Jeffrey D. Laney

A ubiquitin-selective AAA-ATPase mediates transcriptional switching by remodelling a repressor–promoter DNA complex

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 α2 is necessary to evoke the transition between mating-types, and here we dissect the impact of this modification on α2 dynamics at its target promoters. Ubiquitylation of α2 does not alter DNA occupancy by depleting the existing pool of the transcription factor, despite its well-characterized function in directing repressor turnover. Rather, α2 ubiquitylation has a direct role in the rapid removal of the repressor from its DNA targets. This disassembly of α2 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.

Analysis of Protein Ubiquitination

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. Curr. Protoc. Protein Sci. 66:14.5.1‐14.5.13.

Assaying protein ubiquitination in Saccharomyces cerevisiae

Publisher Summary The covalent modification of target proteins by the polypeptide ubiquitin (Ub) is involved in a wide array of cellular processes, ranging from cell cycle progression and receptor-mediated endocytosis to endoplasmic reticulum-associated degradation and cell-type specification. The best understood function of ubiquitination is to tag protein substrates for destruction, with a polymeric chain of Ub molecules being required to target the substrate to the 26S proteasome for hydrolysis. In addition to this common role in protein degradation, a number of examples of nonproteolytic functions for Ub attachment also exist. This chapter describes various methods to determine whether Ub modifies a particular protein in vivo. These assays can be adapted to ask if the substrate is attached to Ub chains and to ascertain the topology (linkages) of the Ub monomers in these polymeric chains. The chapter also discusses the methods for determining the enzymes that are required for substrate ubiquitination.

Degradation of the Saccharomyces cerevisiae mating-type regulator α1: genetic dissection of cis-determinants and trans-acting pathways

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 α to the a phenotype by ensuring the rapid removal of the α2 transcriptional repressor. Transition to the a cell phenotype, however, also requires the inactivation of the α1 transcriptional activator, but the mechanism by which this occurs is currently unknown. Here, we report a central role for the ubiquitin–proteasome system in α1 inactivation. The α1 protein is constitutively short lived and targeted for rapid turnover by multiple ubiquitin-conjugation pathways. Intriguingly, the α-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 α2. Our observations suggest coordinate regulation in the turnover of two master regulatory transcription factors ensures a rapid mating-type switch.

The Short-Lived Matα2 Transcriptional Repressor Is Protected from Degradation In Vivo by Interactions with Its Corepressors Tup1 and Ssn6

ABSTRACT The Matα2 (α2) protein is a transcriptional repressor necessary for the proper expression of cell type-specific genes in Saccharomyces cerevisiae. Like many transcription factors, α2 is rapidly degraded in vivo by the ubiquitin-proteasome pathway. At least two different ubiquitin-dependent pathways target α2 for destruction, one of which recognizes the well-characterized Deg1 degradation determinant near the N terminus of the protein. Here we report that the α2 corepressors Tup1 and Ssn6 modify the in vivo degradation rate of α2. Tup1 modulates the metabolic stability of α2 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 α2 and Tup1 that compromise the α2-Tup1 binding interaction disrupt the ability of Tup1 to stabilize Deg1 proteins. The physical association between Tup1 and α2 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 α2 protein. From these results, we propose that the fraction of α2 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.

Corepressor-directed preacetylation of histone H3 in promoter chromatin primes rapid transcriptional switching of cell-type-specific genes in yeast.

ABSTRACT 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 α2 repressor in the starting state are derepressed to establish the new mating-type-specific gene expression program coincident with the loss of α2 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 α2. 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.

UNIT 14.5 Analysis of Protein Ubiquitination

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.