A heteromeric complex containing the centromere binding factor 1 and two basic leucine zipper factors, Met4 and Met28, mediates the transcription activation of yeast sulfur metabolism.

Abstract

Transcription activation of sulfur metabolism in yeast is dependent on two DNA binding factors, the centromere binding factor 1 (Cbf1) and Met4. While the role of Met4 was clearly established by showing that it acts as a transcription activator, the precise function in transcription of the multi‐functional factor Cbf1 remains more elusive. We report here the identification of a new transcription factor Met28 which participates in the regulation of sulfur metabolism. Cloning and sequencing of MET28 revealed that it encodes a new member of the basic leucine zipper DNA binding factor family. We also demonstrate that Met28 possesses no intrinsic transcription activation capabilities. Studies of the DNA binding characteristics of Met28 led us to identify in gel mobility assays a heteromeric complex containing Cbf1, Met4 and Met28. We further demonstrated that the presence of Cbf1 and Met4 stimulates the binding of Met28 to DNA. ‘Two‐hybrid’ studies allowed us to carry out preliminary investigations on the binary protein‐protein interactions involved in the formation of the Cbf1‐Met4‐Met28 complex. Our results give evidence that the leucine zippers of Met4 and Met28, along with the basic helix‐loop‐helix domain of Cbf1, provide the protein surfaces mediating these interactions. All these results suggest that the multi‐functional factor Cbf1 functions in transcription activation by tethering specific activating factors to the DNA.

Cbf1p Is Required for Chromatin Remodeling at Promoter-proximal CACGTG Motifs in Yeast

“…This appears to be exactly the case at certain MET gene promoters. At the MET16 locus, Cbf1p binding to the CACGTG motif (at Ϫ174 bp) has been extensively characterized and shown to be enhanced by formation of a co-operative Cbf1p⅐Met28p⅐Met4p complex (24,25). We, therefore, examined the chromatin structure at MET16 in a met4 mutant in which Cbf1p binds alone to the CACGTG motif with reduced relative affinity.…”

“…On its own Met4p will not bind DNA (24). At the MET16 promoter, Met4p is recruited as part of a co-operatively bound complex consisting of Met4p, the basic-helix-loop-helix factor Met28p, and Cbf1p, which binds to a single CACGTG motif (24,25). In the absence of Cbf1p the complex does not form at the promoter, and the MET16 gene cannot be activated.…”

The Yeast Ubiquitin Ligase SCFMet30Regulates Heavy Metal Response

“…The importance of Met4 regulation in response to cadmium is underlined by the cadmium hypersensitivity of met4 mutants and mutants in components of the Met4 transcription complexes ( Figure 2C). Surprisingly, met28 mutants were not sensitive to cadmium, even though Met28 is important for expression of several genes involved in sulfur amino acid biosynthesis in response to methionine starvation (Kuras et al, 1996). It is possible that a protein with redundant function is induced in response to cadmium exposure that can replace Met28.…”

“…Both isozyme switching and induction of sulfur amino acid synthesis pathway components in response to cadmium exposure have been shown to depend primarily on the yeast transcription factor Met4 (Fauchon et al, 2002). Microarray analysis showed that cadmium-dependent induction of Ͼ60 genes was severely affected in ⌬met4 mutants (Fauchon et al, 2002 (Kuras et al, 1996) and the DNA-binding factors Cbf1 (Kuras et al, 1997), Met31, and Met32 (Blaiseau et al, 1997). However, only Met4 has transactivating activity and seems to be the focus of regulation (Thomas and Surdin-Kerjan, 1997).…”

“…These include Met28 (Kuras et al, 1996) and the DNA-binding factors Cbf1 (Kuras et al, 1997), Met31, and Met32 (Blaiseau et al, 1997). However, only Met4 has transactivating activity and seems to be the focus of regulation .…”