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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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Replacement of Met4 bZIP by Cbf1 bHLH. (A) Model scheme for association to MET promoters of Met4 and a Met4-Cbf1 chimera containing residues 1–590 of Met4 fused to residues 210–351 of Cbf1. ‘Act’ represents Met4 activation domain and ‘Int’ represents Met4 interaction domain with Met31/32. The three parallel bars represent known protein–protein and protein–DNA interactions. (B) Transcription analysis in WT and cbf1Δ cells expressing the Met4-Cbf1 derivative described in (A) from MET4 endogenous promoter. Cells were cultivated, exposed to cadmium, and RNA levels were quantified by RT real-time PCR as in Figure 1A. For comparison, the maximum level for each gene was set to 100. Error bars indicate average deviations from two independent experiments.
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Figure 7: Replacement of Met4 bZIP by Cbf1 bHLH. (A) Model scheme for association to MET promoters of Met4 and a Met4-Cbf1 chimera containing residues 1–590 of Met4 fused to residues 210–351 of Cbf1. ‘Act’ represents Met4 activation domain and ‘Int’ represents Met4 interaction domain with Met31/32. The three parallel bars represent known protein–protein and protein–DNA interactions. (B) Transcription analysis in WT and cbf1Δ cells expressing the Met4-Cbf1 derivative described in (A) from MET4 endogenous promoter. Cells were cultivated, exposed to cadmium, and RNA levels were quantified by RT real-time PCR as in Figure 1A. For comparison, the maximum level for each gene was set to 100. Error bars indicate average deviations from two independent experiments.

Mentions: To gain more insight into the mechanism of association of Cbf1 with PDC6 in vivo, we used a strain expressing a chimera in which the carboxy-terminal bZIP of Met4 was replaced by the bHLH of Cbf1 (Figure 7A). A previous study showed that expression of this chimera in place of the full length Met4 in a cbf1Δ strain allowed activation of the sulfur amino acid biosynthetic genes and, as a result, supported cell growth on a medium containing sulfate as unique sulfur source (7). We asked whether this chimera would also support activation of PDC6 transcription in response to cadmium. The results showed that PDC6 transcription was diminished by several fold in the cbf1Δ strain expressing the chimera compared to the WT strain expressing the full length Met4 (Figure 7B). For comparison, MET3 was induced at similar levels in both strains, confirming that the chimera was able to activate MET genes in response to cadmium as efficiently as the WT Met4. Two conclusions could be drawn from these results: first, in vivo the TCACGTT sequence present in PDC6 does not bind Cbf1 bHLH as efficiently as the TCACGTG sequence present in MET genes; secondly, assembly of the Met4 activation complex at PDC6 involves a mechanism that is different from the mechanism involved at MET genes.Figure 7.


Transcriptional plasticity through differential assembly of a multiprotein activation complex.

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

Replacement of Met4 bZIP by Cbf1 bHLH. (A) Model scheme for association to MET promoters of Met4 and a Met4-Cbf1 chimera containing residues 1–590 of Met4 fused to residues 210–351 of Cbf1. ‘Act’ represents Met4 activation domain and ‘Int’ represents Met4 interaction domain with Met31/32. The three parallel bars represent known protein–protein and protein–DNA interactions. (B) Transcription analysis in WT and cbf1Δ cells expressing the Met4-Cbf1 derivative described in (A) from MET4 endogenous promoter. Cells were cultivated, exposed to cadmium, and RNA levels were quantified by RT real-time PCR as in Figure 1A. For comparison, the maximum level for each gene was set to 100. Error bars indicate average deviations from two independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2926612&req=5

Figure 7: Replacement of Met4 bZIP by Cbf1 bHLH. (A) Model scheme for association to MET promoters of Met4 and a Met4-Cbf1 chimera containing residues 1–590 of Met4 fused to residues 210–351 of Cbf1. ‘Act’ represents Met4 activation domain and ‘Int’ represents Met4 interaction domain with Met31/32. The three parallel bars represent known protein–protein and protein–DNA interactions. (B) Transcription analysis in WT and cbf1Δ cells expressing the Met4-Cbf1 derivative described in (A) from MET4 endogenous promoter. Cells were cultivated, exposed to cadmium, and RNA levels were quantified by RT real-time PCR as in Figure 1A. For comparison, the maximum level for each gene was set to 100. Error bars indicate average deviations from two independent experiments.
Mentions: To gain more insight into the mechanism of association of Cbf1 with PDC6 in vivo, we used a strain expressing a chimera in which the carboxy-terminal bZIP of Met4 was replaced by the bHLH of Cbf1 (Figure 7A). A previous study showed that expression of this chimera in place of the full length Met4 in a cbf1Δ strain allowed activation of the sulfur amino acid biosynthetic genes and, as a result, supported cell growth on a medium containing sulfate as unique sulfur source (7). We asked whether this chimera would also support activation of PDC6 transcription in response to cadmium. The results showed that PDC6 transcription was diminished by several fold in the cbf1Δ strain expressing the chimera compared to the WT strain expressing the full length Met4 (Figure 7B). For comparison, MET3 was induced at similar levels in both strains, confirming that the chimera was able to activate MET genes in response to cadmium as efficiently as the WT Met4. Two conclusions could be drawn from these results: first, in vivo the TCACGTT sequence present in PDC6 does not bind Cbf1 bHLH as efficiently as the TCACGTG sequence present in MET genes; secondly, assembly of the Met4 activation complex at PDC6 involves a mechanism that is different from the mechanism involved at MET genes.Figure 7.

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