Physical and genetic associations of the Irc20 ubiquitin ligase with Cdc48 and SUMO.

A considerable percentage of the genome is dedicated to the ubiquitin-proteasome system, with the yeast genome predicted to encode approximately 100 ubiquitin ligases (or E3s), and the human genome predicted to encode more than 600 E3s. The most abundant class of E3s consists of RING finger-containing proteins. Although many insights have been obtained regarding the structure and catalytic mechanism of the E3s, much remains to be learned about the function of the individual E3s. Here we characterize IRC20, which encodes a dual RING- and Snf/Swi family ATPase domain-containing protein in yeast that has been implicated in DNA repair. We found that overexpression of IRC20 causes two transcription-associated phenotypes and demonstrate that the Irc20 RING domain possesses ubiquitin E3 activity in vitro. Two mass spectrometry approaches were undertaken to identify Irc20-associated proteins. Wild-type Irc20 associated with Cdc48, a AAA-ATPase that serves as an intermediary in the ubiquitin-proteasome system. A second approach using a RING mutant derivative of Irc20 detected increased association of the Irc20 mutant with SUMO. These findings provide a foundation for understanding the roles of Irc20 in transcription and DNA repair.

Physical and Genetic Interactions Between Uls1 and the Slx5-Slx8 SUMO-Targeted Ubiquitin Ligase.

The Slx5-Slx8 complex is a ubiquitin ligase that preferentially ubiquitylates SUMOylated substrates, targeting them for proteolysis. Mutations in SLX5, SLX8, and other SUMO pathway genes were previously identified in our laboratory as genomic suppressors of a point mutation (mot1-301) in the transcriptional regulator MOT1 To further understand the links between the SUMO and ubiquitin pathways, a screen was performed for high-copy suppressors of mot1-301, yielding three genes (MOT3, MIT1, and ULS1). MOT3 and MIT1 have characteristics of prions, and ULS1 is believed to encode another SUMO-targeted ubiquitin ligase (STUbL) that functionally overlaps with Slx5-Slx8. Here we focus on ULS1, obtaining results suggesting that the relationship between ULS1 and SLX5 is more complex than expected. Uls1 interacted with Slx5 physically in to yeast two-hybrid and co-immunoprecipitation assays, a uls1 mutation that blocked the interaction between Uls1 and Slx5 interfered with ULS1 function, and genetic analyses indicated an antagonistic relationship between ULS1 and SLX5 Combined, our results challenge the assumption that Uls1 and Slx5 are simply partially overlapping STUbLs and begin to illuminate a regulatory relationship between these two proteins.

Gene overexpression: uses, mechanisms, and interpretation.

The classical genetic approach for exploring biological pathways typically begins by identifying mutations that cause a phenotype of interest. Overexpression or misexpression of a wild-type gene product, however, can also cause mutant phenotypes, providing geneticists with an alternative yet powerful tool to identify pathway components that might remain undetected using traditional loss-of-function analysis. This review describes the history of overexpression, the mechanisms that are responsible for overexpression phenotypes, tests that begin to distinguish between those mechanisms, the varied ways in which overexpression is used, the methods and reagents available in several organisms, and the relevance of overexpression to human disease.

A systematic CEN library of the Saccharomyces cerevisiae genome.

Progress in modern genetics is greatly facilitated by systematic resources that enable rapid and comprehensive analysis. Here we report the creation of a nearly complete systematic low-copy (CEN URA3) library of the Saccharomyces cerevisiae genome that complements existing systematic high-copy libraries.

Quality control of a transcriptional regulator by SUMO-targeted degradation.

Slx5 and Slx8 are heterodimeric RING domain-containing proteins that possess SUMO-targeted ubiquitin ligase (STUbL) activity in vitro. Slx5-Slx8 and its orthologs are proposed to target SUMO conjugates for ubiquitin-mediated proteolysis, but the only in vivo substrate identified to date is mammalian PML, and the physiological importance of SUMO-targeted ubiquitylation remains largely unknown. We previously identified mutations in SLX5 and SLX8 by selecting for suppressors of a temperature-sensitive allele of MOT1, which encodes a regulator of TATA-binding protein. Here, we demonstrate that Mot1 is SUMOylated in vivo and that disrupting the Slx5-Slx8 pathway by mutation of the target lysines in Mot1, by deletion of SLX5 or the ubiquitin E2 UBC4, or by inhibition of the proteosome suppresses mot1-301 mutant phenotypes and increases the stability of the Mot1-301 protein. The Mot1-301 mutant protein is targeted for proteolysis by SUMOylation to a much greater extent than wild-type Mot1, suggesting a quality control mechanism. In support of this idea, growth of Saccharomyces cerevisiae in the presence of the arginine analog canavanine results in increased SUMOylation and Slx5-Slx8-mediated degradation of wild-type Mot1. These results therefore demonstrate that Mot1 is an in vivo STUbL target in yeast and suggest a role for SUMO-targeted degradation in protein quality control.

A systematic library for comprehensive overexpression screens in Saccharomyces cerevisiae.

Modern genetic analysis requires the development of new resources to systematically explore gene function in vivo. Overexpression screens are a powerful method to investigate genetic pathways, but the goal of routine and comprehensive overexpression screens has been hampered by the lack of systematic libraries. Here we describe the construction of a systematic collection of the Saccharomyces cerevisiae genome in a high-copy vector and its validation in two overexpression screens.

Regulation of histone modification and cryptic transcription by the Bur1 and Paf1 complexes.

The Bur1-Bur2 and Paf1 complexes function during transcription elongation and affect histone modifications. Here we describe new roles for Bur1-Bur2 and the Paf1 complex. We find that histone H3 K36 tri-methylation requires specific components of the Paf1 complex and that K36 tri-methylation is more strongly affected at the 5' ends of genes in paf1delta and bur2delta strains in parallel with increased acetylation of histones H3 and H4. Interestingly, the 5' increase in histone acetylation is independent of K36 methylation, and therefore is mechanistically distinct from the methylation-driven deacetylation that occurs at the 3' ends of genes. Finally, Bur1-Bur2 and the Paf1 complex have a second methylation-independent function, since bur2delta set2delta and paf1delta set2delta double mutants display enhanced histone acetylation at the 3' ends of genes and increased cryptic transcription initiation. These findings identify new functions for the Paf1 and Bur1-Bur2 complexes, provide evidence that histone modifications at the 5' and 3' ends of coding regions are regulated by distinct mechanisms, and reveal that the Bur1-Bur2 and Paf1 complexes repress cryptic transcription through a Set2-independent pathway.

The BUR1 cyclin-dependent protein kinase is required for the normal pattern of histone methylation by SET2.

BUR1 and BUR2 encode the catalytic and regulatory subunits of a cyclin-dependent protein kinase complex that is essential for normal growth and has a general role in transcription elongation. To gain insight into its specific role in vivo, we identified mutations that reverse the severe growth defect of bur1Delta cells. This selection identified mutations in SET2, which encodes a histone methylase that targets lysine 36 of histone H3 and, like BUR1, has a poorly characterized role during transcription elongation. This genetic relationship indicates that SET2 activity is required for the growth defect observed in bur1Delta strains. This SET2-dependent growth inhibition occurs via methylation of histone H3 on lysine 36, since a methylation-defective allele of SET2 or a histone H3 K36R mutation also suppressed bur1Delta. We have explored the relationship between BUR1 and SET2 at the biochemical level and find that histone H3 is monomethylated, dimethylated, and trimethylated on lysine 36 in wild-type cells, but trimethylation is significantly reduced in bur1 and bur2 mutant strains. A similar methylation pattern is observed in RNA polymerase II C-terminal domain truncation mutants and in an spt16 mutant strain. Chromatin immunoprecipitation assays reveal that the transcription-dependent increase in trimethylated K36 over open reading frames is significantly reduced in bur2Delta strains. These results establish links between a regulatory protein kinase and histone methylation and lead to a model in which the Bur1-Bur2 complex counteracts an inhibitory effect of Set2-dependent histone methylation.

Genetic analysis connects SLX5 and SLX8 to the SUMO pathway in Saccharomyces cerevisiae.

MOT1 encodes an essential ATPase that functions as a general transcriptional regulator in vivo by modulating TATA-binding protein (TBP) DNA-binding activity. Although MOT1 was originally identified both biochemically and in several genetic screens as a transcriptional repressor, a combination of subsequent genetic, chromatin immunoprecipitation, and microarray analysis suggested that MOT1 might also have an additional role in vivo as a transcriptional activator. To better understand the role(s) of MOT1 in vivo, we selected for genomic suppressors of a mot1 temperature-sensitive mutation. This selection identified mutations in SPT15 (TBP) and BUR6, both of which are clearly linked with MOT1 at the functional level. The vast majority of the suppressor mutations, however, unexpectedly occurred in six genes that encode known components of the SUMO pathway and in two other genes with unknown functions, SLX5 and SLX8. Additional results presented here, including extensive synthetic lethality observed between slx5delta and slx8delta and SUMO pathway mutations, suggest that SLX5 and SLX8 are new components or regulators of the SUMO pathway and that SUMO modification might have a general role in transcriptional regulation as part of the TBP regulatory network.

Activation of the Bur1-Bur2 cyclin-dependent kinase complex by Cak1.

Cyclin-dependent kinases (Cdks) were originally identified as regulators of eukaryotic cell cycle progression, but several Cdks were subsequently shown to perform important roles as transcriptional regulators. While the mechanisms regulating the Cdks involved in cell cycle progression are well documented, much less is known regarding how the Cdks that are involved in transcription are regulated. In Saccharomyces cerevisiae, Bur1 and Bur2 comprise a Cdk complex that is involved in transcriptional regulation, presumably mediated by its phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. To investigate the regulation of Bur1 in vivo, we searched for high-copy-number suppressors of a bur1 temperature-sensitive mutation, identifying a single gene, CAK1. Cak1 is known to activate two other Cdks in yeast by phosphorylating a threonine within their conserved T-loop domains. Bur1 also has the conserved threonine within its T loop and is therefore a potential direct target of Cak1. Additional tests establish a direct functional interaction between Cak1 and the Bur1-Bur2 Cdk complex: Bur1 is phosphorylated in vivo, both the conserved Bur1 T-loop threonine and Cak1 are required for phosphorylation and Bur1 function in vivo, and recombinant Cak1 stimulates CTD kinase activity of the purified Bur1-Bur2 complex in vitro. Thus, both genetic and biochemical evidence demonstrate that Cak1 is a physiological regulator of the Bur1 kinase.

Direct stimulation of transcription by negative cofactor 2 (NC2) through TATA-binding protein (TBP).

Negative cofactor 2 (NC2) is an evolutionarily conserved transcriptional regulator that was originally identified as an inhibitor of basal transcription. Its inhibitory mechanism has been extensively characterized; NC2 binds to the TATA-binding protein (TBP), blocking the recruitment of TFIIA and TFIIB, and thereby inhibiting preinitiation complex assembly. NC2 is also required for expression of many yeast genes in vivo and stimulates TATA-less transcription in a Drosophila in vitro transcription system, but the mechanism responsible for the NC2-mediated stimulation of transcription is not understood. Here we establish that yeast NC2 can directly stimulate activated transcription from TATA-driven promoters both in vivo and in vitro, and moreover that this positive role requires the same surface of TBP that mediates the NC2 repression activity. On the basis of these results, we propose a model to explain how NC2 can mediate both repression and activation through the same surface of TBP.