RESUMO
The Matalpha2 (alpha2) protein is a transcriptional repressor necessary for the proper expression of cell type-specific genes in Saccharomyces cerevisiae. Like many transcription factors, alpha2 is rapidly degraded in vivo by the ubiquitin-proteasome pathway. At least two different ubiquitin-dependent pathways target alpha2 for destruction, one of which recognizes the well-characterized Deg1 degradation determinant near the N terminus of the protein. Here we report that the alpha2 corepressors Tup1 and Ssn6 modify the in vivo degradation rate of alpha2. Tup1 modulates the metabolic stability of alpha2 by directly binding to the Deg1-containing region of the protein. TUP1 overexpression specifically stabilizes Deg1-containing proteins but not other substrates of the same ubiquitination enzymes that recognize Deg1. Point mutations in both alpha2 and Tup1 that compromise the alpha2-Tup1 binding interaction disrupt the ability of Tup1 to stabilize Deg1 proteins. The physical association between Tup1 and alpha2 competes with the ubiquitination machinery for access to the Deg1 signal. Finally, we observe that overproduction of both Tup1 and Ssn6, but not either alone, strongly stabilizes the endogenous alpha2 protein. From these results, we propose that the fraction of alpha2 found in active regulatory complexes with Tup1 and Ssn6 is spared from rapid proteolytic destruction and is stabilized relative to the uncomplexed pool of the protein.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transferases Intramoleculares , Proteínas Nucleares/genética , Mutação Puntual , Proteínas/genética , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica , Ubiquitina/metabolismoRESUMO
In response to external environmental stimuli and intrinsic developmental cues, yeast cells reset their gene expression programs and change phenotype. These switches in cellular state require the dismantling of an initial regulatory program, in addition to the induction of different sets of genes to specify the new cell phenotype. Recent experiments examining the role of protein degradation in these transitions have highlighted the importance of inactivating previously utilized regulators and have led to advances in our understanding of how cells change from one phenotypic state to another.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Ubiquitina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Attachment of ubiquitin (Ub) to a protein requires a series of enzymes that recognize the substrate and promote Ub transfer. Several methods are described in this unit for determining if a protein has Ub-transferring activity. They include immunoblotting of immunoprecipitated proteins, affinity purification using His-tagged Ub, assaying for auto-ubiquitination of E3, and assaying ubiquitination of a model substrate protein in vitro and in E. coli cells that express Ub-ligation enzymes. These methods are suitable for a variety of eukaryotic cells, but techniques are specifically described for use with yeast and mammalian cells.
Assuntos
Immunoblotting/métodos , Imunoprecipitação/métodos , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Animais , Cromatografia de Afinidade/métodos , Proteínas Fúngicas/metabolismo , Humanos , Leveduras/metabolismoRESUMO
Switching between alternate states of gene transcription is fundamental to a multitude of cellular regulatory pathways, including those that govern differentiation. In spite of the progress in our understanding of such transitions in gene activity, a major unanswered question is how cells regulate the timing of these switches. Here, we have examined the kinetics of a transcriptional switch that accompanies the differentiation of yeast cells of one mating type into a distinct new cell type. We found that cell-type-specific genes silenced by the alpha2 repressor in the starting state are derepressed to establish the new mating-type-specific gene expression program coincident with the loss of alpha2 from promoters. This rapid derepression does not require the preloading of RNA polymerase II or a preinitiation complex but instead depends upon the Gcn5 histone acetyltransferase. Surprisingly, Gcn5-dependent acetylation of nucleosomes in the promoters of mating-type-specific genes requires the corepressor Ssn6-Tup1 even in the repressed state. Gcn5 partially acetylates the amino-terminal tails of histone H3 in repressed promoters, thereby priming them for rapid derepression upon loss of alpha2. Thus, Ssn6-Tup1 not only efficiently represses these target promoters but also functions to initiate derepression by creating a chromatin state poised for rapid activation.
Assuntos
Cromatina/metabolismo , Proteínas Correpressoras/metabolismo , Regulação Fúngica da Expressão Gênica , Histonas , Regiões Promotoras Genéticas , Transcrição Gênica , Proteínas Correpressoras/genética , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Mating phenotype in the yeast Saccharomyces cerevisiae is a dynamic trait, and efficient transitions between alternate haploid cell types allow the organism to access the advantageous diploid form. Mating identity is determined by cell type-specific transcriptional regulators, but these factors must be rapidly removed upon mating-type switching to allow the master regulators of the alternate state to establish a new gene expression program. Targeted proteolysis by the ubiquitin-proteasome system is a commonly employed strategy to quickly disassemble regulatory networks, and yeast use this approach to evoke efficient switching from the alpha to the a phenotype by ensuring the rapid removal of the alpha2 transcriptional repressor. Transition to the a cell phenotype, however, also requires the inactivation of the alpha1 transcriptional activator, but the mechanism by which this occurs is currently unknown. Here, we report a central role for the ubiquitin-proteasome system in alpha1 inactivation. The alpha1 protein is constitutively short lived and targeted for rapid turnover by multiple ubiquitin-conjugation pathways. Intriguingly, the alpha-domain, a conserved region of unknown function, acts as a degradation signal for a pathway defined by the SUMO-targeted ligase Slx5-Slx8, which has also been implicated in the rapid destruction of alpha2. Our observations suggest coordinate regulation in the turnover of two master regulatory transcription factors ensures a rapid mating-type switch.
Assuntos
Proteínas/metabolismo , Saccharomyces cerevisiae , Ligases/genética , Ligases/metabolismo , Fenótipo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
Switches between different phenotypes and their underlying states of gene transcription occur as cells respond to intrinsic developmental cues or adapt to changing environmental conditions. Post-translational modification of the master regulatory transcription factors that define the initial phenotype is a common strategy to direct such transitions. Emerging evidence indicates that the modification of key transcription factors by the small polypeptide ubiquitin has a central role in many of these transitions. However, the molecular mechanisms by which ubiquitylation regulates the switching of promoters between active and inactive states are largely unknown. Ubiquitylation of the yeast transcriptional repressor alpha2 is necessary to evoke the transition between mating-types, and here we dissect the impact of this modification on alpha2 dynamics at its target promoters. Ubiquitylation of alpha2 does not alter DNA occupancy by depleting the existing pool of the transcription factor, despite its well-characterized function in directing repressor turnover. Rather, alpha2 ubiquitylation has a direct role in the rapid removal of the repressor from its DNA targets. This disassembly of alpha2 from DNA depends on the ubiquitin-selective AAA-ATPase Cdc48. Our findings expand the functional targets of Cdc48 to include active transcriptional regulatory complexes in the nucleus. These data reveal an ubiquitin-dependent extraction pathway for dismantling transcription factor-DNA complexes and provide an archetype for the regulation of transcriptional switching events by ubiquitylation.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica , Ubiquitina/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , DNA Fúngico/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína com ValosinaRESUMO
Attachment of ubiquitin (Ub) to a protein requires a complex of enzymes that recognize the substrate and promote Ub transfer. Sequence motifs present in these enzymes may indicate that other uncharacterized proteins containing these motifs have a biochemical function of Ub-protein ligation, and several in vitro methods are described in this unit for determining if a protein has Ub-transferring activity. They include psmunoblotting of psmunoprecipitated proteins, affinity purification using His-tagged ubiquitin, assaying for auto-ubiquitination of E3, and assaying ubiquitination of a model substrate protein. These methods are suitable for a variety of eukaryotic cells, but techniques are specifically described for use with yeast and mammalian cells.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas/química , Ubiquitina/metabolismo , Animais , Cromatografia de Afinidade , Imuno-Histoquímica , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Leveduras/citologia , Leveduras/metabolismoRESUMO
Developmental transitions in eukaryotic cell lineages revolve around two general processes: the dismantling of the regulatory program specifying an initial differentiated state and its replacement by a new system of regulators. However, relatively little is known about the mechanisms by which a previous regulatory state is inactivated. Protein degradation is implicated in a few examples, but the molecular reasons that a formerly used regulator must be removed are not understood. Many yeast strains undergo a developmental transition in which cells of one mating type differentiate into a distinct cell type by a programmed genetic rearrangement at the MAT locus. We find that Mat(alpha)2, a MAT-encoded transcriptional repressor that is key to creating several cell types, must be rapidly degraded for cells to switch their mating phenotype properly. Strikingly, ubiquitin-dependent proteolysis of alpha2 is required for two mechanistically distinct purposes: It allows the timely inactivation of one transcriptional repressor complex, and it prevents the de novo assembly of a different, inappropriate regulatory complex. Analogous epigenetic mechanisms for reprogramming transcription are likely to operate in many developmental pathways.
Assuntos
Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Proteínas de Homeodomínio/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/crescimento & desenvolvimento , Enzimas de Conjugação de Ubiquitina , Ubiquitina/metabolismo , Proteínas de Transporte/metabolismo , Ligases/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
During late embryogenesis, the expression domains of homeotic genes are maintained by two groups of ubiquitously expressed regulators: the Polycomb repressors and the Trithorax activators. It is not known how the activities of the two maintenance systems are initially targeted to the correct genes. Zeste and GAGA are sequence-specific DNA-binding proteins previously shown to be Trithorax group activators of the homeotic gene Ultrabithorax (Ubx). We demonstrate that Zeste and GAGA DNA-binding sites at the proximal promoter are also required to maintain, but not to initiate, repression of UBX: Furthermore, the repression mediated by Zeste DNA-binding site is abolished in zeste null embryos. These data imply that Zeste and probably GAGA mediate Polycomb repression. We present a model in which the dual transcriptional activities of Zeste and GAGA are an essential component of the mechanism that chooses which maintenance system is to be targeted to a given promoter.