Producción heteróloga de la histona variante macroH2A2 y evaluación de su función epigenética durante el desarrollo embrionario del pez cebra
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Fecha
2014
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
El grado de compactación de la cromatina es determinante e influyente en procesos
fundamentales para la célula como lo son la transcripción génica, la replicación del ADN y
la reparación de éste. Consecuentemente, la modulación de la conformación de la cromatina
es fundamental en la expresión génica La unidad básica de la cromatina es el nucleosoma,
que consiste en 147 pb de ADN sobreenrollados alrededor de un octámero de proteínas
básicas denominadas histonas. Una alteración de la composición del nucleosoma consiste
en incorporar variantes de histonas en determinadas regiones, sustituyendo a las histonas
canónicas. Este reemplazo resulta en un cambio en la arquitectura de la cromatina y por
consiguiente un cambio en la expresión génica, lo cual determina la especialización celular,
la diferenciación de tejidos y la herencia epigenética.
Nuestra atención se centró específicamente en la variante de histona mH2A2, la cual
ha sido propuesta que puede interactuar con componentes de complejos remodeladores de
la cromatina tales como las proteínas del Polycomb-group (PcG). Las proteína
componentes del PcG , han demostrado jugar un papel crucial en la regulación epigenética
de la expresión génica durante las etapas del desarrollo embrionario en diferentes especies.
Teniendo esto en consideración, evaluamos la existencia de una potencial correlación entre
la proteína EZH2 (componente del PoZycomb grupo 2) y la variante de histona mH2A2.
Nuestros resultados experimentales confiaron una expresión trasncripcional de mH2A2
durante el desarrollo embrionario del pez cebra (D. rerio).
De igual forma, intentamos realizar la sobreexpresión de la proteína mH2A2 de pez
cebra por vía recombinante y mediante la utilización de un sistema de expresión heterólogo
bacteriano. A pesar de nuestros esfuerzos, no se logró sobreexpresar en grandes cantidades
mH2A2. Finalmente, ensayos de inmunodetección de la proteína EZH2 en estadíos
embrionarios de 24, 48 y 72 hpf, no fueron concluyentes debido a la inespecificidad del
anticuerpo utilizado. De tal forma, nuestros resultados sugieren que tanto mH2A2 como
EZH2 podrían participar en la regulación de la expresión génica en estadios embrionarios
del desarrollo del pez cebra.
The degree of compaction of chromatin is crucial and influential in fundamental cell processes such as gene transcription, DNA replication and DNA repair. Consequently, the modulation of chromatin conformation is essential for gene expression. The basic unit of chromatin is the nucleosome, which consists of 147 bp of DNA wrapping around an octamer of basic proteins called histones. An alteration of the composition of the nucleosome is to incorporate histone variants in certain regions, replacing canonical histones. This replacement results in a change in the chromatin architecture and therefore a change in gene expression, which determines cell specialization, tissue differentiation and epigenetic inheritance. Our attention focused specifically on mH2A2 histone variant, which has been proposed to interact with components of chromatin remodeling complexes such as proteins of the Polycomb-group (PcG). These factors have been shown to play a crucial role in the epigenetic regulation of gene expression during embryonic development stages in different species. With this in mind, we evaluated the potential existente of a correlation between EZH2 protein (component of the Polycomb group 2) and histone variant rnH2A2. Our experimental results confmed a majority transcriptional mH2A2 expression during embryogenesis of zebrafish (D. rerio). We tried to overexpresss the recombinant protein mH2A2 of zebrafish using a heterologous bacteria1 expression system. Despite many efforts, we did not manage to overexpress mH2A2 in large arnounts. Finally, immunodetection assays of EZH2 protein in embryonic stages 24, 48 and 72 hpf, were inconclusive due to the specificity of the antibody used. So, our results suggest that both rnH2A2 as EZH2 may be involved in regulation of gene expression in embryonic development stages of the zebrafish.
The degree of compaction of chromatin is crucial and influential in fundamental cell processes such as gene transcription, DNA replication and DNA repair. Consequently, the modulation of chromatin conformation is essential for gene expression. The basic unit of chromatin is the nucleosome, which consists of 147 bp of DNA wrapping around an octamer of basic proteins called histones. An alteration of the composition of the nucleosome is to incorporate histone variants in certain regions, replacing canonical histones. This replacement results in a change in the chromatin architecture and therefore a change in gene expression, which determines cell specialization, tissue differentiation and epigenetic inheritance. Our attention focused specifically on mH2A2 histone variant, which has been proposed to interact with components of chromatin remodeling complexes such as proteins of the Polycomb-group (PcG). These factors have been shown to play a crucial role in the epigenetic regulation of gene expression during embryonic development stages in different species. With this in mind, we evaluated the potential existente of a correlation between EZH2 protein (component of the Polycomb group 2) and histone variant rnH2A2. Our experimental results confmed a majority transcriptional mH2A2 expression during embryogenesis of zebrafish (D. rerio). We tried to overexpresss the recombinant protein mH2A2 of zebrafish using a heterologous bacteria1 expression system. Despite many efforts, we did not manage to overexpress mH2A2 in large arnounts. Finally, immunodetection assays of EZH2 protein in embryonic stages 24, 48 and 72 hpf, were inconclusive due to the specificity of the antibody used. So, our results suggest that both rnH2A2 as EZH2 may be involved in regulation of gene expression in embryonic development stages of the zebrafish.
Notas
Tesis (Magíster en Biotecnología)
Palabras clave
Pez Cebra, Embriología, Genética