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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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Transcriptional regulation of PDC6 in S. cerevisiae relatives. (A) Sequence alignment. PDC6 orthologs in S. cerevisiae (YGR087C), S. paradoxus (spar6-g11.1), S. bayanus (sbayc642-g17.1) and C. glabrata (CAGL02937) were aligned with Clustal W. Fully conserved positions are marked by an asterisk. Positions conserved among S. cerevisiae and at least one other species are marked in blue. Positions conserved among several species except S. cerevisiae are marked in green. Positions occupied by different nucleotides are marked in orange. Binding sites for Met31/32 and Cbf1 are marked by red boxes. (B) Saccharomyces cerevisiae (W303-1A), S. paradoxus (CLIB228), S. bayanus (CLIB181) and C. glabrata (CLIB298) strains were grown to early log phase in YPD medium and exposed to 0.5 mM Cd2+. Total RNA was extracted from samples taken just before (−) or 120 min (+) after the addition of cadmium. RNA levels for PDC6 and MET3 orthologs were quantified by RT real-time PCR and normalized to 25S ribosomal RNA. Values represent the average of three independent experiments and error bars indicate average deviations.
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Figure 13: Transcriptional regulation of PDC6 in S. cerevisiae relatives. (A) Sequence alignment. PDC6 orthologs in S. cerevisiae (YGR087C), S. paradoxus (spar6-g11.1), S. bayanus (sbayc642-g17.1) and C. glabrata (CAGL02937) were aligned with Clustal W. Fully conserved positions are marked by an asterisk. Positions conserved among S. cerevisiae and at least one other species are marked in blue. Positions conserved among several species except S. cerevisiae are marked in green. Positions occupied by different nucleotides are marked in orange. Binding sites for Met31/32 and Cbf1 are marked by red boxes. (B) Saccharomyces cerevisiae (W303-1A), S. paradoxus (CLIB228), S. bayanus (CLIB181) and C. glabrata (CLIB298) strains were grown to early log phase in YPD medium and exposed to 0.5 mM Cd2+. Total RNA was extracted from samples taken just before (−) or 120 min (+) after the addition of cadmium. RNA levels for PDC6 and MET3 orthologs were quantified by RT real-time PCR and normalized to 25S ribosomal RNA. Values represent the average of three independent experiments and error bars indicate average deviations.

Mentions: Syntenic orthologs of PDC6 are present in S. paradoxus, S. bayanus and C. glabrata (28,29), three species that diverged from S. cerevisiae after the whole-genome duplication (WGD). Alignment of the promoter regions showed that Met31/32 and Cbf1 binding sites were conserved in S. paradoxus, the closest relative of S. cerevisiae, but not in S. bayanus and C. glabrata, which are more distantly related (Figure 13A). Indeed, S. paradoxus PDC6 possesses the TCACGTT sequence and the first CTGTGGC site, whereas S. bayanus and C. glabrata possess only divergent counterparts. Pattern search using the fuzznuc program (available at http://mobyle.pasteur.fr) confirmed that neither the TCACGTT nor the CTGTGGC sequences were present upstream of S. bayanus and C. glabrata PDC6 orthologs (data not shown). Transcriptional analysis was carried out in S. paradoxus, S. bayanus and C. glabrata strains exposed to cadmium (Figure 13B). The results showed that PDC6 was significantly induced in response to cadmium exposure in S. paradoxus, but almost not in S. bayanus and C. glabrata. Interestingly, the level of PDC6 induction was 10-fold lower in S. paradoxus compared to S. cerevisiae, which is in good agreement with the fact that S. paradoxus PDC6 does not contain the second CTGTGGC site.Figure 13.


Transcriptional plasticity through differential assembly of a multiprotein activation complex.

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

Transcriptional regulation of PDC6 in S. cerevisiae relatives. (A) Sequence alignment. PDC6 orthologs in S. cerevisiae (YGR087C), S. paradoxus (spar6-g11.1), S. bayanus (sbayc642-g17.1) and C. glabrata (CAGL02937) were aligned with Clustal W. Fully conserved positions are marked by an asterisk. Positions conserved among S. cerevisiae and at least one other species are marked in blue. Positions conserved among several species except S. cerevisiae are marked in green. Positions occupied by different nucleotides are marked in orange. Binding sites for Met31/32 and Cbf1 are marked by red boxes. (B) Saccharomyces cerevisiae (W303-1A), S. paradoxus (CLIB228), S. bayanus (CLIB181) and C. glabrata (CLIB298) strains were grown to early log phase in YPD medium and exposed to 0.5 mM Cd2+. Total RNA was extracted from samples taken just before (−) or 120 min (+) after the addition of cadmium. RNA levels for PDC6 and MET3 orthologs were quantified by RT real-time PCR and normalized to 25S ribosomal RNA. Values represent the average of three independent experiments and error bars indicate average deviations.
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Figure 13: Transcriptional regulation of PDC6 in S. cerevisiae relatives. (A) Sequence alignment. PDC6 orthologs in S. cerevisiae (YGR087C), S. paradoxus (spar6-g11.1), S. bayanus (sbayc642-g17.1) and C. glabrata (CAGL02937) were aligned with Clustal W. Fully conserved positions are marked by an asterisk. Positions conserved among S. cerevisiae and at least one other species are marked in blue. Positions conserved among several species except S. cerevisiae are marked in green. Positions occupied by different nucleotides are marked in orange. Binding sites for Met31/32 and Cbf1 are marked by red boxes. (B) Saccharomyces cerevisiae (W303-1A), S. paradoxus (CLIB228), S. bayanus (CLIB181) and C. glabrata (CLIB298) strains were grown to early log phase in YPD medium and exposed to 0.5 mM Cd2+. Total RNA was extracted from samples taken just before (−) or 120 min (+) after the addition of cadmium. RNA levels for PDC6 and MET3 orthologs were quantified by RT real-time PCR and normalized to 25S ribosomal RNA. Values represent the average of three independent experiments and error bars indicate average deviations.
Mentions: Syntenic orthologs of PDC6 are present in S. paradoxus, S. bayanus and C. glabrata (28,29), three species that diverged from S. cerevisiae after the whole-genome duplication (WGD). Alignment of the promoter regions showed that Met31/32 and Cbf1 binding sites were conserved in S. paradoxus, the closest relative of S. cerevisiae, but not in S. bayanus and C. glabrata, which are more distantly related (Figure 13A). Indeed, S. paradoxus PDC6 possesses the TCACGTT sequence and the first CTGTGGC site, whereas S. bayanus and C. glabrata possess only divergent counterparts. Pattern search using the fuzznuc program (available at http://mobyle.pasteur.fr) confirmed that neither the TCACGTT nor the CTGTGGC sequences were present upstream of S. bayanus and C. glabrata PDC6 orthologs (data not shown). Transcriptional analysis was carried out in S. paradoxus, S. bayanus and C. glabrata strains exposed to cadmium (Figure 13B). The results showed that PDC6 was significantly induced in response to cadmium exposure in S. paradoxus, but almost not in S. bayanus and C. glabrata. Interestingly, the level of PDC6 induction was 10-fold lower in S. paradoxus compared to S. cerevisiae, which is in good agreement with the fact that S. paradoxus PDC6 does not contain the second CTGTGGC site.Figure 13.

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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