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. Die Vernetzung des Pflanzenmaterials (1 h) <ol><li> Wachsen die Arabidopsis-Linien in dem Experiment verwendet (Wildtyp - WT - gegen Mutanten und / oder Linien der markierten Version des Proteins von Interesse im Vergleich zu Linien das Tag zum Ausdruck bringen zum Ausdruck zu keinem Protein fusioniert) für 12-18 Tage auf großen Petrischalen (150 mm) mit MS-Agarmedium (1 L: 1x Murashige &amp; Skoog-Salze, 10 g Sucrose, 0,5 g MES, pH 5,7 (KOH), 1% Agar). Alternativ wachsen Pflanzen auf Töpfe mit 3: 1 Mischung aus...

<ol>
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Die hier beschriebene ChIP-Protokoll ist eine reproduzierbare und leistungsfähige Technik, um Wechselwirkungen zwischen Proteinen und spezifischen DNA-Sequenzen in vivo in Arabidopsis-Pflanzen zu analysieren. Eine erfolgreiche Identifizierung von Bindungsstellen für die Proteine ​​von Interesse erfordert eine angemessene Auswahl der Pflanzenorgane oder Entwicklungsstadien, wo die relevanten Wechselwirkungen tatsächlich stattfindet. Darüber hinaus ist es wichtig, eine geeignete Fixierung des Pflanzenmate...

In den letzten Jahren eine Vielzahl von genetischen, molekularen und genomischen Werkzeuge wurden in der Modellpflanze Arabidopsis thaliana entwickelt. Diese Technologie hat sich enorm die Fortschritte im Verständnis, wie die Pflanzenentwicklung reguliert wird erleichtert. Unter den Entwicklungsprozesse untersucht unter Verwendung von Arabidopsis als Modell hat sich die genetische Kontrolle der Blütezeit wurde ausgiebig untersucht. Diese Studien haben gezeigt, dass Pflanzen modulieren, sehr genau den Zeitpunkt der Blüte als Reaktion auf endogene Signale wie Hormone und Alter der Anlage, aber auch auf Umweltsignale, wie beispielsweise Fotoperiode und der Temperatur, die der Blüte synchronisieren Zeit mit den natürlichen Kreislauf der Jahreszeiten 1, 2. Die Isolierung und Charakterisierung von Arabidopsis-Mutanten mit Veränderungen in der Zeit der Blüte wurde Determinante Enthüllung eines komplexen Netzwerks von Genen, die die Blütezeit als Reaktion auf endogene und Umweltfaktoren zu regulieren. Diese genetischen Schaltkreiseauf der Ebene der wenigen Master-Gene, die als Schalter der Blüte handeln integriert, und der genaue Zeitpunkt der Initiierung Blumen hängt von der Balance der Blüte zu fördern und zu verdrängen Aktivitäten, die stromaufwärts von den Blumen Integrator-Gene 1,3 zu arbeiten. Die funktionelle Charakterisierung von Genen, die für ihre Rolle bei der Kontrolle der Blüte Einleitung identifiziert, von der jüngsten Nutzung genomischer Ansätze unterstützt, haben die zentrale Rolle der Transkriptionsregulation bei der Modulation der Blütezeit ergab. In der Tat, viele der Mastergene der Blüte kodieren Transkriptionsfaktoren 4. Darüber hinaus ist eine Anzahl von Chromatin-Remodeling-Protein-Komplexe beeinflussen die Expression der Mastergene der Blüte. Eine Anzahl der Arabidopsis-Mutanten auf ihre veränderten Blühzeit isoliert erwies sich Mutationen in Genen eine Vielzahl von Chromatin Modifikatoren kodiert tragen. Verschiedene Chromatin remodelers die posttranslationale Modifikationen in Histon einzuführene Schwänze, neu zu positionieren Nukleosomen relativ zur DNA oder kanonischen Histone austauschen durch Histon-Varianten sind für die ordnungsgemäße Regulierung der Blüte in Arabidopsis 5,6. Einige dieser Chromatin-Remodeling-Aktivitäten katalysieren die Ablagerung oder Entfernung von kovalenten Modifikationen wie Acetylierung oder Methylierung spezifischer Histon-Resten. Diese Histonmarkierungen werden spezifisch durch spezialisierte Effektoren, die andere Chromatin-Remodeling-Komplexe, Transkriptionsfaktoren oder Komponenten der Transkriptionsmaschinerie zu rekrutieren, um die Transkriptionsaktivität der Blüte Gene regulieren erfasst. Chromatin-Immunopräzipitation (ChIP) ermöglicht die Identifizierung von in vivo-DNA-Bindungsstellen für Proteine ​​von Interesse (Abbildung 1). Dieses Verfahren nutzt die Fähigkeit von bestimmten Chemikalien zu vernetzen, die Proteine ​​mit der DNA. Die resultierenden DNA-Protein-Komplexe können dann unter Verwendung von spezifischen Antikörpern immunpräzipitiert werden against Transkriptionsfaktoren, Chromatin-bindende Proteine ​​oder bestimmte Modifikationen und heterologe Epitope zu dem Protein der Wahl angebracht (im allgemeinen als &quot;Tags&quot; genannt). Die aus diesen Immunpräzipitaten gereinigte DNA kann als Matrize in quantitative PCR (qPCR) Reaktionen verwendet werden, um für die Anreicherung bestimmter Sequenzen von Interesse zu bewerten. Auf diese Weise können die Bindungsstellen von Transkriptionsfaktoren oder die Verteilung Histonmarkierungen in bestimmten Genen 7,8 hergestellt werden. Darüber hinaus kombiniert mit Next Generation Sequencing (NGS), die massiv parallele Sequenzierung ermöglicht, Chip-Technologie ist möglich, die genomweite Identifizierung der Bindungsstellen von Transkriptionsfaktoren sowie Enthüllung epigenomischen Landschaften von Histon-Modifikationen vorgenommen. Ferner ist die gleichzeitige Analyse der Genexpression ermöglicht die Überwachung, wie die Bindung von Transkriptionsregulatoren oder die Abscheidung insbesondere Histonmarkierungen korrelieren mit der Transkriptions activity von Genen 9. Die Verwendung von Chip-Protokolle in Arabidopsis hat es die Bewertung der Auswirkungen, dass eine Vielzahl von Transkriptionsregulatoren haben auf der Chromatin-Organisation des Mastergene der Blüte und wie diese strukturellen Veränderungen beeinflussen die Genexpression 5,6. Vorherige Ergebnisse zeigten, dass die Arabidopsis-Locus EARLY BOLTING im Kurztag (EBS) als Repressor der Blüte und Mutanten in diesem Gen zeigen eine Beschleunigung der Blüte und Hochregulation von dem Master-Gen der Blüte FT wirkt. Darüber hinaus loss-of-function Mutationen im FT vollständig zu unterdrücken, die früh blühende Phänotyp der ebs mutierten Pflanzen, die anzeigt, dass FT wird für die vorzeitige Blüte der ebs Mutanten und dass EBS ist für die Unterdrückung von diesem Meister-Gen der Blüte 10 erforderlich erforderlich , 11. EBS kodiert ein PHD-haltigen Protein, das spezifisch binden können Histon H3 Di- und in th trimethyliertese Lysin 4 Rückstand (H3K4me2 / 3), was auf eine Rolle für die EBS in der Chromatin-vermittelten Repression der FT 12. Die Verwendung des Chips Ansatz gezeigt, dass die Arabidopsis PHD-enthaltendes Protein EBS 10,11 bindet regulatorischen Regionen des Blumen Integrator Gen FT seinen Ausdruck 12 zu unterdrücken. Durch die Verwendung von Chip-Technologie erhalten zusätzliche Daten zeigen, dass dieses Protein ist erforderlich, um niedrige Histonacetylierung, ein Markenzeichen der aktiven Transkription im Chromatin dieser Mastergens der Blüte während Anfangsstadien von Arabidopsis Entwicklung. Diese Beobachtungen zusammen mit genetischen und Genexpressionsdaten, zeigen, dass diese Arabidopsis PHD haltige Protein hat eine zentrale Rolle bei der Feinabstimmung der Blütezeit durch Modulation der Expression des Blumen Integrator Gen FT 12. Die vorliegende Arbeit stellt ein optimiertes Verfahren nützlich, nicht nur für die Analyse von Histonen sondern auch für andereChromatin-assoziierten Proteinen, und mit mehr Effizienz und weniger Versuchszeit. Darüber hinaus zeigt dieser Bericht, wie die Verwendung von Chip-Protokolle hat neue Erkenntnisse über den Zusammenhang zwischen Veränderungen der Chromatinstruktur Modifikationen und Transkriptions Staaten von Pflanzengenen diese Chromatin-vermittelten Mechanismen der Gen-Expressionskontroll Auswirkungen auf den Beginn der Blüte in Arabidopsis vorgesehen ist, und wie.

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			<td>M8250</td>
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			<td>MS (Murashige and Skoog Basal Salt Mixture)</td>
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			<td>Formaldehyde 37%&#160;</td>
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			<td>Use under the fume hood</td>
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			<td>Protease inhibitor mix cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack</td>
			<td>Roche</td>
			<td>5892791001</td>
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			<td>B01010002 (UCD200TO)</td>
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			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
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			<td>Dynabeads&#174; magnetic beads coupled with protein A or protein G</td>
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			<td>Merck Millipore</td>
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			<td>Triton&#8482; X-100 Surfact-Amps&#8482; Detergent Solution</td>
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			<td>(mouse, rat, rabbit&#8230;)-IgG</td>
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			<td>Magnetic rack - DynaMag&#8482;-2</td>
			<td>Life Technologies</td>
			<td>12321D</td>
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			<td>H3K9/14ac polyclonal antibody - Premium&#160;</td>
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			<td>Chelex&#174; 100 Resin</td>
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			<td>Proteinase K</td>
			<td>Life Technologies</td>
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			<td>LightCycler&#174; 480 SYBR Green I Master</td>
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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.

Acht Hauptschritte in diesem Chip-Protokoll zur Identifizierung von in vivo-Protein-DNA-Wechselwirkungen, umfassend das Züchten und Ernten von Pflanzenmaterial, Vernetzen der Chromatin Chromatin Isolierung Chromatin Fragmentierung selektive Isolierung der Komplexe zwischen DNA und vereinzelt werden, das interessierende Protein durch Immunfällung, Proteinverdauung, DNA-Reinigung und qPCR-Analyse (Abbildung 1). Ein entscheidender Schritt in dem Chip-Protokoll ist die Fixierung von DNA-Protein-I...

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

Chromatinimmunpräzipitation ist eine leistungsstarke Technik zur Identifizierung von DNA-Bindungsstellen von Arabidopsis-Proteine ​​in vivo. Dieses Verfahren besteht aus Chromatin Vernetzung und Fragmentierung, Immunopräzipitation mit selektiver Antikörper gegen das Protein von Interesse und qPCR-Analyse der gebundenen DNA. Wir beschreiben eine einfache ChIP Assay für Arabidopsis-Pflanzen optimiert.

Chromatinimmunpräzipitation Assay zur Identifizierung von Arabidopsis-Protein-DNA-Wechselwirkungen<em&gt; In-vivo-</em

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.

Research

JoVE Journal

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