Name: chip methods for dna-protein binding
Uploaded: 2021-04-07T12:12:00.000Z
Duration: 2 min 36 s
Channel: JoVE

K12 Standards

Tennessee Academic Standards

High School

Biology

links-among-engineering-technology-science-and-society

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

Chromatin Immunoprecipitation- ChIP

L’immunoprécipitation de la chromatine, abrégée en ChIP, est une technique permettant d’étudier les interactions protéine-ADN qui régulent l’expression des gènes.
Chez les eucaryotes, l’ADN est enroulé autour des protéines d’histones et forme un complexe connu sous le nom de nucléosome, qui se regroupe ensuite dans une structure connue sous le nom de chromatine pour aider à l’emballage serré de l’ADN dans les cellules.
L’expression des gènes est régulée par des modifications d’histones qui déroulent ou resserrent ces structures ainsi que des protéines qui s’associent à des régions cis-régulatrices de l’ADN.
Dans la ChIP, la chromatine est décomposée et les anticorps qui se lient aux modifications des histones ou aux protéines régulatrices sont utilisés pour isoler les molécules cibles avec l’ADN associé.
La première étape de ce processus consiste à réticuler la protéine à l’ADN à l’aide d’un agent de réticulation, tel que le formaldéhyde, qui immobilise la protéine sur l’ADN en marquant le site de liaison de la protéine. Après réticulation, la chromatine est cisaillée mécaniquement en fragments courts allant de 100 à 200 paires de bases. Ce processus est connu sous le nom de X-ChIP.
Une autre méthode est la N-ChIP, dans laquelle les nucléases digèrent directement l’ADN de la chromatine en fragments courts, sans aucune réticulation préalable.
L’immunoprécipitation est réalisée par l’introduction d’anticorps qui ciblent les protéines régulatrices dans la solution contenant de l’ADN cisaillé ou digéré. Ces anticorps sont liés à des agents qui aident à l’isolement sélectif.
Une méthode courante consiste à lier les anticorps à des billes magnétiques. Un aimant est utilisé pour isoler les anticorps ainsi que les molécules liées.
Le complexe est rincé pour éliminer tous les contaminants vaguement associés. Les molécules cibles sont détachées à l’aide d’un détergent, tel que le SDS.
Dans le cas du X-ChiP, la réticulation est inversée à l’aide de températures élevées, et les régulateurs ou histones associés sont dégradés à l’aide de protéases, laissant derrière eux l’ADN. 
Le séquençage de l’ADN associé peut aider à identifier la séquence cis-régulatrice à laquelle la protéine d’intérêt était liée ou le gène dont l’expression est modulée par une modification particulière des histones.

<ol>
	<li>Gade, P., et Kalvakolanu, D. V. (2012). Le test d’immunoprécipitation de la chromatine comme outil d’analyse de l’activité des facteurs de transcription. Méthodes en biologie moléculaire (Clifton, N.J.), 809, 85-104. https://doi.org/10.1007/978-1-61779-376-9_6</li>
	<li>Nelson, J. D., Denisenko, O. et Bomsztyk, K. (2006). Protocole pour la méthode d’immunoprécipitation rapide de la chromatine (ChIP). Protocoles de la nature, 1(1), 179-185. doi :10.1038/nprot.2006.27</li>
	<li>Das, P. M., Ramachandran, K., VanWert, J. et Singal, R. (2004). Test d’immunoprécipitation de la chromatine. Biotechniques, 37(6), 961-969.</li>
</ol>

immunoprécipitation de la chromatine - chip

L'immunoprécipitation de la chromatine, ou ChIP, est une technique basée sur les anticorps utilisée pour identifier les sites de l'ADN qui se lient aux facteurs de transcription d'intérêt ou aux protéines histones. Cela aide également à déterminer le type de modifications des histones telles que l’acétylation, la phosphorylation ou la méthylation.

Types de puces

La puce peut être divisée en deux types : X-ChIP et N-ChIP. X-ChIP implique la réticulation in vivo des histones et des protéines régulatrices à l'ADN, la fragmentation de l'ADN par sonication et l'isolement des complexes protéine-ADN en les immunoprécipitant avec des anticorps spécifiques. D’autre part, N-ChIP n’implique pas de réticulation de l’ADN aux protéines et la digestion est réalisée à l’aide de nucléases.

Analyse en aval des interactions ADN - protéines

Après avoir isolé l’ADN d’intérêt, des techniques telles que la PCR, les puces à ADN ou le Southern blot peuvent être utilisées pour l’analyse. Alternativement, cet ADN peut être utilisé pour un séquençage profond, connu sous le nom de ChIP-Seq. La puce peut être modifiée à l'aide d'autres méthodologies pour différentes analyses telles que ChIA-PET, une technique qui combine les principes de la puce avec la capture de conformation chromosomique pour détecter les interactions chromatiniennes à longue portée médiées via une protéine d'intérêt ; enChIP, une technique qui utilise le système CRISPR/Cas9 pour cibler des régions génomiques spécifiques et RIP-ChIP/RIP-Seq, une modification de ChIP utilisée pour analyser les interactions protéine-ARN.

Comparaison entre N-ChIP et X-ChIP

N-ChIP et X-ChIP ont leurs propres avantages et inconvénients. N-ChIP entraîne une liaison plus forte aux anticorps et une immunoprécipitation efficace et hautement spécifique. Cependant, N-ChIP ne convient que dans le cas de protéines étroitement liées telles que les histones, car les facteurs de transcription peuvent se détacher au cours du traitement. De plus, toute la chromatine digérée par les nucléases n’est pas solubilisée, ce qui entraîne l’absence de certaines fractions de l’échantillon. X-ChIP est une excellente méthodologie pour étudier les facteurs de transcription qui ne sont pas étroitement liés à l'ADN en raison de son étape de réticulation. Le test X-ChIP est également plus sensible que le N-ChIP et nécessite moins d’échantillons ainsi que d’anticorps. Les inconvénients de la puce X incluent une difficulté possible de fragmentation en raison d'une réticulation excessive et de faux positifs dus à la réticulation des interactions transitoires entre les protéines de l'ADN.

Immunoprécipitation de la chromatine - ChIP

L’immunoprécipitation de la chromatine, ou ChIP, est une technique à base d’anticorps utilisée pour identifier les sites de l’ADN qui se lient à des facteurs de transcription d’intérêt ou ...

Education

Enseignement

JoVE Core

Molecular Biology

Biologie moléculaire

Unit 2

Unité 2

Analyzing Gene Expression and Function

Analyse de l'expression et de la fonction géniques

KEY TERMS AND CONCEPTS

TERMES ET CONCEPTS CLÉS

chip methods for dna-protein binding

Chromatin immunoprecipitation, or ChIP, is an antibody-based method for finding where proteins bind to DNA. It is used to identify DNA sites bound by transcription factors or histone proteins. ChIP also helps show which histone changes are present, including acetylation, phosphorylation, and methylation.

ChIP is commonly divided into two forms: X-ChIP and N-ChIP. X-ChIP first cross-links proteins to DNA inside the cell, then breaks the DNA into pieces by sonication. The protein-DNA complexes are then isolated by immunoprecipitation with specific antibodies. N-ChIP does not use cross-linking. Instead, it uses nucleases to digest the chromatin.

After the DNA of interest is isolated, it can be analyzed in several ways. PCR, microarrays, and Southern blot can be used to study the captured DNA. The sample can also be sent for deep sequencing, which is called ChIP-Seq.

ChIP can also be adapted for other kinds of analysis. ChIA-PET combines ChIP with chromosome conformation capture to detect long-range chromatin interactions that are mediated by a protein of interest. enChIP uses the CRISPR/Cas9 system to target specific genomic regions. RIP-ChIP and RIP-Seq are used to study protein-RNA interactions.

N-ChIP and X-ChIP each have strengths and limits. N-ChIP gives stronger antibody binding and efficient, highly specific immunoprecipitation. It works best with tightly bound proteins such as histones, but transcription factors may be lost during processing. Some nuclease-digested chromatin may also remain insoluble, so parts of the sample can be missed.

X-ChIP is useful for studying transcription factors that do not bind tightly to DNA because it includes a cross-linking step. It is also more sensitive than N-ChIP and uses less sample and fewer antibodies. Its drawbacks include possible problems with fragmentation if cross-linking is too strong and false positives from transient DNA-protein interactions.

ChIP Methods for DNA-Protein Binding

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key terms and concepts

Explore how ChIP detects DNA-protein binding, compares X-ChIP and N-ChIP, and shows how captured DNA can be analyzed by PCR, sequencing, and more.

ChIP Methods for DNA-Protein Binding | JoVE K12

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BIO1.ETS2.3

ChIP Methods for DNA-Protein Binding

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