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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
Published on: July 25, 2019
Actividad de la histona H2A deubiquitinasa del complejo represivo del policombo PR-DUB
Johanna C Scheuermann1, Andrés Gaytán de Ayala Alonso, Katarzyna Oktaba
1European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Nature
|May 4, 2010
Resumen
El complejo de deubiquitinasa represiva Polycomb (PR-DUB), que contiene Calypso y ASX, elimina la monoubiquitinación H2A. Esta deubiquitinación es crucial para el silenciamiento del gen del grupo Polycomb y la represión del gen HOX en Drosophila.
Área de la Ciencia:
- La epigenética y la regulación transcripcional.
- La modificación de la cromatina y el silenciamiento de genes.
- Biología del desarrollo Biología del desarrollo.
Sus antecedentes:
- Las proteínas del grupo del policombo (PcG) son repressores transcripcionales esenciales.
- Las proteínas PcG funcionan en complejos multiproteicos para modificar la cromatina.
- El complejo represivo policomboso 1 (PRC1) y los factores asociados al dRing (dRAF) monoubiquitinate histona H2A.
Objetivo del estudio:
- Para caracterizar el gen calipso no caracterizado de Drosophila PcG.
- Para identificar la función de la proteína Calypso y su complejo asociado.
- Para aclarar el papel de la deubiquitinación H2A en el silenciamiento de genes mediado por Polycomb.
Principales métodos:
- Purificación bioquímica de los complejos proteicos de Drosophila.
- Reconstitución de Drosophila recombinante y complejos humanos.
- Análisis de mutantes de Drosophila que carecen de componentes PR-DUB.
- Ensayos in vitro e in vivo para la deubiquitinación H2A y la represión del gen HOX.
Principales resultados:
- El gen calipso no caracterizado de Drosophila PcG codifica BAP1.
- Calypso forma un complejo con ASX, llamado deubiquitinasa represiva Polycomb (PR-DUB).
- PR-DUB tiene deubiquitinatos H2A pero no H2B.
- Los mutantes que carecen de PR-DUB muestran un aumento de la monoubiquitinación H2A y una disminución de la represión del gen HOX.
Conclusiones:
- El silenciamiento de genes de policombos implica un equilibrio entre la ubiquitinación H2A (por PRC1/dRAF) y la deubiquitinación (por PR-DUB).
- La actividad catalítica de Calypso y la presencia de ASX son esenciales para la función PR-DUB.
- PR-DUB juega un papel crítico en el mantenimiento del silenciamiento del gen HOX durante el desarrollo.
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