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 central role of floral repressors.</a> Plant Science. 181 (4), 364-378 (2011).</li><li>Pajoro, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+(r)evolution+of+gene+regulatory+networks+controlling+Arabidopsis+plant+reproduction:+a+two-decade+history.">The (r)evolution of gene regulatory networks controlling Arabidopsis plant reproduction: a two-decade history.</a> J Exp Bot. 65 (17), 4731-4745 (2014).</li><li>He, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+regulation+of+flowering.">Chromatin regulation of flowering.</a> Trends Plant Sci. 17 (9), 556-562 (2012).</li><li>Jarillo, J. A., Pineiro, M., Cubas, P., Martinez-Zapater, J. 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F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , (1989).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+Chain+Reaction:+Basic+Protocol+Plus+Troubleshooting+and+Optimization+Strategies.">Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies.</a> J. Vis. Exp. , e3998 (2012).</li><li>Wong, M. L., Medrano, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+for+mRNA+quantitation.">Real-time PCR for mRNA quantitation.</a> Biotechniques. 39 (1), 75-85 (2005).</li><li>Dedon, P. C., Soults, J. A., Allis, C. D., Gorovsky, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+formaldehyde+fixation+and+immunoprecipitation+technique+for+studying+protein-DNA+interactions.">A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions.</a> Anal Biochem. 197 (1), 83-90 (1991).</li><li>Yu, C. 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D., Bonyadi, R., Gifford, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fluorescence-activated+cell+sorting+in+studying+plant+development+and+environmental+responses.">The use of fluorescence-activated cell sorting in studying plant development and environmental responses.</a> Int J Dev Biol. 57 (6-8), 545-552 (2013).</li><li>Deal, R. B., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+INTACT+method+for+cell+type-specific+gene+expression+and+chromatin+profiling+in+Arabidopsis+thaliana.">The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana.</a> Nat Protoc. 6 (1), 56-68 (2011).</li><li>Zeng, P. Y., Vakoc, C. R., Chen, Z. C., Blobel, G. A., Berger, S. 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The authors declare that they have no competing financial interests.

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

20.1K Views.  Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)-Universidad Politécnica de Madrid. 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 occurr...