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

A imunoprecipitação da cromatina, abreviada como ChIP, é uma técnica para estudar as interações proteína-DNA que regulam a expressão gênica.
Nos eucariotos, o DNA é enrolado em proteínas histonas e forma um complexo conhecido como nucleossomo, que se agrupa em uma estrutura conhecida como cromatina para ajudar no empacotamento apertado do DNA nas células.
A expressão gênica é regulada por meio de modificações de histonas que desenrolam ou apertam essas estruturas, bem como proteínas que se associam a regiões cis-regulatórias no DNA.
No ChIP, a cromatina é quebrada e os anticorpos que se ligam a modificações de histonas ou proteínas reguladoras são usados para isolar as moléculas-alvo com o DNA associado.
O primeiro passo nesse processo é reticular a proteína ao DNA com a ajuda de um agente de reticulação, como o formaldeído. Isso imobiliza a proteína no DNA, marcando o local de ligação da proteína. Após a reticulação, a cromatina é mecanicamente cortada em fragmentos curtos que variam de 100 a 200 pares de bases. Esse processo é conhecido como X-ChIP.
Um método alternativo é o N-ChIP, no qual as nucleases digerem diretamente o DNA da cromatina em fragmentos curtos, sem qualquer reticulação prévia.
A imunoprecipitação é realizada pela introdução de anticorpos que têm como alvo as proteínas reguladoras na solução que contém DNA cisalhado ou digerido. Esses anticorpos estão ligados a agentes que ajudam no isolamento seletivo.
Um método comum é ligar os anticorpos a esferas magnéticas. Um ímã é usado para isolar os anticorpos junto com quaisquer moléculas ligadas.
O complexo é enxaguado para lavar quaisquer contaminantes fracamente associados. As moléculas-alvo são destacadas com a ajuda de um detergente, como o SDS.
No caso do X-ChiP, a reticulação é revertida com a ajuda de temperaturas elevadas, e os reguladores ou histonas associados são degradados com a ajuda de proteases, deixando para trás o DNA. 
O sequenciamento do DNA associado pode ajudar a identificar a sequência cis-regulatória à qual a proteína de interesse estava ligada ou o gene cuja expressão é modulada por uma modificação específica da histona.

<ol>
	<li>Gade, P., & Kalvakolanu, D. V. (2012). Ensaio de imunoprecipitação da cromatina como ferramenta para analisar a atividade do fator de transcrição. Métodos em biologia molecular (Clifton, N.J.), 809, 85–104. https://doi.org/10.1007/978-1-61779-376-9_6</li>
	<li>Nelson, J. D., Denisenko, O., & Bomsztyk, K. (2006). Protocolo para o método de imunoprecipitação rápida da cromatina (ChIP). Protocolos da Natureza, 1(1), 179–185. DOI:10.1038/nprot.2006.27</li>
	<li>Das, P. M., Ramachandran, K., VanWert, J., & Singal, R. (2004). Ensaio de imunoprecipitação da cromatina. Biotécnicas, 37(6), 961-969.</li>
</ol>

imunoprecipitação de cromatina - chip

A imunoprecipitação da cromatina, ou ChIP, é uma técnica baseada em anticorpos usada para identificar locais no DNA que se ligam a fatores de transcrição de interesse ou proteínas histonas. Também ajuda a determinar o tipo de modificações das histonas, como acetilação, fosforilação ou metilação. <h4>Tipos de Chip</h4> O ChIP pode ser dividido em dois tipos - X-ChIP e N-ChIP. O X-ChIP envolve a ligação cruzada in vivo de histonas e proteínas reguladoras ao DNA, fragmentando o DNA por sonicação e isolando os complexos proteína-DNA por imunoprecipitação deles com anticorpos específicos. Por outro lado, o N-ChIP não envolve reticulação de DNA a proteínas e a digestão é realizada por meio de nucleases. <h4>Análise downstream de interações DNA-proteína</h4> Após isolar o DNA de interesse, técnicas como PCR, microarrays ou Southern Blot podem ser utilizadas para análise. Alternativamente, este DNA pode ser usado para sequenciamento profundo, conhecido como ChIP-Seq. O ChIP pode ser modificado usando outras metodologias para diferentes análises, como ChIA-PET, uma técnica que combina os princípios do ChIP com captura de conformação cromossômica para detectar interações de cromatina de longo alcance mediadas por uma proteína de interesse; enChIP, uma técnica que emprega o sistema CRISPR/Cas9 para atingir regiões genômicas específicas e RIP-ChIP/RIP-Seq, uma modificação do ChIP usada para analisar interações proteína-RNA. <h4>Comparação entre N-ChIP e X-ChIP</h4> N-ChIP e X-ChIP têm suas próprias vantagens e desvantagens. N-ChIP resulta em ligação mais forte de anticorpos e imunoprecipitação eficiente e altamente específica. No entanto, o N-ChIP é adequado apenas no caso de proteínas fortemente ligadas, como as histonas, pois os fatores de transcrição podem se separar durante o processamento. Além disso, nem toda a cromatina digerida com nuclease é solubilizada, resultando na perda de certas frações da amostra. X-ChIP é uma excelente metodologia para estudar fatores de transcrição que não estão fortemente ligados ao DNA devido à sua etapa de reticulação. O ensaio X-ChIP também é mais sensível que o N-ChIP e requer menores quantidades de amostras e também de anticorpos. As desvantagens do chip X incluem possível dificuldade na fragmentação devido ao excesso de reticulação e falsos positivos devido à reticulação de interações transitórias entre proteínas de DNA.

Imunoprecipitação de cromatina - ChIP

A imunoprecipitação da cromatina, ou ChIP, é uma técnica baseada em anticorpos usada para identificar locais no DNA que se ligam a fatores de transcrição de interesse ou ...

Education

JoVE Core

Molecular Biology

Biologia Molecular

Unit 2

Unidade 2

Analyzing Gene Expression and Function

Analisando a Expressão e a Função Gênica

KEY TERMS AND CONCEPTS

TERMOS-CHAVE E CONCEITOS

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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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...

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transgenic plants

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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

molecular biology

JoVE Core Biologia Molecular mostra os processos moleculares da célula através de vídeos de animação claros e concisos. Além disso, nos vídeos de Cientistas em ação, a aplicação desses conceitos é demonstrada por meio de experimentos reais em laboratórios modernos.

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BIO1.ETS2.3

ChIP Methods for DNA-Protein Binding

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