Los autores desean agradecer el laboratorio del Dr. Zhao Keji (NIH / NHLBI) por su ayuda en la provisión de resultados de la secuenciación. También nos gustaría dar las gracias al proyecto del genoma UCSC para el uso de Genoma del navegador para visualizar la secuencia asignada lee. Este trabajo ha sido financiado por el Camino R00HD055052 NIH para el Premio de la Independencia y R01HD065816 de NICHD, la Fundación Lucile Parkard, y la Universidad Johns Hopkins, la puesta en marcha fondos para XC

(El procedimiento completo dura chip aproximadamente dos días. Preparación de las bibliotecas de chip para la secuenciación de alto rendimiento requiere unos 2-3 días). 1. Disecar y preparar el tejido para el Experimento chip (aproximadamente 1 millón de células) <ol><li> Disecar el tejido de interés (por ejemplo, 200 pares de Drosophila testículos) en PBS frío inhibidor de la proteasa + 1x (disolver una pastilla de cóctel de inhibidores de la proteasa en 1,5 ml de PBS 1x para obtener la solución stock de 7x) + PMSF (concentración final de 100 mg / mL). Lavar dos veces tejido y resuspender el tejido en 200 l de la misma solución de PBS con inhibidores. </li><li> Para fijar la muestra, añadir 5,5 l de formaldehído al 37% (formaldehído caliente en 37 ° C baño de agua durante 1 minuto antes de usar). Se incuba a 37 ° C durante 15 minutos, vórtice cada 5 minutos entre ellos. </li><li> Colocar la muestra en hielo durante el tejido para asentarse durante 2 minutos. Lavar 2 veces con 450 l de PBS (con inhibidores de la proteasa y PMSF). Ejemplo de ahora puede ser la tiendad a -20 ° C. Repetir varias disección para obtener suficiente material para CHIP. </li></ol> 2. Preparar sobrenadante con conjugación de la proteína-ADN para chip de ensayo <ol><li> Combina la muestra suficiente de paso en un tubo de 1,3 ml 1,75 eppendorf. </li><li> Retire PBS de muestra de tejido. Añadir 200 l de tampón de lisis (50 mM Tris-HCl, pH 7,6, 1 mM de CaCl2, 0,2% de Triton X-100 o NP-40, 5 butirato mM, y 1x cóctel inhibidor de la proteinasa). Añadir una solución fresca de stock PMSF a tampón de lisis (PMSF concentración final de 0,5 mM). Homogeneizar con azul homogeneizador (Cat # Fisher 749.521 a 1590) hasta que los tejidos se disocian por completo sin ningún tipo de agregación, se incuban a temperatura ambiente (temperatura ambiente) durante 10 minutos. </li><li> Sonicación: sonicador uso micropunta (Misonix HS-XL200) a la potencia 20. Soníquelos durante 10 segundos y el resto en hielo durante 50 seg. Repetir 4-5 veces (tiempo de sonicación óptima se debe determinar empíricamente, como ya sonicación producirá fragmentos más pequeños. Para chip utilizando anticuerpos contra Histonmodificaciones e, normalmente sonicar la cromatina a aproximadamente 200-300 pares de bases, porque el factor de transcripción o de un regulador de la cromatina (por ejemplo, las proteínas Polycomb), que normalmente sonicar cromatina 200-1000 pb. Sin embargo, el tamaño de los fragmentos óptima debe ser probada empíricamente. NOTA: Mantenga siempre el tubo en hielo incluso durante sonicando PARA PREVENIR LA MUESTRA DE CALENTANDO! </li><li> Diluir la muestra mediante la adición de 1,8 ml tampón RIPA (10 mM Tris-HCl, pH 7,6, EDTA 1 mM, SDS al 0,1%, 0,1% de Na-desoxicolato, 1% de Triton X-100, con inhibidores de proteasa y PMSF a la misma concentración como se describe anteriormente). Ahorra un 40 l como el control de entrada mediante la adición de 2 l de NaCl 5M y se incuba a 65 ° C durante la noche (O / N) para neutralizar la reticulación. </li><li> Para conjugar anticuerpos a perlas, añadir 40 l Proteína A perlas a un tubo eppendorf de 1,5 ml, a continuación, añadir 600 l de PBS y roca a 4 ° C durante 2 minutos, se aplican a perlas de imán y eliminar el sobrenadante. Añadir 100 l de PBS y el anticuerpo de interés (la cantidad de anticuerpo debe ser determinado Empircamente). Incubar a temperatura ambiente durante 1 hora o 4 ° C durante 4 horas. </li><li> Eliminar sobrenadante de perlas de imán y añadir 1 ml de extracto de cromatina desde el paso de 2,4 a las perlas. Gire a 4 º CO / N. </li><li> Aplique la muestra chip para eliminar el imán y el sobrenadante. Lavar las perlas con el tampón siguiente a 4 ° C durante 10 minutos cada uno: 2 veces con 1 ml de tampón RIPA [1.89 &#39;RIPA buffer&#39; + 315 ml de inhibidores de la proteasa l 7x + 20 l PMSF]; 2x con 1 ml de tampón RIPA + 0,3 M de NaCl [1,89 ml tampón RIPA + 220 l NaCl 3 M]; 2x con 1 ml de tampón de LiCl (0,25 M LiCl, 0,5% de NP40, 0,5% NaDOC); 1x con 1 ml de 1x TE + 0,2% de Triton X-100; 1x con 1 ml de 1x TE. </li><li> Para invertir reticulación, resuspender las perlas en 100 l de tampón TE + 3 l SDS al 10% + 5 l de proteinasa K (20 mg / ml). Se incuba a 65 ° CO / N. </li><li> Aplicar perlas de imán y transferir sobrenadante a un tubo nuevo. El sobrenadante contiene la muestra de ADN. Lavar perlas con 100 l de TE + 0,5 MNaCl y combinar el sobrenadante dos. </li><li> El fenol-cloroformo extraer la muestra de ADN. Añadir 200 l de fenol: Chlorofrom: IAA (25:24:1) y la mezcla de vórtice. Girar a 14k durante 5 minutos a temperatura ambiente. Por otra parte, el ADN puede ser extraído por medio de Qiagen kit de purificación de PCR. </li><li> Se transfiere la capa sobrenadante / acuosa en un nuevo tubo, y añadir acrilamida lineal hasta una concentración final de 20 mcg / ml (Alternativamente 1 l de glucógeno en 20 mg / ml por cada ml de sobrenadante 1 también se puede utilizar. Glucógeno se utiliza para qPCR posterior o los análisis de PCR, mientras que la acrilamida lineal se utiliza para la secuenciación de los ensayos.) Añadir 20 l de 3M NaOAc y 500 l de EtOH 100%. Mezclar bien y se incuba a 80 ° C durante 10 minutos. Girar a la velocidad máxima durante 20 minutos a 4 ° C. </li><li> Remover el sobrenadante y lavar con 300 l de EtOH 70%. Aire seco y volver a suspender la muestra en 50 l de buffer TE. Ejemplo de ahora se puede almacenar a -20 ° C. La muestra puede ser utilizado para PCR cuantitativa (qPCR) de ensayo (Figura 1). </li></ol> 3a. Analizar chip-ed ADN utilizando qPCR <ol><li> Diluir el ADN genómico a la concentración siguiente para hacer una curva estándar: sin diluir, 1/10, 1/100, 1/1000, 1/5000. </li><li> Para cada pareja de cebadores, creado qPCR en dos ejemplares en una placa de 96 pocillos con el ADN genómico de 3a.1 paso a paso, de control de entrada y de chips de ADN-ed: 10 l 2x SYBR mezcla de PCR (Fermentas, K0222); 1 l de 10 mM Cebador; 1 l 10 mM primer inversa; x l chip-ed ADN (control de entrada, o ADN genómico); y l libre de nucleasa H 2 O para ajustar el volumen de reacción a 20 l. </li><li> Girar hacia abajo la placa de 96 pocillos con mini placa de control de giro (Labnet). </li><li> Realizar qPCR con ABI 7300 PCR en tiempo real del sistema (u otros sistemas de PCR en tiempo real) con la siguiente condición: Etapa 1: 50 ° C durante 2 min, 1 ciclo; Etapa 2: 95 ° C durante 10 min, 1 ciclo; Etapa 3: 95 ° C durante 15 s, 60 ° C durante 1 min, 40 cycles; Etapa 4 (fase de disociación): 95 ° C durante 15 s, 60 ° C durante 1 min, 95 ° C durante 15 s. </li><li> Compruebe la curva de disociación: un pico en la gráfica la disociación térmica sugiere una amplificación de la única reacción de PCR. Más de un pico indica no específica del producto con el par de cebadores, los datos qPCR no debe ser utilizado. </li><li> Determinar la fase lineal de amplificación exponencial de reacción de PCR para cada conjunto de cebadores, mediante el cálculo de la fórmula que resuelve cantidad de ADN de acuerdo con el Ct (ciclo umbral) de valor, que se basa en la curva estándar utilizando la serie de ADN genómico diluido en 3a. 1. </li><li> Si el valor de Ct de chip-ed del ADN y control de entrada están dentro del rango lineal del valor Ct, calcular el enriquecimiento de chip-ed del ADN frente a la entrada, de acuerdo con el factor de dilución de chip-ed del ADN y control de entrada en el punto 2.4. </li></ol> 3b. Amplificar chip-ed del ADN para la secuenciación de alto rendimiento. <ol><li> Fin reparación de chip-ed mezcla de ADN (UseEpicentro final del ADN Kit de reparación), creado en el hielo: 1-34 ADN genómico de la etapa l 2,12 (0,1 a 5 mg) 5 l Fin 10x tampón reparación 5 l 2,5 mM dNTP mezcla 5 l de ATP 10 mM x l H 2 O para ajustar el volumen de reacción a 49 l 1 l de fin de reparación enzima mezcla (polimerasa de ADN de T4 polinucleótido quinasa de T4 +) Incubar durante 45 minutos a temperatura ambiente. Purificar la reacción utilizando Qiagen reacción MinElute Limpieza de equipo. Eluir en 30 tampón de elución l (EB). </li><li> Añadir A voladizos de al extremo 3 &#39;: 30 l de producto de ADN eluido de 3b.1 paso 5 Buffer l NEB 10x # 2 10 l 1 mM dATP 2 l DH 2 O 3 l 5 U / l fragmento Klenow (3 &#39;→ 5&#39; exo-) Incubar durante 30 minutos a 37 ° C. Purificar la reacción utilizando MinElute kit ReactionCleanup. Eluir en 10 l EB. </li><li> Solexa enlazador ligadura: 10 l de ADN eluido de 3b.2 paso 10 l ddH2O 2.5 &amp;mu; l 10x tampón T4 ADN ligasa Un adaptador de l oligo mezcla de Illumina (1/10-diluted en stock) 2,5 l ligasa de ADN T4 (400 unidades / l) Incubar durante 1 hora a temperatura ambiente. Purificar la reacción utilizando MinElute kit ReactionCleanup. Eluir en 20 l EB. Ejemplo de ahora se puede almacenar a -20 ° C. </li><li> Ejecutar la muestra de ADN eluido de 3b.3 paso utilizando el aparato de E-gel. Aislar la banda 300-500 pb en el gel y se purifica utilizando el kit Qiagen Gel Extraction (este paso elimina los ~ 125 pb auto-ligó enlazadores de la ligadura de adaptador oligo). Eluir EB en 12 l. </li><li> Amplificar la biblioteca usando dos a dos finales (PE) de Illumina cebadores: 10,5 l de ADN eluido de 3b.4 paso 12,5 l de mezcla maestra (2X Phusion HF, Finnzymes) 1 l de PE1 cebador de PCR 1 1 l de PE2 cebador de PCR 2 PCR condiciones: 98 ° C durante 30 segundos (98 ° C 10 seg, 65 ° C 30 seg, 72 ° C 30 segundos), se repiten 20 ciclos 72 ° C durante 5 minutos. </li><li> Las muestras de 3B.5 paso puede ser utilizado para chip-ss después de seleccionar el ADN de tamaño adecuado (300-500 pb) usando el estándar de 2% en gel de agarosa. Ejemplo de ahora se puede almacenar a -20 ° C. Lo mejor es aislar cada muestra en un gel de chip único para evitar la contaminación. Se pueden enviar muestras para la secuenciación de alto rendimiento. </li></ol> 3c. Solexa análisis de canalización <ol><li> La secuenciación de 25-pb lee se obtuvieron del Analizador de Illumina Genoma (GA) tubería. </li><li> Todas las lecturas fueron alineados para el genoma de Drosophila (dm3) con el eland (alineación local eficiente de los datos de nucleótidos), permitiendo un máximo de dos desajustes con la secuencia de referencia. </li><li> Lecturas asignadas únicamente se conservaron para su análisis posterior. Para múltiples lecturas idénticas, hasta tres copias fueron retenidas para reducir la posibilidad de sesgos de amplificación por PCR. </li><li> La salida de la tubería de GA se convierten en datos extensibles navegador (BED) archivos. </li><li> Para generarlos archivos de la peluca para subir a la UCSC navegador para la visualización, se utilizó un scrip de python que se ha descrito en una publicación anterior 3 de 4 pb como el tamaño de la ventana y de 160 pb como el tamaño de los fragmentos de ADN. </li><li> Para clasificar los genes de Drosophila en los genes silenciosos y expresado, hemos utilizado un número digital llamado RPKM (lee / por kilobase fusionó región exonic / por millón de lecturas asignadas). Los genes con RPKM = 0 se clasifica en el grupo en silencio y con los genes RPKM ≥ 1 se clasificaron en los grupos expresados, los cuales pueden ser clasificados en tres subgrupos, como los genes con bajos niveles de expresión, moderado y alto, y cada grupo tiene aproximadamente el mismo número de los genes. </li><li> Para comparar el nivel de modificación de las histonas y la expresión génica, se utilizó un script en Python que se ha descrito en una publicación anterior 3. </li></ol> 4. Los resultados representativos Ejemplos de resultados qPCR con chip-bam (bolsa de canicas) mutante<html> testículos se muestran en la Figura 1 4. En bam testículos, se produce un fallo en la transición de espermatogonias proliferativa a la diferenciación de espermatocitos 5, 6. Usamos BAM testículos como una fuente de células germinales no diferenciadas, que están enriquecidos en este tipo de tejido. Diferenciación de los genes necesarios para la diferenciación de los espermatozoides, como factor de transcripción específico de 87 hombres (mst87F), Don Juan (dj), y la cebolla difusa (FZO) no se expresan en Bam testículos. Estos genes son altamente enriquecido con la represión H3K27me3 modificación de las histonas 7 (Figura 1A), pero carecen de la participación activa de modificación de las histonas H3K4me3 8 (fig. 1B), una firma de la cromatina que se llama &#39;monovalente&#39; 7. Enriquecimiento de cualquiera de las H3K27me3 o H3K4me3 se determina por la normalización de una ciclina expresada constitutivamente un gen (Cyca) 4. contenido &quot;&gt; chip-ss análisis utilizando el mismo conjunto de anticuerpos (es decir, represiva y activa H3K27me3 H3K4me3) con Bam testículos mutantes (Figura 2) validó los resultados qPCR muestra en la Figura 1. Para los tres genes probados diferenciación terminal mst87F, dj y FZO , sus regiones genómicas son altamente enriquecido con H3K27me3 pero no H3K4me3 (Figura 2A-2C). En cambio, el gen expresado constitutivamente Cyca tiene H3K4me3 importante, pero poco H3K27me3 vinculante cerca de su sitio de inicio de la transcripción (TSS) (Figura 2D). Por otra parte, los perfiles de chip de H3K27me3 y H3K4me3 cerca de las DST de cuatro clases de genes expresados ​​diferencialmente son consistentes con la función de cada modificación de las histonas. Como se muestra en la Figura 3A, el enriquecimiento de H3K27me3 aguas abajo de la DST está inversamente correlacionada con el nivel de expresión génica. Los genes silenciosos tienen la mayor H3K27me3 mientras que elgenes altamente expresados ​​no vinculante H3K27me3. Estos datos son consistentes con el papel represivo de H3K27me3 sobre la expresión génica. En contraste, el enriquecimiento de H3K4me3 alrededor de los DST mostró la correlación opuesta con el nivel de expresión génica (Figura 3B), consistente con el papel activo de H3K4me3 sobre la expresión génica. <img alt="Figura 1" src="/files/ftp_upload/3745/3745fig1.jpg" /> Figura 1. QPCR análisis de chip-ed ADN utilizando anticuerpos contra cualquiera de la represivo H3K27me3 modificación de las histonas (A) o activa H3K4me3 modificación de las histonas (B) en los testículos mutantes enriquecida con células no diferenciadas BAM. (A) En Bam testículos, los genes de diferenciación (Mst87F, dj, FZO) se enriquecen con la modificación de las histonas H3K27me3 represivo. (B) los genes de diferenciación se agotan con H3K4me3 marca activa. El nivel de chips de ADN (DNA chip / entrada) en el gen diana (Mst87F, dj o FZO) se normalizó primero en el lev<html> El chip de ADN en el gen de control de Cyca. Las barras de error indican la desviación estándar de tres réplicas biológicas independientes. <img alt="Figura 2" src="/files/ftp_upload/3745/3745fig2.jpg" /> Figura 2. UCSC genoma navegador instantáneas de H3K27me3 H3K4me3 y enriquecimiento a través de las regiones genómicas completas de (A) Mst87F (B) y dj (C) FZO, (d) los genes Cyca. El círculo H3K27me3 enriquecido región (D) refleja el estado de la cromatina un gen CG7264 superposición, que es humilde se expresa en los testículos bam (RPKM = 1), pero altamente expresado en los testículos de tipo salvaje (RPKM = 130) 8 (chip-ss datos de 7). Las sondas utilizadas en el análisis de PCR cuantitativa de los resultados de chip en la Figura 1 se marcan en la parte inferior de cada parcela. <a href="http://www.jove.com/files/ftp_upload/3745/3745fig2large.jpg" target="_blank">Haga clic aquí para ver más grande la figura</a> . tp_upload/3745/3745fig3.jpg &quot;/&gt; Figura 3. Chip-y siguientes perfiles utilizando H3K27me3 y H3K4me3 en Bam testículos 7. Los cuatro grupos de genes (7.509 genes) fueron clasificados de acuerdo a sus niveles de expresión génica basado en RNA-Seq 8 resultados. Las clases representativas de los genes se trazan para el enriquecimiento de una modificación de las histonas en particular, el uso de secuencias de 3 KB de aguas arriba a aguas abajo 3kb de sus sitios de inicio de transcripción (DST). Esto genera un perfil de enriquecimiento de (A) H3K27me3 (K27) y (B) H3K4me3 (K4) y siguientes chip-análisis en cada grupo. <a href="http://www.jove.com/files/ftp_upload/3745/3745fig3large.jpg" target="_blank">Haga clic aquí para ver más grande la figura</a> .

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No tenemos nada que revelar.

La versatilidad de los análisis de chip discutidos en este protocolo se puede utilizar en diferentes tejidos, lo que proporciona una oportunidad para estudiar el estado de la cromatina en un sistema biológicamente relevantes. Experimentos chip usando células cultivadas procedentes de sistemas son convenientes para llevar a cabo porque gran cantidad de células se pueden obtener fácilmente. Sin embargo, las células cultivadas no reflejan necesariamente las células en un entorno multi-celular. Mediante el desarrollo...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Completa el cóctel de inhibidores de la proteasa del Mini </td><td> Roche </td><td> 11836153001 </td><td></td></tr><tr><td> El formaldehído (37%) </td><td> Supelco </td><td> 47083-U </td><td></td></tr><tr><td> PMSF </td><td> Sigma </td><td> 78830 </td><td></td></tr><tr><td> Kontes pellets maja </td><td> Fischer Scientific </td><td> K749521-1590 </td><td></td></tr><tr><td> PCR kit de purificación de </td><td> Qiagen </td><td> 28104 </td><td></td></tr><tr><td> Poliacrilamida lineal </td><td> Sigma </td><td> 56575-1ML </td><td></td></tr><tr><td> El glucógeno </td><td> Qiagen </td><td> 158930 </td><td></td></tr><tr><td> SYBR verde / ROX qPCR Master Mix </td><td> Fermentas </td><td> K0223 </td><td></td></tr><tr><td> Mini placa de control de giro </td><td> Labnet </td><td> Z723533 </td><td></td></tr><tr><td> PCR en tiempo real del sistema </td><td> Applied Biosystem </td><td> 4351101 </td><td></td></tr><tr><td> Procesador de ultrasonidos pequeño volumen </td><td> Misonix </td><td> HS-XL2000 </td><td> Modelo descontinuado </td></tr><tr><td> Dynabeads, Proteína A </td><td> Invitrogen </td><td> 100-01D </td><td></td></tr><tr><td> Dynamag imán </td><td> Invitrogen </td><td> 123-21D </td><td></td></tr><tr><td> Fenol: Chlorofrom: AIA </td><td> Invitrogen </td><td> 15593-049 </td><td></td></tr><tr><td> Epicentro final del ADN kit de reparación </td><td> Epicentro Biotecnologías </td><td> ER0720 </td><td></td></tr><tr><td> MinElute Reaction equipo de limpieza </td><td> Qiagen </td><td> 28204 </td><td></td></tr><tr><td> Fragmento de Klenow (3 &#39;→ 5&#39; exo-) </td><td> New England Biolabs </td><td> M0212S </td><td></td></tr><tr><td> ADN ligasa de T4 </td><td> Promega Corporation </td><td> M1794 </td><td></td></tr><tr><td> Adaptador de oligonucleótidos </td><td> Illumina </td><td> PE-400-1001 </td><td></td></tr><tr><td> Vinculados Primer Fin de 1,0 y 2,0 </td><td> Illumina </td><td> 1001783 1001 784 </td><td></td></tr><tr><td> E-Gel Electorphoresis sistema </td><td> Invitrogen </td><td> G6512ST </td><td></td></tr><tr><td> 2X Phusion HF Mastermix </td><td> Finnzymes </td><td> F-531 </td><td></td></tr></tbody></table>

chromatin immunoprecipitation (chip) using drosophila tissue

Epigenética sigue siendo un campo de rápido desarrollo que estudia cómo el estado de la cromatina contribuye a la expresión diferencial de genes en distintos tipos de células en diferentes etapas de desarrollo. La regulación epigenética contribuye a un amplio espectro de procesos biológicos, incluyendo la diferenciación celular durante el desarrollo embrionario y la homeostasis en la edad adulta. Una estrategia de crítica en los estudios epigenéticos es examinar cómo diferentes modificaciones de las histonas y los factores de la cromatina regula la expresión génica. Para abordar esta, inmunoprecipitación de cromatina (CHIP) se utiliza ampliamente para obtener una instantánea de la asociación de factores particulares con el ADN en las células de interés. Técnica chip comúnmente utiliza células cultivadas como material de partida, que puede obtenerse en abundancia y homogeneidad para generar datos reproducibles. Sin embargo, hay varias advertencias: En primer lugar, el medio para hacer crecer células en placas de Petri es diferente de la que in vivo, por lo tanto puedeno reflejan el estado de la cromatina endógena de las células en un organismo vivo. En segundo lugar, no todos los tipos de células pueden ser cultivadas ex vivo. Hay sólo un número limitado de líneas celulares, de los cuales la gente puede obtener suficiente material para chip de ensayo. Aquí se describe un método para hacer experimentos chip usando tejidos de Drosophila. El material de partida se disecciona tejido de un animal vivo, con lo que puede reflejar con precisión el estado de la cromatina endógeno. La capacidad de adaptación de este método con muchos tipos diferentes de tejidos permitirá a los investigadores para hacer frente a una gran cantidad biológicamente más preguntas relevantes con respecto a la regulación epigenética en vivo 1 y 2. La combinación de este método con secuenciación de alto rendimiento (chip-ss) también permitirá a los investigadores para obtener un panorama epigenómico.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP using Drosophila tissue at JoVE.com

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recientemente alto rendimiento de la tecnología de secuenciación ha aumentado considerablemente la sensibilidad de la cromatina immunoprecipitation (CHIP) y el experimento se le solicite su aplicación con células purificadas o tejidos disecados. Aquí delinear un método para usar la técnica con el chip<em&gt; Drosophila</em&gt; Tejido, que puede abordar el estado de la cromatina endógena en un sistema bien caracterizado biológica.

Inmunoprecipitación de cromatina (CHIP), utilizando Drosophila Tejido

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

chromatin-immunoprecipitation-chip-using-drosophila-tissue

Research

JoVE Journal

Biology

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

25.0K vistas.  Johns Hopkins University. Recientemente alto rendimiento de la tecnología de secuenciación ha aumentado considerablemente la sensibilidad de la cromatina immunoprecipitation (CHIP) y el experimento se le solicite su aplicación con células purificadas o tejidos disecados. Aquí delinear un método para usar la técnica con el chip Drosophila Tejido, que puede abordar el estado de la cromatina endógena en un sistema bien caracterizado biológica.

Video: Chromatin Immunoprecipitation ChIP using Drosophila tissue

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Los patrones de las células madre embrionarias usando el Flip Chip Bio

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Investigación

Biología

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Un ensayo de la cromatina de tejido cerebral humano

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Inmunoprecipitación de la cromatina de células estaminales embrionarias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

Inmunoprecipitación de ADN metilado

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

Inmunoprecipitación de cromatina (CHIP) para el ensayo dinámico de modificación de las histonas en la expresión génica activado en las células humanas

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Ensayo de inmunoprecipitación de la cromatina de los genes específicos de tejido utilizando etapa temprana de embriones de ratón

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Comparación cuantitativa de Cis-Elemento regulador (CRE) Las actividades de transgénicos Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Immunoprecipitation cromatina eficiente utilizando limitación de las cantidades de biomasa

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

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.

Generación de Plantilla ADN Immunoprecipitation cromatina alta calidad para la secuenciación de alto rendimiento (chip-ss)

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.

El Enfoque ChroP Combina chip y la espectrometría de masas para diseccionar Locus específicos proteómicos Paisajes de la cromatina

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