The E2 Ubiquitin Conjugase Rad6 Is Required for the ArgR/Mcm1 Repression ofARG1Transcription

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 ϳ10-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.In response to the role of Rad6/Ubc2 as an E2 ubiquitin conjugase, mutations in its gene affect multiple cellular processes. Rad6 acts with Rad18 in pathways of DNA repair (3-5, 46) and with the E3 ubiquitin ligase Ubr1 in the pathway leading to the degradation of multiubiquitinated protein substrates via the 26S proteasome (22,48,69). Independently of Rad18 and Ubr1, Rad6 is required for transcriptional silencing at telomeres and the HM loci (34). The ability of Rad6 to ubiquitinate histones H2A, H2B, and H3 in vitro (29,30,39) and H2B in vivo (59) has led to the suggestion that its ability to regulate gene expression results from changes in chromatin structure. This idea is supported by findings that disruption of rad6 results in changes in the sites of integration of retrotransposons (47,56) and that a strain with a K123R mutation in the principal ubiquitination site of histone H2B has the same sporulation defect as a strain with rad6 deleted (59).The ARG1 promoter provides a valuable system to study the role of factors involved in the activation and repression of transcription. ARG1 encodes argininosuccinate synthetase, which is required in a pathway that also includes ARG2, ARG5,6, ARG8, ARG3, and ARG4 for the biosynthesis of arginine. Transcription of this group of genes is subject to general amino acid control mediated by the activator protein Gcn4 (14,20,32). ARG1, as well as ARG5,6, ARG8, and ARG3, is also subject to repression by arginine (10,14,16,20,38,50). Arginine repression requires a DNA binding comp...

The cap binding complex influences H2B ubiquitination by facilitating splicing of the SUS1 pre-mRNA

“…ARG1 is induced when cells are grown in media lacking arginine (SC-Arg); but in YPD, the expression of ARG1 is repressed (Turner et al 2002). Both induction and repression of ARG1 expression are tightly regulated by H2B ubiquitination.…”

Rad6-Bre1-mediated histone H2B ubiquitylation modulates the formation of double-strand breaks during meiosis

“…Although we favor this possibility, the rad6 might affect DSB formation indirectly. The rad6 mutant is partially deficient in transcriptional silencing (26) and in transcription (7,8), thus affecting overall transcription status in a cell. A change of the transcription of genes involved in meiotic recombination might affect DSB formation in rad6 cells.…”

“…Histone H2B ubiquitylation requires a specific E3 partner, Bre1 (5,6). The ubiquitylation of histone H2B is involved in the silencing of gene expression as well as transcription (5)(6)(7)(8). Rad6 is recruited to promoter regions of constitutively transcribed genes in a Bre1-dependent manner (6).…”

Effectors of Lysine 4 Methylation of Histone H3 in Saccharomyces cerevisiae Are Negative Regulators of PHO5 and GAL1-10

“…repressed ϳ10-fold in the absence of Rad6 (40). Similarly, GAL10 is expressed at much higher levels in a rad6 null strain; however, the corresponding histone H2B lysine 123 to the arginine (K123R) mutant did not derepress GAL10 transcription (29).…”