The E2 Ubiquitin Conjugase Rad6 Is Required for the ArgR/Mcm1 Repression of ARG1 Transcription

Abstract

Transcription of the Saccharomyces cerevisiae ARG1 gene is under the control of both positive and negative elements. Activation of the gene in minimal medium is induced by Gcn4. Repression occurs in the presence of arginine and requires the ArgR/Mcm1 complex that binds to two upstream arginine control (ARC) elements. With the recent finding that the E2 ubiquitin conjugase Rad6 modifies histone H2B, we examined the role of Rad6 in the regulation of ARG1 transcription. We find that Rad6 is required for repression of ARG1 in rich medium, with expression increased ∼10-fold in a rad6 null background. Chromatin immunoprecipitation analysis indicates increased binding of TATA-binding protein in the absence of Rad6. The active-site cysteine of Rad6 is required for repression, implicating ubiquitination in the process. The effects of Rad6 at ARG1 involve two components. In one of these, histone H2B is the likely target for ubiquitination by Rad6, since a strain expressing histone H2B with the principal ubiquitination site converted from lysine to arginine shows a fivefold relief of repression. The second component requires Ubr1 and thus likely the pathway of N-end rule degradation. Through the analysis of promoter constructs with ARC deleted and an arg80 rad6 double mutant, we show that Rad6 repression is mediated through the ArgR/Mcm1 complex. In addition, analysis of an ada2 rad6 deletion strain indicated that the SAGA acetyltransferase complex and Rad6 act in the same pathway to repress ARG1 in rich medium.

We thank Carol Hannam, David Litchfield, Greg Gloor, Colin Coros, and George Chaconas for their contributions to the manuscript. Plasmids and strains were kindly provided by Susan Liebman and Mary Ann Osley.

This work was supported by a grant from the Canadian Institutes of Health Research to C.J.B. A.R.R. was supported by an NSERC Studentship, and H.P. was supported by an Ontario Graduate Scholarship.