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 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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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 ...

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