Dissection of combinatorial control by the Met4 transcriptional complex.

Met4 is the transcriptional activator of the sulfur metabolic network in Saccharomyces cerevisiae. Lacking DNA-binding ability, Met4 must interact with proteins called Met4 cofactors to target promoters for transcription. Two types of DNA-binding cofactors (Cbf1 and Met31/Met32) recruit Met4 to promoters and one cofactor (Met28) stabilizes the DNA-bound Met4 complexes. To dissect this combinatorial system, we systematically deleted each category of cofactor(s) and analyzed Met4-activated transcription on a genome-wide scale. We defined a core regulon for Met4, consisting of 45 target genes. Deletion of both Met31 and Met32 eliminated activation of the core regulon, whereas loss of Met28 or Cbf1 interfered with only a subset of targets that map to distinct sectors of the sulfur metabolic network. These transcriptional dependencies roughly correlated with the presence of Cbf1 promoter motifs. Quantitative analysis of in vivo promoter binding properties indicated varying levels of cooperativity and interdependency exists between members of this combinatorial system. Cbf1 was the only cofactor to remain fully bound to target promoters under all conditions, whereas other factors exhibited different degrees of regulated binding in a promoter-specific fashion. Taken together, Met4 cofactors use a variety of mechanisms to allow differential transcription of target genes in response to various cues.

Determinants of the ubiquitin-mediated degradation of the Met4 transcription factor.

In yeast, the Met4 transcription factor and its cofactors Cbf1, Met28, Met31, and Met32 control the expression of sulfur metabolism and oxidative stress response genes. Met4 activity is tuned to nutrient and oxidative stress conditions by the SCF(Met30) ubiquitin ligase. The mechanism whereby SCF(Met30)-dependent ubiquitylation of Met4 controls Met4 activity remains contentious. Here, we have demonstrated that intracellular cysteine levels dictate the degradation of Met4 in vivo, as shown by the ability of cysteine, but not methionine or S-adenosylmethionine (AdoMet), to trigger Met4 degradation in an str4Delta strain, which lacks the ability to produce cysteine from methionine or AdoMet. Met4 degradation requires its nuclear localization and activity of the 26 S proteasome. Analysis of the regulated degradation of a fully functional Met4-Cbf1 chimera, in which Met4 is fused to the DNA binding domain of Cbf1, demonstrates that elimination of Met4 in vivo can be triggered independently of both its normal protein interactions. Strains that harbor the Met4-Cbf1 fusion as the only source of Cbf1 activity needed for proper kinetochore function exhibit high rates of methionine-dependent chromosomal instability. We suggest that SCF(Met30) activity or Met4 utilization as a substrate may be directly regulated by intracellular cysteine concentrations.