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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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Mutagenesis of PDC6 promoter. (A) Schematic diagram showing base changes introduced in PDC6 promoter. (B) pdc6Δ strains containing at the URA3 locus a WT allele of PDC6 or mutant derivatives containing either a A-triplet upstream the two CTGTGGC motifs, or TCACGTG instead of TCACGTT, or both changes, were grown to early log phase in YPD medium and exposed to 0.5 mM Cd2+. Total RNA was extracted from samples taken at the time points indicated. RNA levels for PDC6 and MET3 were quantified by RT-real time PCR and normalized to 25S ribosomal RNA. Values represent the average of two independent experiments and error bars indicate average deviations. (C) MET32 and met32Δ strains containing the WT allele of PDC6 or the derivative with the AAACTGTGGC motifs were manipulated as in (B).
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Figure 12: Mutagenesis of PDC6 promoter. (A) Schematic diagram showing base changes introduced in PDC6 promoter. (B) pdc6Δ strains containing at the URA3 locus a WT allele of PDC6 or mutant derivatives containing either a A-triplet upstream the two CTGTGGC motifs, or TCACGTG instead of TCACGTT, or both changes, were grown to early log phase in YPD medium and exposed to 0.5 mM Cd2+. Total RNA was extracted from samples taken at the time points indicated. RNA levels for PDC6 and MET3 were quantified by RT-real time PCR and normalized to 25S ribosomal RNA. Values represent the average of two independent experiments and error bars indicate average deviations. (C) MET32 and met32Δ strains containing the WT allele of PDC6 or the derivative with the AAACTGTGGC motifs were manipulated as in (B).

Mentions: We next mutated the promoter of PDC6 to transform the binding sites for Met31/32 and Cbf1 into canonical sites, either separately or altogether (Figure 12A). As shown in Figure 12B, these mutations resulted in acceleration of PDC6 transcription in response to cadmium, especially in the case of Met31/32 binding sites, so that mRNA levels at 45 min in the mutated strains were similar to those at 90 min in the WT (Figure 12B). However, mRNA levels at 15 min remained low, indicating that the mutations did accelerate PDC6 transcription but were not sufficient to make the transcriptional kinetics of PDC6 similar to that of MET genes. One likely explanation for this observation is that binding of Cbf1 and Met31/32 to their target promoters and recruitment of Met4 involve sequences adjacent to the AAACTGTGGC and TCACGTG sites. The existence of additional promoter elements recognized by some repressive factors cannot be completely ruled out either.Figure 12.


Transcriptional plasticity through differential assembly of a multiprotein activation complex.

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

Mutagenesis of PDC6 promoter. (A) Schematic diagram showing base changes introduced in PDC6 promoter. (B) pdc6Δ strains containing at the URA3 locus a WT allele of PDC6 or mutant derivatives containing either a A-triplet upstream the two CTGTGGC motifs, or TCACGTG instead of TCACGTT, or both changes, were grown to early log phase in YPD medium and exposed to 0.5 mM Cd2+. Total RNA was extracted from samples taken at the time points indicated. RNA levels for PDC6 and MET3 were quantified by RT-real time PCR and normalized to 25S ribosomal RNA. Values represent the average of two independent experiments and error bars indicate average deviations. (C) MET32 and met32Δ strains containing the WT allele of PDC6 or the derivative with the AAACTGTGGC motifs were manipulated as in (B).
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Related In: Results  -  Collection

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Figure 12: Mutagenesis of PDC6 promoter. (A) Schematic diagram showing base changes introduced in PDC6 promoter. (B) pdc6Δ strains containing at the URA3 locus a WT allele of PDC6 or mutant derivatives containing either a A-triplet upstream the two CTGTGGC motifs, or TCACGTG instead of TCACGTT, or both changes, were grown to early log phase in YPD medium and exposed to 0.5 mM Cd2+. Total RNA was extracted from samples taken at the time points indicated. RNA levels for PDC6 and MET3 were quantified by RT-real time PCR and normalized to 25S ribosomal RNA. Values represent the average of two independent experiments and error bars indicate average deviations. (C) MET32 and met32Δ strains containing the WT allele of PDC6 or the derivative with the AAACTGTGGC motifs were manipulated as in (B).
Mentions: We next mutated the promoter of PDC6 to transform the binding sites for Met31/32 and Cbf1 into canonical sites, either separately or altogether (Figure 12A). As shown in Figure 12B, these mutations resulted in acceleration of PDC6 transcription in response to cadmium, especially in the case of Met31/32 binding sites, so that mRNA levels at 45 min in the mutated strains were similar to those at 90 min in the WT (Figure 12B). However, mRNA levels at 15 min remained low, indicating that the mutations did accelerate PDC6 transcription but were not sufficient to make the transcriptional kinetics of PDC6 similar to that of MET genes. One likely explanation for this observation is that binding of Cbf1 and Met31/32 to their target promoters and recruitment of Met4 involve sequences adjacent to the AAACTGTGGC and TCACGTG sites. The existence of additional promoter elements recognized by some repressive factors cannot be completely ruled out either.Figure 12.

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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