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Transcriptional plasticity through differential assembly of a multiprotein activation complex.

Cormier L, Barbey R, Kuras L - Nucleic Acids Res. (2010)

Bottom Line: Study of Cbf1 and Met31/32 association with PDC6 allowed us to find a new mechanism of recruitment of Met4, which allows PDC6 being differentially regulated compared to sulfur amino acid biosynthetic genes.Our findings provide a new example of mechanism allowing transcriptional plasticity within a regulatory network thanks to a definite toolbox comprising a unique master activator and several dedicated DNA-binding cofactors.We also show evidence suggesting that integration of PDC6 to the Met4 regulon may have occurred recently in the evolution of the Saccharomyces cerevisiae lineage.

View Article: PubMed Central - PubMed

Affiliation: CNRS, Centre de Génétique Moléculaire, Gif-sur-Yvette, France.

ABSTRACT
Cell adaptation to the environment often involves induction of complex gene expression programs under the control of specific transcriptional activators. For instance, in response to cadmium, budding yeast induces transcription of the sulfur amino acid biosynthetic genes through the basic-leucine zipper activator Met4, and also launches a program of substitution of abundant glycolytic enzymes by isozymes with a lower content in sulfur. We demonstrate here that transcriptional induction of PDC6, which encodes a pyruvate decarboxylase isoform with low sulfur content, is directly controlled by Met4 and its DNA-binding cofactors the basic-helix-loop-helix protein Cbf1 and the two homologous zinc finger proteins Met31 and Met32. Study of Cbf1 and Met31/32 association with PDC6 allowed us to find a new mechanism of recruitment of Met4, which allows PDC6 being differentially regulated compared to sulfur amino acid biosynthetic genes. Our findings provide a new example of mechanism allowing transcriptional plasticity within a regulatory network thanks to a definite toolbox comprising a unique master activator and several dedicated DNA-binding cofactors. We also show evidence suggesting that integration of PDC6 to the Met4 regulon may have occurred recently in the evolution of the Saccharomyces cerevisiae lineage.

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In vitro associations of Met31 and Met32 with PDC6 promoter. (A) Gel shift assays. The indicated amounts of E. coli cell extracts expressing polyhistidine-tagged Met31 and Met32 (lanes 2–6 and 7–11, respectively) or not (lane 12) were incubated with equimolar amounts of the indicated 32P-labeled DNA fragments. The MET3 fragment contains AAACTGTGGC. The PDC6 fragments contain both CTGTGG sites (second panel) and either of them (third and fourth panel). Fragments were resolved by 5% polyacrylamide gel electrophoresis and visualized by PhosphorImager analysis. (B) Western blot. Extracts used in (A) containing hisMet31 or hisMet32 were separated by 12% SDS–polyacrylamide gel electrophoresis and analyzed by immunoblotting with a monoclonal antibody to the his-tag (Novagen). The calculated molecular weights of hisMet31 and hisMet32 are 23 226.5 and 25 441, respectively.
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Figure 4: In vitro associations of Met31 and Met32 with PDC6 promoter. (A) Gel shift assays. The indicated amounts of E. coli cell extracts expressing polyhistidine-tagged Met31 and Met32 (lanes 2–6 and 7–11, respectively) or not (lane 12) were incubated with equimolar amounts of the indicated 32P-labeled DNA fragments. The MET3 fragment contains AAACTGTGGC. The PDC6 fragments contain both CTGTGG sites (second panel) and either of them (third and fourth panel). Fragments were resolved by 5% polyacrylamide gel electrophoresis and visualized by PhosphorImager analysis. (B) Western blot. Extracts used in (A) containing hisMet31 or hisMet32 were separated by 12% SDS–polyacrylamide gel electrophoresis and analyzed by immunoblotting with a monoclonal antibody to the his-tag (Novagen). The calculated molecular weights of hisMet31 and hisMet32 are 23 226.5 and 25 441, respectively.

Mentions: To gain information on Met31 binding to PDC6 and better characterize Met32 binding, gel shift assays were carried out using Escherichia coli cell extracts containing or not polyhistidine-tagged Met31 and Met32 proteins (hisMet31 and hisMet32; Figure 4). The result clearly showed hisMet31 and hisMet32 binding to the PDC6 fragment encompassing the two TGTGGC sites (Figure 4, second panel). However binding to PDC6 was much less efficient compared to MET3, even though MET3 contains only one CTGTGGC motif (Figure 4, first panel). Gel shift assays with shorter PDC6 fragments containing either of the two CTGTGGC sites revealed that Met32 was not able to bind to site #2 (Figure 4, fourth panel), which was consistent with the ChIP results in Figure 3C. Moreover, the fraction of fragment with site #1 shifted in the presence of hisMet31 was significantly higher than the fraction shifted in the presence of hisMet32 (Figure 4, third panel). Since gels shift were carried out with similar amounts of hisMet31 and hisMet32 (see western blot in Figure 4B), we concluded that Met31 and Met32 had differential affinities for PDC6.Figure 4.


Transcriptional plasticity through differential assembly of a multiprotein activation complex.

Cormier L, Barbey R, Kuras L - Nucleic Acids Res. (2010)

In vitro associations of Met31 and Met32 with PDC6 promoter. (A) Gel shift assays. The indicated amounts of E. coli cell extracts expressing polyhistidine-tagged Met31 and Met32 (lanes 2–6 and 7–11, respectively) or not (lane 12) were incubated with equimolar amounts of the indicated 32P-labeled DNA fragments. The MET3 fragment contains AAACTGTGGC. The PDC6 fragments contain both CTGTGG sites (second panel) and either of them (third and fourth panel). Fragments were resolved by 5% polyacrylamide gel electrophoresis and visualized by PhosphorImager analysis. (B) Western blot. Extracts used in (A) containing hisMet31 or hisMet32 were separated by 12% SDS–polyacrylamide gel electrophoresis and analyzed by immunoblotting with a monoclonal antibody to the his-tag (Novagen). The calculated molecular weights of hisMet31 and hisMet32 are 23 226.5 and 25 441, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: In vitro associations of Met31 and Met32 with PDC6 promoter. (A) Gel shift assays. The indicated amounts of E. coli cell extracts expressing polyhistidine-tagged Met31 and Met32 (lanes 2–6 and 7–11, respectively) or not (lane 12) were incubated with equimolar amounts of the indicated 32P-labeled DNA fragments. The MET3 fragment contains AAACTGTGGC. The PDC6 fragments contain both CTGTGG sites (second panel) and either of them (third and fourth panel). Fragments were resolved by 5% polyacrylamide gel electrophoresis and visualized by PhosphorImager analysis. (B) Western blot. Extracts used in (A) containing hisMet31 or hisMet32 were separated by 12% SDS–polyacrylamide gel electrophoresis and analyzed by immunoblotting with a monoclonal antibody to the his-tag (Novagen). The calculated molecular weights of hisMet31 and hisMet32 are 23 226.5 and 25 441, respectively.
Mentions: To gain information on Met31 binding to PDC6 and better characterize Met32 binding, gel shift assays were carried out using Escherichia coli cell extracts containing or not polyhistidine-tagged Met31 and Met32 proteins (hisMet31 and hisMet32; Figure 4). The result clearly showed hisMet31 and hisMet32 binding to the PDC6 fragment encompassing the two TGTGGC sites (Figure 4, second panel). However binding to PDC6 was much less efficient compared to MET3, even though MET3 contains only one CTGTGGC motif (Figure 4, first panel). Gel shift assays with shorter PDC6 fragments containing either of the two CTGTGGC sites revealed that Met32 was not able to bind to site #2 (Figure 4, fourth panel), which was consistent with the ChIP results in Figure 3C. Moreover, the fraction of fragment with site #1 shifted in the presence of hisMet31 was significantly higher than the fraction shifted in the presence of hisMet32 (Figure 4, third panel). Since gels shift were carried out with similar amounts of hisMet31 and hisMet32 (see western blot in Figure 4B), we concluded that Met31 and Met32 had differential affinities for PDC6.Figure 4.

Bottom Line: Study of Cbf1 and Met31/32 association with PDC6 allowed us to find a new mechanism of recruitment of Met4, which allows PDC6 being differentially regulated compared to sulfur amino acid biosynthetic genes.Our findings provide a new example of mechanism allowing transcriptional plasticity within a regulatory network thanks to a definite toolbox comprising a unique master activator and several dedicated DNA-binding cofactors.We also show evidence suggesting that integration of PDC6 to the Met4 regulon may have occurred recently in the evolution of the Saccharomyces cerevisiae lineage.

View Article: PubMed Central - PubMed

Affiliation: CNRS, Centre de Génétique Moléculaire, Gif-sur-Yvette, France.

ABSTRACT
Cell adaptation to the environment often involves induction of complex gene expression programs under the control of specific transcriptional activators. For instance, in response to cadmium, budding yeast induces transcription of the sulfur amino acid biosynthetic genes through the basic-leucine zipper activator Met4, and also launches a program of substitution of abundant glycolytic enzymes by isozymes with a lower content in sulfur. We demonstrate here that transcriptional induction of PDC6, which encodes a pyruvate decarboxylase isoform with low sulfur content, is directly controlled by Met4 and its DNA-binding cofactors the basic-helix-loop-helix protein Cbf1 and the two homologous zinc finger proteins Met31 and Met32. Study of Cbf1 and Met31/32 association with PDC6 allowed us to find a new mechanism of recruitment of Met4, which allows PDC6 being differentially regulated compared to sulfur amino acid biosynthetic genes. Our findings provide a new example of mechanism allowing transcriptional plasticity within a regulatory network thanks to a definite toolbox comprising a unique master activator and several dedicated DNA-binding cofactors. We also show evidence suggesting that integration of PDC6 to the Met4 regulon may have occurred recently in the evolution of the Saccharomyces cerevisiae lineage.

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