Name: chromatin immunoprecipitation assay for the identification of arabidopsis protein-dna interactions in vivo
Uploaded: 2016-01-14T16:00:00.000+00:00
Duration: 12 min 36 s
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. 
	CAUTION! Formaldehyde is toxic by inhalation, in contact with skin and if swallowed, and should be handled in a chemical hood wearing suitable personal protective equipment. Steps 1.2 &#8211; 1.6 should be performed under the fume hood.</li>
	<li>Make little holes in the lid of the 50 ml tubes with a laboratory burner-heated needle. Add 40 ml of 1x PBS buffer and 1.08 ml of formaldehyde to a final concentration of 1% (stock 37% solution). Collect 1.5 g of plant material in those 50 ml tubes. Keep the tubes on ice during crosslinking.</li>
	<li>Close the 50 ml tubes with the lid. Place the tubes in a desiccator and apply vacuum. Vacuum infiltrate the tissue for 10 min, then release the vacuum carefully, to prevent churning up the solution. Mix the sample. Repeat twice. After infiltration, observe the plant material and confirm that it becomes slightly translucent and tend to sink to the bottom of the tube (Figure 2).</li>
	<li>Add 2.5 ml of 2 M Glycine to achieve a final concentration of 0.125 M. Apply vacuum for 5 min. Glycine acts as a competitive inhibitor of formaldehyde crosslinking.</li>
	<li>Discard the PBS formaldehyde-glycine solution by inverting the closed 50 ml tube with holes in the lid.</li>
	<li>Rinse the plantlets twice with 1x PBS and once with water discarding the washing solution as described in step 1.5. Dry them on a paper towel and freeze in liquid nitrogen. 
	NOTE: Frozen tissue can be kept at &#8211;80 &#176;C for weeks.</li>
</ol>
2. Preparation of the Antibodies (1 day)
NOTE: For immunoprecipitation, the use of magnetic beads conjugated with antibodies via a protein G or protein A linker with high affinity for the constant domain of the antibody heavy chain is recommended. The DNA-protein complexes can attach unspecifically to the surface of the beads-antibody conjugates. For that reason, it is necessary to perform controls without specific antibodies to quantify the non-specific background binding.
<ol>
	<li>For each immunoprecipitation, prepare 15 &#181;l of magnetic beads in a 1.5 ml microcentrifuge tube. The amount of beads depends on the quantity of the chromatin used for immunoprecipitation and also on the antibodies.</li>
	<li>To wash, add 1 ml of Chromatin Immunoprecipitation (ChIP) dilution buffer to the beads, mix by rotation and place the 1.5 ml microcentrifuge tube in a magnetic rack. Wait around 1 min to let the beads attach to the magnet. With the 1.5 ml microcentrifuge tubes still in the rack, remove the supernatant by pipetting. Repeat twice. 
	NOTE: For each plant line two immunoprecipitation sets are needed: one with the beads and antibody (Ab) and a second no antibody (No-Ab) control with magnetic beads only.</li>
	<li>Resuspend the beads in 200 &#181;l of ChIP dilution buffer. Add the required amount of the specific Ab to one of the two tubes prepared for each plant line (the exact dilution differs for each Ab and has to be optimized experimentally or, in the case of commercial antibodies, follow the instructions of the manufacturer). Use this sample for immunoprecipitation. Add the same amount of unspecific IgG to the other tube, which will be used as negative control.</li>
	<li>Incubate O/N on a rotating wheel at 4 &#176;C to let the antibody attach to the beads.</li>
</ol>
3. Chromatin Extraction (4 hr)
<ol>
	<li>Grind thoroughly the frozen plant material in liquid nitrogen using mortar and pestle until the powder becomes homogenous and light green. Transfer the powder to a new 50 ml tube.</li>
	<li>For steps 3.2 &#8211; 3.6, work under the fume hood. Add 30 ml of Extraction buffer 1 (ExB 1), and mix well to soak the tissue powder. From this step on, keep the samples at 4&#160;&#176;C at all times. Frozen tissue and liquid nitrogen leftovers will cause the buffer to freeze. Make sure to thaw the frozen tissue completely by inverting the tubes 4-5 times every 2 min.</li>
	<li>Clear the slurry by passing it through a filtration tissue with pore size of 22-25 &#181;m into a new 50 ml tube and centrifuge it at 1,000 x g for 20 min at 4 &#176;C. 
	NOTE: In this step, the nuclei and all the organelles will pellet. ExB 1 contains enough sucrose to provide high density and prevent the disruption of organelle structure.</li>
	<li>Gently remove the supernatant by decantation. At this stage, observe a green pellet of around 2 ml.</li>
	<li>Resuspend the pellet in 5 ml of ExB 2. Resuspension of the pellet will be difficult at this point. Centrifuge at 1,000 x g for 10 min at 4 &#176;C. 
	NOTE: ExB 2 contains 1% Triton X-100 to help burst the chloroplasts open and remove chlorophyll.</li>
	<li>Resuspend the pellet in 300 &#181;l of Extraction Buffer 3 (ExB 3).</li>
	<li>In a clean microfuge tube, add 600 &#181;l of ExB 3. Take the resuspended pellet from step 3.6 and carefully layer it on top of the clean ExB 3.</li>
	<li>Spin for 1 hr at 16,000 x g at 4 &#176;C. 
	NOTE: This step helps removing the detergent from the sample.</li>
	<li>Remove the supernatant by aspirating with a pipette. Resuspend the nuclear pellet in 300 &#181;l of sonication buffer by slowly pipetting up and down to avoid bubble formation, which may affect sonication efficiency. Carefully pipette all the suspension out and transfer it to a clean 1.5 ml microcentrifuge tube. 
	NOTE: This buffer contains SDS, to lyse the nuclei and release the chromatin.</li>
	<li>Sonicate the suspension to lyse the nuclei and randomly shear the chromatin into small fragments. Use sonication conditions that render chromatin enriched in fragments between 200-800 bp (20-30 cycles with the settings 30 sec ON / 30 sec OFF at 4 &#176;C for the sonication device available described in the Table of materials/reagents). Check the efficiency of sonication by electrophoretic separation of the obtained DNA fragments on an agarose gel13, loading 2 &#181;l of sonicated chromatin (Figure 3). 
	CAUTION! Make sure to wear ear protection equipment during the sonication step in case the ultrasound device is not contained in a soundproof cabinet. 
	Crucial step! The chromatin fragmentation largely depends on the sonication equipment. The sonication conditions should be adjusted to achieve enrichment in the appropriate DNA sizes. See Figure 3 for an example of how to optimize chromatin fragmentation.</li>
	<li>Spin the solution once at 12,000 x g for 10 min at 4 &#176;C. Transfer the supernatant by pipetting to a clean 1.5 ml microcentrifuge tube. Discard the pellet. Take 1/10 of the supernatant to a new 1.5 ml microcentrifuge tube, mark it as Input and freeze at -20 &#176;C.</li>
	<li>Dilute the chromatin 10x with ChIP dilution buffer to dilute SDS to final concentration of 0.1%. 
	Note: Samples can be frozen and stored at -20 &#176;C for several weeks.</li>
</ol>
4. Immunoprecipitation of the Protein of Interest and Rescue of DNA (1 day, 3 hr)
<ol>
	<li>Wash the beads coated with the Ab from step 2.4 three times with 1 ml of ChIP dilution buffer to remove excess of antibody as described in step 2.2. Resuspend in 200 &#181;l of ChIP dilution buffer. Add 50 &#181;l of isolated chromatin. Incubate O/N on a rotating wheel at 4 &#176;C. 
	NOTE: Check the concentration of the chromatin in microvolume UV-Vis spectrophotometer. 50 &#181;l of isolated chromatin should contain more than 10 &#181;g of DNA.</li>
	<li>For steps 4.2-4.5 work in 4 &#176;C. Wash the beads twice in 1 ml of Low Salt Washing buffer as described in step 2.2.</li>
	<li>Wash the beads once in 1 ml of High Salt Washing buffer.</li>
	<li>Wash the beads once in 1 ml of LiCl Washing buffer.</li>
	<li>Wash the beads twice in 1 ml of TE buffer. After the last wash, make sure to fully remove the TE buffer left in the tube by aspiration with a pipette.</li>
	<li>Take out the INPUT samples (step 3.9). Transfer 5 &#181;l of the INPUT to a new 1.5 ml microcentrifuge tube and continue as with the ChIP samples. Reverse crosslinking by adding 200 &#181;l of 5% chelating ion exchange resin, and incubate for 10 min at 95 &#176;C shaking every 2-3 min. Spin down at 16,000 x g for 30 sec and carefully transfer the supernatant into a new microcentrifuge tube by pipetting. 
	NOTE: While the traditional method for crosslinking reversal involves an O/N incubation with NaCl, the incubation with a chelating ion resin at 95&#160;&#176;C allows for efficient decrosslinking and elution of DNA in 10 min, therefore making the protocol one day shorter.</li>
	<li>Add 2 &#181;l of proteinase K (10 mg/ml) and incubate at 37 &#176;C for 30 min to digest proteins and release DNA.</li>
	<li>Stop the reaction by incubating at 95 &#176;C for 10 min.</li>
	<li>Quickly spin at 16,000 x g for 30 sec and transfer supernatant to a new tube by pipetting.</li>
	<li>Clean the DNA isolated using a standard DNA fragments purification kit. 
	NOTE: Proceed according to the manufacturer&#39;s instructions.</li>
	<li>Transfer the DNA binding column from the kit to a new 1.5 ml microcentrifuge tube. Elute the purified DNA by adding 20 &#181;l of DNase-free water to the center of the column and spinning at 16,000 x g for 60 sec. Stop point: Samples can be stored at -80 &#176;C for several weeks.</li>
</ol>
5. Measuring Abundance of Binding Sites in the Immunoprecipitated DNA by qPCR (4 hr)
NOTE: DNA isolated from the precipitated chromatin has to be analyzed to determine which DNA fragments have been ChIP-ed from the total chromatin, due to its binding to the protein of interest.
<ol>
	<li>Design primers for the genomic regions of interest with a melting temperature (Tm) of 60 &#176;C and a GC content of 30%-80%. As chromatin is fragmented by sonication, the length of amplified sequence should not be longer than 150-200 nucleotides (Table 1). 
	NOTE: A useful tool for primer design is Primer 3 program (http://bioinfo.ut.ee/primer3-0.4.0/). Additional guidelines for primer design are available in previous reports8,14,15.</li>
	<li>Use a BLAST program to make sure that the primers are specific (especially promoters can share similar TF binding sequences) and test the primer efficiency using 1:10 dilutions in water of the input DNA (step 4.9). Certain regions of the genome will be purified better than others. It is therefore important to design PCR primers and check them on the diluted input DNA.</li>
	<li>Dilute the purified DNA 1:10 with water and pipette 5 &#181;l of the diluted DNA in a PCR tube for each reaction. 
	NOTE: Dilute only the amount needed, as DNA can attach to the walls of the 1.5 ml microcentrifuge tubes. Multiple thawing-freezing cycles increase the number of attached DNA molecules. For each primer pair, amplification in the input, Ab-ChIP and No-Ab ChIP has to be analyzed.</li>
	<li>To complete the PCR mix, add Sybr Green PCR master mix to a final concentration of 1x, and 0.5 &#181;M of each primer. Fill up to 20 &#181;l with DNase-free water.</li>
	<li>Perform the qPCR reaction following the instructions of the Sybr Green PCR master mix manufacturer.</li>
</ol>
6. Data Analysis
NOTE: Among the existing ways of analyzing ChIP-data, two are most commonly used. The first of them is the fold enrichment method, also named as &#8216;relative enrichment&#8217;, &#39;signal over background&#39; or &#39;relative to the no-antibody control&#39;. The second method is named as &#8220;% of input&#8221;.
<ol>
	<li>Data analysis by fold enrichment method.
	<ol>
		<li>Create a spreadsheet with samples names and raw Ct values that have been obtained after qPCR reaction.</li>
		<li>For each sample, subtract from its raw Ct value the raw Ct value obtained for the corresponding No-Ab control. NOTE: After this mathematical operation, Ct value for No-Ab sample will equal 0.</li>
		<li>Calculate fold enrichment by exponentiation operation with base 2 and exponent (power) equal to the negative remainder obtained in step 6.1.2 (Table 2). 
		fold enrichment = 2 - (Ct(sample) - Ct(No-Ab))&#160; 
		NOTE: In this method the abundance of a specific DNA fragment in the ChIP-ed sample is compared with the abundance of this fragment in No-Ab control. The assumption of this method is that the level of background signal is reproducible between different primer sets, samples, and replicate experiments. However, this &#180;noise&#180; signal levels do vary between primer sets, samples, and experiments.</li>
	</ol>
	</li>
	<li>Data analysis by % of input method.
	<ol>
		<li>Create a spreadsheet with samples names and raw Ct values that have been obtained for them after qPCR reaction.</li>
		<li>Adjust raw Ct values for input samples by subtracting a value of logarithm base 2 from the fraction of total extracted chromatin that was taken as an input. 
		NOTE: In this protocol the subtracted value equals 3.32, as a 10% of total chromatin was taken as an input in this protocol. Log2(10) equals 3.32.</li>
		<li>Calculate % of input by multiplying by 100 the value of exponentiation operation with base 2 and exponent (power) equal to remainder of adjusted input values subtracted from raw Ct values of the sample (Table 3). 
		% of input = 100*2 (adjusted input &#8211; Ct(sample)) 
		NOTE: With this procedure, the relation between specific DNA abundance in the ChIP-ed sample to the total isolated chromatin (input sample) is calculated. Further details on ChIP data analysis can be found in Haring et al.8.</li>
	</ol>
	</li>
</ol>

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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><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%&amp;nbsp;</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 magnetic beads coupled with protein A or protein G</td><td>Life Technologies</td><td>10003D/10001D</td><td>Check manufacturer&amp;rsquo;s manual for antibody affinity</td></tr><tr><td>Miracloth</td><td>Merck Millipore</td><td>475855</td><td></td></tr><tr><td>Triton X-100 Surfact-Amps Detergent Solution</td><td>Life Technologies</td><td>85112</td><td></td></tr><tr><td>(mouse, rat, rabbit&amp;hellip;)-IgG</td><td>Diagenode</td><td>C15400001, C15420001, C15410206</td><td></td></tr><tr><td>Magnetic rack - DynaMag-2</td><td>Life Technologies</td><td>12321D</td><td></td></tr><tr><td>H3K9/14ac polyclonal antibody - Premium&amp;nbsp;</td><td>Diagenode</td><td>C15410200-10</td><td></td></tr><tr><td>Chelex 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 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...

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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 ...

chromatin-immunoprecipitation-assay-for-identification-arabidopsis

Research

JoVE Journal

Biology

20.3K Views.  Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)-Universidad Politécnica de Madrid. 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.

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

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Biochemistry

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...

Immunology and Infection

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...

Chromatin Immunoprecipitation Assay: A Technique to Study Protein-DNA Interactions in Cells

Source: Fuchs, A. R.,et al. <a href="https://www.jove.com/t/58270">A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia.</a> J. Vis. Exp. (2018).
This video describes protocol for chromatin immunoprecipitation (ChIP) assay, which is a classic technique to demonstrate protein-DNA interactions.This technique is used to identify target genes in human chronic lymphocytic leukemia cells.

Source: Fuchs, A. R.,et al. A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia. J. Vis. Exp. ...

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

Summary

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Chapters in this video

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

mRNA Interactome Capture from Plant Protoplasts

Detection of Histone Modifications in Plant Leaves

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Identification of Post-translational Modifications of Plant Protein Complexes

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

Chromatin Immunoprecipitation Assay: A Technique to Study Protein-DNA Interactions in Cells