Este trabalho foi apoiado por um prêmio de estudioso de V (V2014-004) e um estudioso V plus award (D2016-030) da Fundação para pesquisa do câncer de V e um americano câncer sociedade institucional bolsa de investigação (ACS IRG Grant 58-006-53) para o centro da Universidade de Purdue para câncer Pesquisa. E. G. P. foi apoiado pelo prêmio Borch pós doação ao departamento de farmacologia Molecular e Química Medicinal de Universidade de Purdue.

1. preparações <ol><li>preparar 100 mL de solução hipotônica Buffer r.: 0,3 M de sacarose, o 60 mM KCl, 60 mM Tris pH 8.0, 2 mM EDTA e 0,5% NP-40. Loja a 4 ° c. 
	Nota: Algumas linhas celulares, tais como HEK293T, exigem condições menos rigorosas de Lise. Se núcleos lyse facilmente, utilize d. modificado Buffer r: 25 mM HEPES pH 7,6, 25 mM KCl, 5 mM MgCl 2, 0,05 mM EDTA, 0,1% NP-40 e 10% de glicerol.</li>
	<li>Prepare uma solução stock de 250 mL de solução de x mRIPA 2: pH de Tris 100mm 8.0, 2% NP-40 e 0,5% de sódio Deoxycholate do.</li>
	<li>Preparar um 100 mL de solução de NaCl 5 de M.</li>
	<li>Usando o x mRIPA 2 e soluções de NaCl 5m, preparar 50 mL de solução de x mRIPA 1 para cada uma das seis sal concentrações: 0 mM, 100mm, 200mm, 300mm, 400mm e 500 mM de NaCl. Antes de iniciar o experimento, cool todas as soluções na Ice.</li>
	Linhas sob condições desejadas de células <li>crescer. 
	Nota: Ao determinar os efeitos de derrubando uma proteína, SSE para a linha "knockdown" deve ser comparado com sua células. Estes SSE deve ser executadas lado a lado. Para obter exemplos usando inibidores químicos ou estímulos, consulte seções 3-5.</li>
</ol> 2. Extração de sal sequencial básica <ol><li>colheita 8 milhões de células de cada condição e lavar duas vezes com 5 mL de PBS frio gelo. 
	Nota: É fundamental ter igual número de células que possuem concentrações de proteína equivalente. O número exato de células pode precisar de ser otimizado para linhas de célula individual ou proteínas específicas. É importante não ter muitos, como o aumento nas concentrações de proteína impedirá a observação da curva de eluição, que depende de ligação do equilíbrio. No entanto, com muito poucas células não pode haver proteína suficiente para detectar a curva, e a pelota de cromatina é mais difícil de isolar e podem ser perdidos entre fracções.</li>
	<li>Ressuspender as células em 1 mL de tampão A + inibidores da protease, transferir para tubos de centrífuga de micro de 1,5 mL e top giram sobre inferior a 4 ° c durante 10 min.</li>
	<li>Isolar os núcleos por centrifugação a 6000 x g por 5 min a 4 ° c. 
	Nota: Após esta etapa o envelope nuclear ainda deve estar intacto. Se o envelope nuclear está intacto, a pelota re-irá suspender totalmente; no entanto, quando o envelope é lysed e a cromatina é liberada, a pelota não irá re-suspender. Se o envelope nuclear não está intacto após incubação de tampão A, usar modificado A. Buffer</li>
	Inibidores de <li>Adicionar 200 µ l de mRIPA 0 mM NaCl + protease cada pellet de núcleos antes de re-suspender o sedimento. Homogeneizar a amostra cada pipetando 15 vezes com uma pipeta de 1 mL. A pelota deve Ressuspender facilmente, no entanto, os núcleos serão lyse como ele é pipetado e a amostra se tornará espesso e pegajoso e difícil de colocar.
	<ol><li>a fim de elaborar a pelota na ponta, toque final da ponta da pipeta contra o fundo do tubo. A pelota não irá dissolver totalmente uma vez que o DNA é liberado do núcleo, mas pipetagem quebrará cima</li>
	</ol></li> <li>Quando todas as amostras têm sido homogeneizadas, incubar todas as amostras no gelo por 3 min. 
	Nota: O tempo de incubação pode precisar de ser otimizada dependendo da proteína de interesse.</li>
	<li>Pellet de cromatina isolado por centrifugação das amostras por 3 min a 6500 x g a 4 ° c.</li>
	<li>Transferência de sobrenadante para uma centrífuga limpa 1,5 mL tubo. Esta será a fração de 0 mM. Esta solução de mRIPA de 0 mM pode atuar como uma etapa de lavagem para lise dos núcleos e remover qualquer proteínas soltas não associadas com cromatina.</li>
	Inibidores de <li>Adicionar 200 µ l de mRIPA 100 mM NaCl + protease cada pellet de cromatina antes de re-suspender o sedimento. Romper a pelota de cromatina pipetando a pelota e descer 15 vezes. 
	Nota: É fundamental para ser consistente com o número de vezes que a pelota em pipetado.</li>
	<li>Incubar no gelo por 3 min. Esta etapa de incubação permite que todas as amostras alcançar um estado de equilíbrio, que é particularmente importante quando existem múltiplos exemplos.</li>
	<li>Centrifugar 6500 x g por 3 min a 4 ° c e transferência sobrenadante para um tubo limpo 1,5 mL.</li>
	<li>Repetir 2.6-2.10 para as concentrações de sal restantes. 
	Nota: Após 400 mM NaCl, mRIPA a pelota deve ser clara e como e não vai ficar no fundo do tubo. O sedimento pode ser colocado na tampa do tubo de centrífuga enquanto o sobrenadante isolado.</li>
	<li>Adicionar 70 µ l de 4x do carregamento de proteína tingir de cada fração e carregar 30 µ l de cada fração em um gel do acrilamido do SDS para análise ocidental do borrão. 
	Nota: Para determinar o perfil de ligação da proteína, é fundamental para carregar volumes equivalentes das concentrações de proteína igual lisado, ao invés de carregamento.</li>
	<li>Realizar um western blot padrão transferindo para uma membrana e usar os anticorpos primários para proteínas de interesse.</li>
	<li>Para dosar a proteína eluída da cromatina, incubar o borrão em anticorpos secundários de fluorescência infravermelho IRDye e borrão de imagem com uma câmera. Enquanto podem ser utilizados outros métodos de desenvolvimento, nós recomendamos fluorescência ou imagiologia por infravermelhos, como é mais quantitativa na natureza.</li>
	<li>Uso ImageJ ou software similar para calcular a intensidade para a proteína eluída em cada concentração de sal. Pela representação gráfica da intensidade da banda contra a concentração de sal, pode ser determinado o padrão de eluição de sua proteína do interesse.</li>
</ol> 3. Extração de sal sequencial na presença de um &#34; leitor &#34; inibidor <ol><li>colheita dois conjuntos de células (4 milhões) e isolar os núcleos como um padrão SSE.</li>
	<li>Re-suspender ambos os conjuntos em 200 µ l de mRIPA 0 mM NaCl e incubar durante 3 min. Isso permitirá que a remoção de qualquer proteína livre nos núcleos.</li>
	<li>Adicionar 200 µ l mRIPA 0 mM de NaCl para cada pellet. Adicionar o inibidor (2 µ l (+) JQ1 a 1 mm) de uma amostra e DMSO para o conjunto de controle.</li>
	<li>Agitar o sedimento por pipetagem 15 vezes e incubar no gelo por 5 min.</li>
	<li>Centrifugar 6500 x g por 3 min a 4 ° c e transferência sobrenadante para um tubo limpo 1,5 mL.</li>
	<li>Repetir 3.3-4 para todas as concentrações de sal e realizar um western blot padrão.</li>
</ol> 4. Extração de sal sequencial na presença de um &#34; escritor &#34; inibidor <ol><li>tratar as células com o inibidor (10 µM SAHA) ou DMSO por 3 h.</li>
	<li>Colher células 4 milhões para cada tratamento.</li>
	<li>Realizar um SSE padrão para as amostras com o inibidor adicionado para todos os buffers.</li>
</ol> 5. Sequencial sal extração seguinte dano do ADN <ol><li>tratar células com 1 doxorrubicina µM por 1 h.</li>
	<li>Alterar os meios de comunicação sobre as células e lhes permitem recuperar para 3h.</li>
	<li>Colher 8 milhões de células e executar o SSE básico.</li>
</ol> 6. Extração de sal não-sequencial <ol><li>colher 12 milhões de células e lave com PBS.</li>
	<li>Dividir células que existem 2 milhões de células por tubo de centrífuga micro.</li>
	<li>Ressuspender em 500 µ l de tampão A e incubar durante 10 min.</li>
	<li>Isolar os núcleos por centrifugação a 6000 x g..</li>
	<li>Re-suspender aglomerados em um 200 µ l de cada um dos buffers de mRIPA + NaCl.</li>
	<li>Homogeneizar cada amostra pipetando 15 vezes com uma ponta de pipeta de 1 mL.</li>
	<li>Incubar no gelo por 3 min.</li>
	<li>Isolar a cromatina por centrifugação a 6500 x g e transferir o sobrenadante para limpar os tubos. Adicionar 70 µ l de corante a carregar e executar 30 µ l em um gel de SDS-page para análise ocidental do borrão.</li>
</ol>

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</ol>

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Caracterização das interações da proteína e cromatina através da extração de sal é um método comum que tem sido utilizado por décadas de14,15; no entanto, isso não foi sistematicamente otimizado antes de revelar sua utilidade completa. Vamos demonstrar como este método sequencial fornece uma maneira rápida e barata de distinguir entre alterações na ligação de cromatina quando a proteína ou o ambiente é alterado. SSE é altamente adaptável e o...

Regulamento de ADN em células eucarióticas é um sistema complexo e sofisticado que é rigidamente controlado por uma variedade de proteínas que coordenar as respostas aos estímulos intracelulares e extracelulares. DNA é acondicionada em torno do histone octamers para os nucleossomas formulário, que podem ser livremente distribuídos ao longo do DNA ou compactados em bobinas apertado1. Este arranjo estrutural do DNA e histonas é conhecido como cromatina, que é regulada por uma rede de proteínas que ler, escrever e apagar post modificações translacional (PTM) em histonas2. Alguns histona PTMs, tais como a acetilação, mudar a carga de aminoácidos que são depositados, alterando as interações entre histonas e DNA2. Histona PTMs servem também para recrutar reguladores transcricionais, empresas de reestruturação da cromatina, maquinaria de reparo de dano de DNA e maquinaria de replicação do DNA para regiões específicas do genoma3.
A maioria dos métodos para o estudo de interações de cromatina ou sonda interações de pequena escala ou envolvem grandes análises de todo o genoma. Estudos de ligação in vitro , frequentemente, utilizam domínios individuais recombinantes com peptídeos de histona ou DNA, em ensaios, tais como ensaios de turno electroforese mobilidade (EMSA), Calorimetria de Titulação Isotérmica, polarização de fluorescência e peptídeo pulldowns. Porque estes ensaios geralmente se concentram em um domínio de proteínas individuais, eles facilitam a compreensão de uma pequena peça do puzzle, mas não nos permitem compreender a natureza cooperativa de proteínas de vários domínios, muito menos seu papel em várias proteínas complexos. Outra camada de complexidade é adicionada pela composição heterogênea dos mamíferos mais complexos de cromatina-modificando. Esta heterogeneidade de proteína, em combinação com a natureza dinâmica da paisagem da cromatina, torna desafiador para recapitular o na vivo vinculando interações de proteínas da cromatina a cromatina em vitro.
Métodos in vivo têm feito avanços significativos; no entanto, eles são muitas vezes caro, demorado e tecnicamente desafiador. Imunoprecipitação da cromatina, seguida por sequenciamento (ChIP-seq) é muito útil para determinar a localização de proteínas e modificações do histone através do genoma, porém requer substancial otimização4. As proteínas são frequentemente quitosana a cromatina para preservar as interações; no entanto, isto pode produzir interações artificiais e pode causar o epítopo mascaramento5. Além disso, as moleculas (IP) requerem anticorpos altamente específicos e otimização extensiva de DNA cisalhamento e condições IP por ChIP-qPCR usando um sítio de ligação conhecida, que muitas vezes não é disponível a priori. Após a otimização das condições de ChIP, processamento das amostras é dispendioso e requer várias semanas ou meses para sequenciar e analisar. Embora este método é inestimável para identificar a localização da cromatina ligado proteínas através do genoma, o custo e compromisso de torná-lo proibitivo para usar esse método para gerar hipóteses sobre como pequenas mudanças podem afetar Propriedades de associação global .
Neste artigo, descrevemos como um ensaio de extração de sal sequencial (SSE) pode ser usado para examinar os perfis de ligação global de proteínas da cromatina-limite e distinguir como as alterações em uma proteína, complexo, ou global PTM perfil podem alterar interações. Apesar de extração de sal é uma técnica comum e amplamente utilizada, demonstraremos como esse método sequencial é altamente reprodutível e versátil. SSE nos permite caracterizar como uma única subunidade de um complexo ou até mesmo um único domínio contribui para afinidade geral do complexo da cromatina em massa. SSE pode também ser usado para determinar se a ligação de uma proteína é influenciada pelas alterações na paisagem de cromatina, fornecendo hipóteses interessantes para o direcionamento de marca de histona que podem ser confirmados usando ChIP-seq e outros estudos de ampla do genoma.
Adaptamos, originalmente, esse método de Wu et al., para examinar a função de Polybromo1 (PBRM1) na ligação do PBAF cromatina remodeler6,7. Usando esta técnica, determinou o papel de PBRM1 para ligação de cromatina dentro a cromatina PBAF remodelação complexa e determinado a contribuição relativa das seis bromodomains individuais para esta função7.
Aqui descrevemos como otimizar esse método para explorar a ligação da cromatina em diferentes tipos de células, para avaliar a afinidade obrigatória relativa da cromatina semelhante modificando complexos, para examinar o deslocamento de uma proteína de cromatina por um inibidor químico, e para determinar os efeitos da ligação de cromatina após alterações à paisagem da cromatina.

<table><tbody><tr><td>Sodium Chloride, Crystal 2.5 Kg AR (ACS)</td><td>Avantor/Macron</td><td>7581-06</td><td></td></tr><tr><td>Sucrose, Crystal 500 G AR&amp;uml; (ACS)</td><td>Avantor/Macron</td><td>8360-04</td><td></td></tr><tr><td>Potassium Chloride, Granular 500 G AR&amp;uml; (ACS)</td><td>Avantor/Macron</td><td>6858-04</td><td></td></tr><tr><td>Trizma(R) base,Primary Standard and Buffer, &gt;=99.9% (titration), crystalline</td><td>Sigma-Aldrich</td><td>T1503-1kg</td><td></td></tr><tr><td>EDTA (Ethylenedinitrilo) Tetraacetic Acid, Disodium Salt, Dihydrate 125 G AR (ACS)</td><td>Avantor/Macron</td><td>4931-02</td><td></td></tr><tr><td>NONIDET P-40 SUBSTRATE, 100mL UN3082</td><td>Amresco</td><td>E109-100ML</td><td></td></tr><tr><td>HEPES Buffer Solution (1M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Magnesium Chloride, 6-Hydrate, Crystal 500 G AR&amp;uml; (ACS)</td><td>Avantor/Macron</td><td>5958-04</td><td></td></tr><tr><td>GLYCEROL, 1L</td><td>Amresco</td><td>0854-1L</td><td></td></tr><tr><td>DEOXYCHOLIC ACID SODIUM SALT, 100g</td><td>Amresco</td><td>0613-100G</td><td></td></tr><tr><td>Eppendorf Research plus pipette</td><td>Fisher Scientific</td><td>13-690-032</td><td></td></tr><tr><td>Eppendorf 5424 R refrigerated microcentrifuge</td><td>Eppendorf</td><td>5424 R</td><td></td></tr><tr><td>Secondary mouse IgG HRP-linked</td><td>Cell Signaling</td><td>7076</td><td></td></tr><tr><td>Secondary rabbit IgG HRP-linked</td><td>Cell Signaling</td><td>7074</td><td></td></tr><tr><td>BMI-1 antibody</td><td>Millipore</td><td></td><td></td></tr><tr><td>PBRM1 antibody</td><td>Bethyl</td><td>A301-591A</td><td></td></tr><tr><td>ARID1a antibody</td><td>Santa Cruz</td><td>sc-32761</td><td></td></tr><tr><td>BRD4 antibody</td><td>Bethyl</td><td>A301-9852a</td><td></td></tr><tr><td>(+) JQ1</td><td>Cayman Chemical Company</td><td>11187</td><td></td></tr><tr><td>SAHA</td><td>Cayman Chemical Company</td><td>10009929</td><td></td></tr><tr><td>Doxorubicin</td><td>AK Scientific</td><td>25316-40-9</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Macron</td><td>4948-02</td><td></td></tr><tr><td>Mini Trans-Blot Cell</td><td>BioRad</td><td>1703930</td><td></td></tr><tr><td>Mini Gel Tank</td><td>ThermoFisher</td><td>A25977</td><td></td></tr><tr><td>Albumin, Bovine (BSA)</td><td>Amresco</td><td>0332-100g</td><td>Used in as a 5% solution in PBS-T as blocking solution for Western blots</td></tr><tr><td>Bolt MOPS SDS Running Buffer (20x)</td><td>Life Technologies</td><td>B0001</td><td></td></tr><tr><td>Bolt LDS Sample Buffer (4x)</td><td>Life Technologies</td><td>B0007</td><td></td></tr><tr><td>PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa</td><td>ThermoFisher</td><td>26619</td><td></td></tr><tr><td>Methyl Alcohol, Anhydrous</td><td>Macron</td><td>3041-10</td><td></td></tr><tr><td>Trypsin kEDTA, 1X</td><td>Cellgro</td><td>25-053-CI</td><td></td></tr><tr><td>SODIUM AZIDE, 250g UN1687</td><td>Amresco</td><td>0639-250G</td><td></td></tr><tr><td>SODIUM DODECYL SULFATE (SDS)500g UN1325</td><td>Amresco</td><td>0227-500G</td><td></td></tr><tr><td>Glycine,for electrophoresis, &gt;=99%</td><td>Sigma-Aldrich</td><td>G8898-1kg</td><td></td></tr><tr><td>BigTop Microcentrifuge Tubes, Polypropylene</td><td>VWR</td><td>20170-333</td><td></td></tr><tr><td>1000 &amp;micro;L Pipet Tips</td><td>VWR</td><td>83007-382</td><td></td></tr><tr><td>NuPAGE Bis-Tris Precast Gels 4-12%</td><td>Invitrogen</td><td>NW04125BOX</td><td></td></tr><tr><td>Leupeptin Hemisulfate, 5 MG</td><td>RPI Research Products</td><td>22035-0.005</td><td>Used for Protease inhibitor solution.</td></tr><tr><td>APROTININ, 10 MG</td><td>RPI Research Products</td><td>20550-0.01</td><td>Used for Protease inhibitor solution.</td></tr><tr><td>PEPSTATIN A, 5 MG</td><td>RPI Research Products</td><td>30100-0.005</td><td>Used for Protease inhibitor solution.</td></tr><tr><td>OVCA429 cells</td><td>Gift from Karen Cowden Dahl. Ph.D.</td><td></td><td></td></tr><tr><td>HEK293T</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>HeLa cells</td><td>ATCC</td><td>CCL-2</td><td></td></tr><tr><td>McCoy&#039;s 5A media</td><td>Corning Mediatech</td><td>10-050-CV</td><td></td></tr><tr><td>DMEM</td><td>Corning Mediatech</td><td>10-013-CV</td><td></td></tr><tr><td>Minimum Essential Medium (MEM), Powder</td><td>Corning Mediatech</td><td>50-011-PC</td><td></td></tr><tr><td>MEM NEAA (100X) non-essential amino acid</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>FB-11: Fetal Bovine Serum, U.S. Source</td><td>Omega Scientific</td><td>FB-11</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>sodium pyruvate</td><td>Life Technologies</td><td>11360070</td><td></td></tr><tr><td>VWR Power Source</td><td>VWR</td><td>13-690-032</td><td></td></tr></tbody></table>

sequential salt extractions for the analysis of bulk chromatin binding properties of chromatin modifying complexes

Elucidação das propriedades de ligação de proteínas da cromatina-direcionamento pode ser muito desafiador devido à natureza complexa da cromatina e a heterogeneidade dos mamíferos mais complexos de cromatina-modificando. Para superar estes obstáculos, adaptámos um ensaio de extração de sal sequencial (SSE) para avaliar as afinidades de ligação relativo dos complexos ligados a cromatina. Este ensaio fácil e simples pode ser usado por não-especialistas para avaliar a diferença relativa na vinculação afinidade de dois complexos relacionados as mudanças na afinidade de um complexo quando uma subunidade é perdida ou um domínio individuais é inativado e a mudança na afinidade de ligação após alterações à paisagem da cromatina. Suspendendo sequencialmente re-cromatina em massa no aumento da quantidade de sal,... somos capazes de perfil de eluição de uma proteína específica da cromatina. Usando esses perfis, somos capazes de determinar como as alterações em um complexo de cromatina-modificando ou alterações no ambiente de cromatina afetam as interações de ligação. SSE de acoplamento com outros ensaios in vitro e em vivo , podemos determinar os papéis de domínios individuais e proteínas sobre a funcionalidade de um complexo em uma variedade de ambientes de cromatina.

Neste artigo, vamos demonstrar as vantagens e aplicações do método comumente usados extração sequencial de sal (SSE) que nós adaptamos do literatura6. Na Figura 1, comparamos a reprodutibilidade do SSE para extrair proteínas não sequencial, detectando os padrões de eluição de ARID1a e PBRM1. Consistentemente, observamos que ARID1a, uma subunidade BAF, elutes principalmente em 200 mM NaCl e PBRM1, uma subunidade PBAF exclusiv...

Watch this Scientific Journal Video about Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes at JoVE.com

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Extração de sal sequencial das proteínas da cromatina vinculada é uma ferramenta útil para determinar as propriedades de vinculação de grandes complexos de proteínas. Esse método pode ser empregado para avaliar o papel das subunidades individuais ou domínios na afinidade total de uma proteína complexa em massa de cromatina.

Extrações de sal sequenciais para a análise da cromatina em massa vincular propriedades de cromatina modificando complexos

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

sequential-salt-extractions-for-analysis-bulk-chromatin-binding

Research

JoVE Journal

Genetics

8.3K visualizações.  Purdue University. Extração de sal sequencial das proteínas da cromatina vinculada é uma ferramenta útil para determinar as propriedades de vinculação de grandes complexos de proteínas. Esse método pode ser empregado para avaliar o papel das subunidades individuais ou domínios na afinidade total de uma proteína complexa em massa de cromatina.

Vídeo: Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Análise em tempo real do fator de transcrição, ligação, transcrição, tradução e volume de negócios para exibir eventos globais durante a ativação celular

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Genética

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

Análise quantitativa de proteomas cromatina na Doença

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Medicina

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

Ensaios para validação de inibidores de acetiltransferase histone

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

Pesquisa do câncer

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

Cromatina Análise de Interação com Emparelhados Fim-Tag Sequencing (Chia-PET) para mapeamento Interações cromatina e Compreensão regulação da transcrição

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Biologia

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Geração de alta Chromatin Template DNA Immunoprecipitation Qualidade para seqüenciamento de alto rendimento (CHIP-seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

A Abordagem ChroP Combina chip e Espectrometria de Massa dissecar proteômica Paisagens Locus específicas da cromatina

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

Identificação de MyoD interactome Usando purificação de afinidade em tandem Acoplado a Espectrometria de Massa

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5&#39; DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

O Método ChIP-exo: Identificando Protein-DNA Interações com perto da base Pair Precision

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA ...

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single strands, each requiring distinct modes of replication: continuous (leading) and discontinuous (lagging). Understanding the interactions of chromatin-associated proteins with replicating DNA strands in vivo is crucial for elucidating the mechanisms of chromatin assembly and DNA repair coupled to DNA replication. This protocol presents the enrichment and sequencing of protein-associated nascent DNA (eSPAN) method, designed to quantify relative protein levels on nascent leading and lagging DNA strands at replication forks. The eSPAN procedure starts with chromatin immunoprecipitation (ChIP, in yeast) or cleavage under targets and tagmentation (CUT&amp;Tag; in mammalian cells) of a protein of interest, followed by enrichment of the protein-associated nascent DNA by bromodeoxyuridine (BrdU) immunoprecipitation. Strand-specific next-generation sequencing is applied to isolated ssDNA. This technique can be used to determine whether a protein is enriched at leading or lagging replication forks. The eSPAN provides genome-wide strand-preference of chromatin-associated proteins, including histones at replication forks.

Enrichment and sequencing of protein-associated nascent DNA (eSPAN) was developed to detect the relative abundance of a chromatin-associated protein on two replicating DNA strands, thereby revealing molecular insight into chromatin replication and its coupled processes. This protocol describes eSPAN procedures in yeast and mouse embryonic stem (ES) cells.

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single ...

Chromatin Immunoprecipitation

Histones are proteins that help organize DNA in eukaryotic nuclei by serving as &#8220;scaffolds&#8221; around which DNA can be wrapped, forming a complex called &#8220;chromatin&#8221;. These proteins can be modified through the addition of chemical groups, and these changes affect gene expression. Researchers use a technique called chromatin immunoprecipitation (ChIP) to better understand which DNA regions associate with specific histone modifications or other gene regulatory proteins. Antibodies are used to isolate the protein of interest, and the bound DNA is extracted for analysis. Here, JoVE presents the principles behind ChIP, discussing specific histone modifications and their relationship to gene expression and DNA organization. We then review how to perform a ChIP protocol, and explore the ways scientists are currently using this technique.

Imunoprecipitação de Cromatina

Histones are proteins that help organize DNA in eukaryotic nuclei by serving as &#8220;scaffolds&#8221; around which DNA can be wrapped, forming a complex ...