RESUMEN
Spn1/Iws1 is an essential eukaryotic transcription elongation factor that is conserved from yeast to humans as an integral member of the RNA polymerase II elongation complex. Several studies have shown that Spn1 functions as a histone chaperone to control transcription, RNA splicing, genome stability, and histone modifications. However, the precise role of Spn1 is not understood, and there is little understanding of why it is essential for viability. To address these issues, we have isolated 8 suppressor mutations that bypass the essential requirement for Spn1 in Saccharomyces cerevisiae. Unexpectedly, the suppressors identify several functionally distinct complexes and activities, including the histone chaperone FACT, the histone methyltransferase Set2, the Rpd3S histone deacetylase complex, the histone acetyltransferase Rtt109, the nucleosome remodeler Chd1, and a member of the SAGA coactivator complex, Sgf73. The identification of these distinct groups suggests that there are multiple ways in which Spn1 bypass can occur, including changes in histone acetylation and alterations in other histone chaperones. Thus, Spn1 may function to overcome repressive chromatin by multiple mechanisms during transcription. Our results suggest that bypassing a subset of these functions allows viability in the absence of Spn1.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Factores de Elongación Transcripcional , Cromatina , Proteínas de Unión al ADN/genética , Histona Acetiltransferasas/genética , Chaperonas de Histonas/genética , Histona Desacetilasas/genética , Histona Metiltransferasas/genética , Histonas/genética , Nucleosomas , Factores de Elongación de Péptidos/genética , ARN Polimerasa II/genética , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Supresión Genética , Factores de Transcripción/genética , Transcripción Genética , Factores de Elongación Transcripcional/genéticaRESUMEN
Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. Three conserved and essential chaperones, Spt6, Spn1/Iws1, and FACT, associate with elongating RNA polymerase II and interact with each other physically and/or functionally; however, there is little understanding of their individual functions or their relationships with each other. In this study, we selected for suppressors of a temperature-sensitive spt6 mutation that disrupts the Spt6-Spn1 physical interaction and that also causes both transcription and chromatin defects. This selection identified novel mutations in FACT. Surprisingly, suppression by FACT did not restore the Spt6-Spn1 interaction, based on coimmunoprecipitation, ChIP, and mass spectrometry experiments. Furthermore, suppression by FACT bypassed the complete loss of Spn1. Interestingly, the FACT suppressor mutations cluster along the FACT-nucleosome interface, suggesting that they alter FACT-nucleosome interactions. In agreement with this observation, we showed that the spt6 mutation that disrupts the Spt6-Spn1 interaction caused an elevated level of FACT association with chromatin, while the FACT suppressors reduced the level of FACT-chromatin association, thereby restoring a normal Spt6-FACT balance on chromatin. Taken together, these studies reveal previously unknown regulation between histone chaperones that is critical for their essential in vivo functions.
Asunto(s)
Cromatina/metabolismo , Regulación de la Expresión Génica/genética , Chaperonas de Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción Genética/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Chaperonas de Histonas/genética , Mutación , Nucleosomas/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
Enhancers are DNA sequences composed of transcription factor binding sites that drive complex patterns of gene expression in space and time. Until recently, studying enhancers in their genomic context was technically challenging. Therefore, minimal enhancers, the shortest pieces of DNA that can drive an expression pattern that resembles a gene's endogenous pattern, are often used to study features of enhancer function. However, evidence suggests that some enhancers require sequences outside the minimal enhancer to maintain function under environmental perturbations. We hypothesized that these additional sequences also prevent misexpression caused by a transcription factor binding site mutation within a minimal enhancer. Using the Drosophila melanogastereven-skipped stripe 2 enhancer as a case study, we tested the effect of a Giant binding site mutation (gt-2) on the expression patterns driven by minimal and extended enhancer reporter constructs. We found that, in contrast to the misexpression caused by the gt-2 binding site deletion in the minimal enhancer, the same gt-2 binding site deletion in the extended enhancer did not have an effect on expression. The buffering of expression levels, but not expression pattern, is partially explained by an additional Giant binding site outside the minimal enhancer. Deleting the gt-2 binding site in the endogenous locus had no significant effect on stripe 2 expression. Our results indicate that rules derived from mutating enhancer reporter constructs may not represent what occurs in the endogenous context.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Mutación , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. We investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped (eve). While Hunchback directly represses the eve stripe 3+7 enhancer, we found that in the eve stripe 2+7 enhancer, Hunchback repression is prevented by nearby sequences-this phenomenon is called counter-repression. We also found evidence that Caudal binding sites are responsible for counter-repression, and that this interaction may be a conserved feature of eve stripe 2 enhancers. Our results alter the textbook view of eve stripe 2 regulation wherein Hb is described as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted. We discuss how counter-repression may influence eve stripe 2 regulation and evolution.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Sitios de Unión/genética , Proteínas de Unión al ADN/genética , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero , Elementos de Facilitación Genéticos/genética , Femenino , Proteínas de Homeodominio/metabolismo , MasculinoRESUMEN
The National Institutes of Health (NIH) encourages trainees to make Individualized Development Plans to help them prepare for academic and nonacademic careers. We describe our approach to building an Individualized Development Plan, the reasons we find them useful and empowering for both PIs and trainees, and resources to help other labs implement them constructively.