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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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Analysis of PDC6 transcription under various conditions. (A) PDC6 transcription in response to sulfur limitation. WT cells were grown to early log phase into B-medium supplemented with 0.5 mM methionine, collected by filtration, and transferred into B-medium with no sulfur source at time zero. Samples were taken at the time points indicated and total RNA was extracted and quantified by RT-real time PCR. Values were normalized to 25S ribosomal RNA and represent the average of two independent experiments. Error bars indicate average deviations. (B) PDC6 transcription in response to Met4 hyperactivation. met4::GAL1-MET4 Δmet30 (CC932-8B) cells were grown in YP-raffinose medium to early log phase and supplemented with galactose at the zero time point. (C) PDC6 and MET3 transcription upon exposure to various heavy metals. Wild-type cells were grown to early-log phase in YPD medium and exposed to 0.5 mM CdCl2, 1 mM CuCl2, 1 mM ZnCl2, 2 mM CoCl2, 50 µM HgCl2, 0.5 mM MnCl2 or 30 µM AgNO3 for 90 min.
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Figure 2: Analysis of PDC6 transcription under various conditions. (A) PDC6 transcription in response to sulfur limitation. WT cells were grown to early log phase into B-medium supplemented with 0.5 mM methionine, collected by filtration, and transferred into B-medium with no sulfur source at time zero. Samples were taken at the time points indicated and total RNA was extracted and quantified by RT-real time PCR. Values were normalized to 25S ribosomal RNA and represent the average of two independent experiments. Error bars indicate average deviations. (B) PDC6 transcription in response to Met4 hyperactivation. met4::GAL1-MET4 Δmet30 (CC932-8B) cells were grown in YP-raffinose medium to early log phase and supplemented with galactose at the zero time point. (C) PDC6 and MET3 transcription upon exposure to various heavy metals. Wild-type cells were grown to early-log phase in YPD medium and exposed to 0.5 mM CdCl2, 1 mM CuCl2, 1 mM ZnCl2, 2 mM CoCl2, 50 µM HgCl2, 0.5 mM MnCl2 or 30 µM AgNO3 for 90 min.

Mentions: To further establish the role of Met4 in PDC6 regulation, we analyzed PDC6 transcription in sulfur limitation (Figure 2). The results showed that PDC6 transcription was strongly activated when cells were transferred to minimal medium depleted in sulfur (Figure 2A). We also monitored PDC6 transcription in a strain expressing Met4 from the inducible GAL1 promoter and lacking Met30, the negative regulator of Met4. As already reported, expression of Met4 in a met30Δ background allows induction of MET genes in rich medium (25). We found that Met4 expression in the absence of Met30 was also sufficient to induce PDC6 transcription in rich media (Figure 2B). Altogether, these results demonstrate that PDC6 activation is not limited to cadmium exposure but occurs in other conditions that induce MET genes. By contrast, no activation was observed in the presence of metals that do not induce MET genes, such as copper, cobalt, manganese, mercury, silver or zinc (Figure 2C).Figure 2.


Transcriptional plasticity through differential assembly of a multiprotein activation complex.

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

Analysis of PDC6 transcription under various conditions. (A) PDC6 transcription in response to sulfur limitation. WT cells were grown to early log phase into B-medium supplemented with 0.5 mM methionine, collected by filtration, and transferred into B-medium with no sulfur source at time zero. Samples were taken at the time points indicated and total RNA was extracted and quantified by RT-real time PCR. Values were normalized to 25S ribosomal RNA and represent the average of two independent experiments. Error bars indicate average deviations. (B) PDC6 transcription in response to Met4 hyperactivation. met4::GAL1-MET4 Δmet30 (CC932-8B) cells were grown in YP-raffinose medium to early log phase and supplemented with galactose at the zero time point. (C) PDC6 and MET3 transcription upon exposure to various heavy metals. Wild-type cells were grown to early-log phase in YPD medium and exposed to 0.5 mM CdCl2, 1 mM CuCl2, 1 mM ZnCl2, 2 mM CoCl2, 50 µM HgCl2, 0.5 mM MnCl2 or 30 µM AgNO3 for 90 min.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Analysis of PDC6 transcription under various conditions. (A) PDC6 transcription in response to sulfur limitation. WT cells were grown to early log phase into B-medium supplemented with 0.5 mM methionine, collected by filtration, and transferred into B-medium with no sulfur source at time zero. Samples were taken at the time points indicated and total RNA was extracted and quantified by RT-real time PCR. Values were normalized to 25S ribosomal RNA and represent the average of two independent experiments. Error bars indicate average deviations. (B) PDC6 transcription in response to Met4 hyperactivation. met4::GAL1-MET4 Δmet30 (CC932-8B) cells were grown in YP-raffinose medium to early log phase and supplemented with galactose at the zero time point. (C) PDC6 and MET3 transcription upon exposure to various heavy metals. Wild-type cells were grown to early-log phase in YPD medium and exposed to 0.5 mM CdCl2, 1 mM CuCl2, 1 mM ZnCl2, 2 mM CoCl2, 50 µM HgCl2, 0.5 mM MnCl2 or 30 µM AgNO3 for 90 min.
Mentions: To further establish the role of Met4 in PDC6 regulation, we analyzed PDC6 transcription in sulfur limitation (Figure 2). The results showed that PDC6 transcription was strongly activated when cells were transferred to minimal medium depleted in sulfur (Figure 2A). We also monitored PDC6 transcription in a strain expressing Met4 from the inducible GAL1 promoter and lacking Met30, the negative regulator of Met4. As already reported, expression of Met4 in a met30Δ background allows induction of MET genes in rich medium (25). We found that Met4 expression in the absence of Met30 was also sufficient to induce PDC6 transcription in rich media (Figure 2B). Altogether, these results demonstrate that PDC6 activation is not limited to cadmium exposure but occurs in other conditions that induce MET genes. By contrast, no activation was observed in the presence of metals that do not induce MET genes, such as copper, cobalt, manganese, mercury, silver or zinc (Figure 2C).Figure 2.

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.

Show MeSH
Related in: MedlinePlus