RESUMEN
Eukaryotic cells package their genomes around histone octamers. In response to DNA damage, checkpoint activation in yeast induces core histone degradation resulting in 20%-40% reduction in nucleosome occupancy. To gain insight into this process, we developed a new approach to analyze the chromatin-associated proteome comprehensively before and after damage. This revealed extensive changes in protein composition after Zeocin-induced damage. First, core histones and the H1 homolog Hho1 were partially lost from chromatin along with replication, transcription, and chromatin remodeling machineries, while ubiquitin ligases and the proteasome were recruited. We found that the checkpoint- and INO80C-dependent recruitment of five ubiquitin-conjugating factors (Rad6, Bre1, Pep5, Ufd4, and Rsp5) contributes to core and linker histone depletion, reducing chromatin compaction and enhancing DNA locus mobility. Importantly, loss of Rad6/Bre1, Ufd4/TRIP12, and Pep5/VPS11 compromise DNA strand invasion kinetics during homology-driven repair. Thus we provide a comprehensive overview of a functionally relevant genome-wide chromatin response to DNA damage.
Asunto(s)
Ensamble y Desensamble de Cromatina , Reparación del ADN , ADN de Hongos/metabolismo , Histonas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , ADN de Hongos/genética , Histonas/genética , Complejo de la Endopetidasa Proteasomal/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The histone variant H2A.Z is deposited into chromatin by specific machinery and is required for genome functions. Using a linker-mediated complex strategy combined with yeast genetic complementation, we demonstrate evolutionary conservation of H2A.Z together with its chromatin incorporation and functions. This approach is applicable to the evolutionary analyses of proteins that form complexes with interactors.
Asunto(s)
HistonasRESUMEN
Quantitative control of histones and histone variants during cell cycle is relevant to their epigenetic functions. We found that the level of yeast histone variant H2A.Z in the G2/M-phase is actively kept low by the ubiquitin proteasome system and SUMO-targeted ubiquitin ligases. Overexpression of H2A.Z induced defects in mitotic progression, suggesting functional importance of this quantitative control.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Histonas/genética , Proteína SUMO-1/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular , Histonas/metabolismo , Mitosis , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Targeted protein degradation using the auxin-inducible degron (AID) system is garnering attention in the research field of Caenorhabditis elegans, because of the rapid and efficient target depletion it affords, which can be controlled by treating the animals with the phytohormone auxin. However, the current AID system has drawbacks, i.e., leaky degradation in the absence of auxin and the requirement for high auxin doses. Furthermore, it is challenging to deplete degron-fused proteins in embryos because of their eggshell, which blocks auxin permeability. Here, we apply an improved AID2 system utilizing AtTIR1(F79G) and 5-phenyl-indole-3-acetic acid (5-Ph-IAA) to C. elegans and demonstrated that it confers better degradation control vs the previous system by suppressing leaky degradation and inducing sharp degradation using 1,300-fold lower 5-Ph-IAA doses. We successfully degraded the endogenous histone H2A.Z protein fused to an mAID degron and disclosed its requirement in larval growth and reproduction, regardless of the presence of maternally inherited H2A.Z molecules. Moreover, we developed an eggshell-permeable 5-Ph-IAA analog, 5-Ph-IAA-AM, that affords an enhanced degradation in laid embryos. Our improved system will contribute to the disclosure of the roles of proteins in C. elegans, in particular those that are involved in embryogenesis and development, through temporally controlled protein degradation.