoring how the binding of transcriptional regulators or the deposition of particular histone marks correlate with the transcriptional activity of genes9.
The use of ChIP protocols in Arabidopsis has allowed assessing the impact that a variety of transcriptional regulators have on the chromatin organization of master genes of flowering and how these structural changes influence gene expression5,6. Previous results showed that the Arabidopsis locus EARLY BOLTING IN SHORT DAYS (EBS) acts as a repressor of flowering and mutants in this gene show an acceleration of flowering and upregulation of the master gene of flowering FT. In addition, loss-of-function mutations in FT fully suppress the early flowering phenotype of the ebs mutant plants, indicating that FT is required for the premature flowering of ebs mutants and that EBS is necessary for the repression of this master gene of flowering10,11. EBS encodes a PHD-containing protein that can specifically bind histone H3 di- and trimethylated in the lysine 4 residue (H3K4me2/3), suggesting a role for EBS in the chromatin-mediated repression of FT12. The use of the ChIP approach demonstrated that the Arabidopsis PHD-containing protein EBS10,11 binds regulatory regions of the floral integrator gene FT to repress its expression12. Additional data obtained through the use of ChIP technology showed that this protein is required to maintain low levels of histone acetylation, a hallmark of active transcription, in the chromatin of this master gene of flowering during initial stages of Arabidopsis development. These observations, together with genetic and gene expression data, demonstrate that this Arabidopsis PHD-containing protein has a central role in the fine tuning of flowering time by modulating the expression of the floral integrator gene FT12. The work presented here provides an optimized method useful not only for the analysis of histones but also for other chromatin associated proteins, and with increased efficiency and reduced experimental time. Furthermore, this report illustrates how the use of ChIP protocols has provided new insights into the relationship between changes in chromatin modifications and transcriptional states of plant genes, and how these chromatin-mediated mechanisms of gene expression control impact on the onset of flowering in Arabidopsis.

<table border="0">
	<tbody>
		<tr>
			<td>MES</td>
			<td>Sigma</td>
			<td>M8250</td>
			<td></td>
		</tr>
		<tr>
			<td>MS (Murashige and Skoog Basal Salt Mixture)</td>
			<td>Sigma</td>
			<td>M5524</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 37%&#160;</td>
			<td>Sigma</td>
			<td>F8775-25</td>
			<td>Use under the fume hood</td>
		</tr>
		<tr>
			<td>Protease inhibitor mix cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack</td>
			<td>Roche</td>
			<td>5892791001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioruptor Standard sonication device</td>
			<td>Diagenode</td>
			<td>B01010002 (UCD200TO)</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>50046</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads&#174; magnetic beads coupled with protein A or protein G</td>
			<td>Life Technologies</td>
			<td>10003D/10001D</td>
			<td>Check manufacturer&#8217;s manual for antibody affinity</td>
		</tr>
		<tr>
			<td>Miracloth</td>
			<td>Merck Millipore</td>
			<td>475855</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#8482; X-100 Surfact-Amps&#8482; Detergent Solution</td>
			<td>Life Technologies</td>
			<td>85112</td>
			<td></td>
		</tr>
		<tr>
			<td>(mouse, rat, rabbit&#8230;)-IgG</td>
			<td>Diagenode</td>
			<td>C15400001, C15420001, C15410206</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic rack - DynaMag&#8482;-2</td>
			<td>Life Technologies</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K9/14ac polyclonal antibody - Premium&#160;</td>
			<td>Diagenode</td>
			<td>C15410200-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Chelex&#174; 100 Resin</td>
			<td>Bio-Rad</td>
			<td>142-2832</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Life Technologies</td>
			<td>17916</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Myc Tag Antibody, clone 4A6</td>
			<td>Millipore</td>
			<td>05-724</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler&#174; 480 SYBR Green I Master</td>
			<td>Roche</td>
			<td>4707516001</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromatin immunoprecipitation assay for the identification of arabidopsis protein-dna interactions in vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Eight main steps can be singled out in this ChIP protocol for the identification of in vivo protein-DNA interactions, including growing and harvesting of plant material, cross-linking of chromatin, chromatin isolation, chromatin fragmentation, selective isolation of the complexes between DNA and the protein of interest by immunoprecipitation, protein digestion, DNA purification, and qPCR analysis (Figure 1). A crucial step in the ChIP protocol is the fixation of DNA-protein interactions in a cro...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo at JoVE.com

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

The overall goal of this procedure is to unveil interactions between proteins and DNA in the chromatin of arabidopsis cells. This method can help answer key questions in the plant biology field, such as which genes are regulated by a given transcription factor, or what system modifications are associated with a transcriptional status of genes. The main advantage of this technique is that through the cross-linking step, it is possible to fix and capture changes in chromatin organization occurring during plant development.Though this method can provide a insight into protein interaction in arabidopsis thaliana, it also can be applied to different plant orders and adapted to other plant species. Demonstrating this procedure will be Dorota Komar and Alfonso Mouriz, two PhD students in our laboratory. After growing arabidopsis according to the text protocol, collect 1.5 grams of plant material in 50 milliliter tubes, and keep them on ice during cross-linking.Then, use a laboratory burner-heated needle to make little holes in the lids of the tubes. Add 40 milliliters of 1x pbs and 1.08 milliliters of formaldehyde to the tubes. Screw the lids on the tubes, and place the tubes in a desiccator.Vacuum infiltrate for 10 minutes, then carefully release the vacuum to prevent churning up the solution. After mixing the sample, repeat the vacuum and mixing steps. Upon infiltration, observe the plant material and confirm that it becomes slightly translucent and sinks to the bottom of the tube.Add 2.5 milliliters of two molar glycine to a final concentration of 0.125 molar and apply the vacuum for five minutes. After releasing the vacuum, discard the solution by inverting the tubes and allowing it to drip through the holes in the lids. Use pbs to rinse the plantlets twice, and rinse with water once.Then dry the plantlets on a paper towel before using liquid nitrogen to freeze. Per each immunoprecipitation, prepare 15 microliters of magnetic beads in a 1.5 milliliter microcentrifuge tube. Add one milliliter of chromatin immunoprecipitation, or chip dilution buffer, to the beads, mix by rotation, and place the 1.5 milliliter microcentrifuge tube in a magnetic rack.After allowing the beads to attach to the magnet for about a minute, use a pipetman to remove the supernatant before repeating the wash. Re-suspend the beads in 200 microliters of chip dilution buffer. For immunoprecipitation add the required amount of the specific antibody to one of the two tubes prepared for each plant blind.A key factor in successful chip experiment is the chosen antibody for the immunoprecipitation of the protein of interest. Antibodies raised against plant transcription factor can be useful for chip experiments, but the serum has to be thoroughly tested. For example, antibodies checked only in Western blot analysis are not necessarily valid for chip.As a negative control, add the same amount of non-specific IGG to the second tube. Incubate the tubes at four degrees Celsius overnight on a rotating wheel to allow the antibody to attach to the beads. Using a mortar and pestle, thoroughly grind the frozen plant material in liquid nitrogen until the powder becomes homogeneous and light green.Transfer the powder to a new 50 milliliter tube. Under a fume hood, add 30 milliliters of extraction buffer one, and mix well to soak the tissue powder. From this step on, keep the samples at four degrees Celsius at all times, and make sure that the tissue is completely thawed before moving forward.Clear the slurry by passing it through a filtration tissue with a pore size of 22 to 25 micrometers into a new 50 milliliter tube. Then centrifuge at 1, 000 times g and four degrees Celsius for 20 minutes. Gently decant the supernatant from the pellet, which should be about two milliliters in volume.With five milliliters of extraction buffer two, re-suspend the pellet. Then centrifuge at 1, 000 times g and four degrees Celsius for 10 minutes. After decanting the supernatant, re-suspend the pellet in 300 microliters of extraction buffer three.In a separate tube, add 600 microliters of extraction buffer three and then carefully layer the re-suspended pellet on top of the clean buffer. Centrifuge the tube for one hour at 16, 000 times g at four degrees Celsius, followed by aspiration of the supernatant with a pipette. Next, use 300 microliters of sonication buffer to slowly re-suspend the nuclear pellet, avoiding bubble formation, which may affect sonication efficiency.Carefully transfer the suspension to a clean, 1.5 milliliter tube. Sonicate the suspension to lyse the nuclei and randomly share the chromatin into 200 to 600 base pair fragments. Verify the DNA fragment sizes on an agarose gel, then spin the solution at 12, 000 times g and four degrees Celsius for 10 minutes.Transfer the supernatant to a fresh 1.5 milliliter tube, and discard the pellet. Aliquot one-tenth of the supernatant into a new tube, and label it as input'before freezing at minus 20 degrees Celsius. Finally, make a ten-fold dilution of the chromatin in chip dilution buffer to achieve a final SDS concentration of 0.1 percent.Samples can then be frozen and stored at minus 20 degrees Celsius for several weeks. After incubating the beads with antibodies overnight, use one milliliter of chip dilution buffer to wash the beads three times, then re-suspend the beads in one milliliter of the diluted chromatin, and incubate the tube overnight on a rotating wheel at four degrees Celsius. The following day, place the beads on the magnetic rack at four degrees Celsius, and after removing the solution, use one milliliter of low-salt washing buffer to wash the beads twice, then use high-salt washing buffer to wash the beads once.After using one milliliter of lithium chloride washing buffer to wash the beads once, use one milliliter of TE buffer to wash the beads twice. Aspirate to thoroughly remove the TE wash buffer. Once the input samples are thawed, transfer five microliters of each into new 1.5 milliliter tubes, and moving forward, treat similarly to the chip samples.Reverse the cross-linking by adding 200 microliters of five percent chelating ion exchange resin and incubate at 95 degrees Celsius for 10 minutes, shaking every two to three minutes. Spin down at 16, 000 times g for 30 seconds, and carefully transfer the supernatant into new microfuge tubes. Add two microliters of 10 milligrams per milliliter protonase K, and incubate at 37 degrees Celsius for 30 minutes to digest proteins and release DNA.To stop the reaction, incubate at 95 degrees Celsius for 10 minutes, Quickly spin at 16, 000 times g for 30 seconds, and transfer the supernatant to a new tube. After cleaning the DNA according to the text protocol, dilute one to ten with water, and use five microliters per reaction to measure the abundance of binding sites by qPCR. Analyze the data according to the text protocol.Aribidopsis leaves are covered in wax layers and trichomes favor the formation of air bubbles, both of which make it difficult for cross-linking reagents to penetrate the plant tissue. Shown here are cross-linked seedling samples that were vacuum cross-linked to increase fixation efficiency. This figure demonstrates that an increased number of sonication cycles progressively reduces the average size of the DNA in chip samples, reaching the optimum enrichment in the range of 200 to 600 base pairs after 20 to 30 cycles, as demonstrated here.As seen in this figure, four regions important for the transcriptional regulation of the FT locus, a master gene of flowering, have been chosen for the analysis. Chip results demonstrate that the EBS protein combined one of the four regions tested, a regulatory sequence close to the transcriptional start site of FT.Finally, chip assays performed using an antibody directed against histone h3 acetylated in lysines nine and 14, which is correlated with transcriptionally active chromatin, revealed higher abundance of H3K9/14ac in mutant DBS versus wild type plants in all four genomic fragments of FT tested, consistent with the observed up-regulation of this master gene of flowering in this mutant. Once mastered, this technique can be done in two days if it is performed properly.Following this procedure, together with other measures like gene expression analysis, can contribute to answering other questions, like:What is the biological role of the binding of the protein to DNA? Is it at transcriptional activation or repression? And Which histone modifications are associated?After it's development, this technique paved the way for researchers in the field of plant epigenetics to explore chromatin states in our reductive cells. And this can be extended to other plant species. After watching this video, you should have a good understanding of how to analyse the binding of your aribidopsis protein of interest to DNA in vivo.Don't forget that working with 2-methoxyethanol, formaldehyde or sonicator reagents, pathogens and stem mutations can be extremely hazardous, and precautions such as protective clothes, gloves or hearing protection should be always taken while using this procedure.

chromatin-immunoprecipitation-assay-for-identification-arabidopsis

Research

JoVE Journal

Biology

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

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 ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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 ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).