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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 association of Cbf1 with PDC6 promoter. (A) Schematic of PDC6 showing the DNA fragments used in the gel shift assay. ‘M’ boxes represent the CTGTGGC motifs. ‘T’ boxes represent the TATA elements. (B) Gel shift assays with large DNA fragments. The 32P-labeled DNA fragments indicated were incubated with 0, 50 and 200 ng of a recombinant derivative of Cbf1 containing the bHLH domain fused to a polyhistidine tag (hisCbf1BD). Fragments were resolved by 5% polyacrylamide gel electrophoresis and visualized by PhosphorImager analysis. The MET16 fragment covers positions from –126 to –270 and contains TCACGTG. (C) Gel shift assays with 40 bp DNA fragments. The 32P-labeled DNA fragments indicated were incubated with 0, 200, 100, 50 and 25 ng of hisCbf1BD (only 0 and 200 ng in the case of mut PDC6). The PDC6 fragment covers positions from –350 to –389 and contains the TCACGTT sequence. The MET16 fragment covers positions from –155 to –194 and contains the TCACGTG sequence. mut PDC6 contains TCCAGTT instead of TCACGTT.
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Figure 5: In vitro association of Cbf1 with PDC6 promoter. (A) Schematic of PDC6 showing the DNA fragments used in the gel shift assay. ‘M’ boxes represent the CTGTGGC motifs. ‘T’ boxes represent the TATA elements. (B) Gel shift assays with large DNA fragments. The 32P-labeled DNA fragments indicated were incubated with 0, 50 and 200 ng of a recombinant derivative of Cbf1 containing the bHLH domain fused to a polyhistidine tag (hisCbf1BD). Fragments were resolved by 5% polyacrylamide gel electrophoresis and visualized by PhosphorImager analysis. The MET16 fragment covers positions from –126 to –270 and contains TCACGTG. (C) Gel shift assays with 40 bp DNA fragments. The 32P-labeled DNA fragments indicated were incubated with 0, 200, 100, 50 and 25 ng of hisCbf1BD (only 0 and 200 ng in the case of mut PDC6). The PDC6 fragment covers positions from –350 to –389 and contains the TCACGTT sequence. The MET16 fragment covers positions from –155 to –194 and contains the TCACGTG sequence. mut PDC6 contains TCCAGTT instead of TCACGTT.

Mentions: The absence of TCACGTG sequence upstream of PDC6 raised the question whether Cbf1 could bind PDC6 by itself or was recruited through interactions with Met4 and/or other factors involved in PDC6 activation. To address this point, we first performed gel shift assays using a recombinant derivative of Cbf1 containing the bHLH domain (residues 210–351) fused to a polyhistidine tag (hisCbf1BD; Figure 5). This derivative was incubated with four overlapping DNA fragments covering PDC6 from positions −96 to −620 (Figure 5A), and with a fragment of MET16 containing the TCACGTG sequence as a control. The results in Figure 5B showed that hisCbf1BD did associate with PDC6 by its own. The fact that fragments II and III of PDC6 produced band shifts of similar intensities encouraged us to examine more carefully the overlapping region, which led us to notice the sequence TCACGTT between positions −368 and −374. To determine whether Cbf1 was able to bind this sequence, we performed additional gel shift assays with two short 40 base-pair DNA fragments containing either the TCACGTT sequence of PDC6 or a mutated version. For comparison, a MET16 fragment containing the bona fide TCACGTG site was included. The results did show hisCbf1BD association with the WT PDC6, but not with the mutant (Figure 5C). Moreover, hisCbf1BD affinity for PDC6 was comparable to its affinity for MET16 (Figure 5C).Figure 5.


Transcriptional plasticity through differential assembly of a multiprotein activation complex.

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

In vitro association of Cbf1 with PDC6 promoter. (A) Schematic of PDC6 showing the DNA fragments used in the gel shift assay. ‘M’ boxes represent the CTGTGGC motifs. ‘T’ boxes represent the TATA elements. (B) Gel shift assays with large DNA fragments. The 32P-labeled DNA fragments indicated were incubated with 0, 50 and 200 ng of a recombinant derivative of Cbf1 containing the bHLH domain fused to a polyhistidine tag (hisCbf1BD). Fragments were resolved by 5% polyacrylamide gel electrophoresis and visualized by PhosphorImager analysis. The MET16 fragment covers positions from –126 to –270 and contains TCACGTG. (C) Gel shift assays with 40 bp DNA fragments. The 32P-labeled DNA fragments indicated were incubated with 0, 200, 100, 50 and 25 ng of hisCbf1BD (only 0 and 200 ng in the case of mut PDC6). The PDC6 fragment covers positions from –350 to –389 and contains the TCACGTT sequence. The MET16 fragment covers positions from –155 to –194 and contains the TCACGTG sequence. mut PDC6 contains TCCAGTT instead of TCACGTT.
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Figure 5: In vitro association of Cbf1 with PDC6 promoter. (A) Schematic of PDC6 showing the DNA fragments used in the gel shift assay. ‘M’ boxes represent the CTGTGGC motifs. ‘T’ boxes represent the TATA elements. (B) Gel shift assays with large DNA fragments. The 32P-labeled DNA fragments indicated were incubated with 0, 50 and 200 ng of a recombinant derivative of Cbf1 containing the bHLH domain fused to a polyhistidine tag (hisCbf1BD). Fragments were resolved by 5% polyacrylamide gel electrophoresis and visualized by PhosphorImager analysis. The MET16 fragment covers positions from –126 to –270 and contains TCACGTG. (C) Gel shift assays with 40 bp DNA fragments. The 32P-labeled DNA fragments indicated were incubated with 0, 200, 100, 50 and 25 ng of hisCbf1BD (only 0 and 200 ng in the case of mut PDC6). The PDC6 fragment covers positions from –350 to –389 and contains the TCACGTT sequence. The MET16 fragment covers positions from –155 to –194 and contains the TCACGTG sequence. mut PDC6 contains TCCAGTT instead of TCACGTT.
Mentions: The absence of TCACGTG sequence upstream of PDC6 raised the question whether Cbf1 could bind PDC6 by itself or was recruited through interactions with Met4 and/or other factors involved in PDC6 activation. To address this point, we first performed gel shift assays using a recombinant derivative of Cbf1 containing the bHLH domain (residues 210–351) fused to a polyhistidine tag (hisCbf1BD; Figure 5). This derivative was incubated with four overlapping DNA fragments covering PDC6 from positions −96 to −620 (Figure 5A), and with a fragment of MET16 containing the TCACGTG sequence as a control. The results in Figure 5B showed that hisCbf1BD did associate with PDC6 by its own. The fact that fragments II and III of PDC6 produced band shifts of similar intensities encouraged us to examine more carefully the overlapping region, which led us to notice the sequence TCACGTT between positions −368 and −374. To determine whether Cbf1 was able to bind this sequence, we performed additional gel shift assays with two short 40 base-pair DNA fragments containing either the TCACGTT sequence of PDC6 or a mutated version. For comparison, a MET16 fragment containing the bona fide TCACGTG site was included. The results did show hisCbf1BD association with the WT PDC6, but not with the mutant (Figure 5C). Moreover, hisCbf1BD affinity for PDC6 was comparable to its affinity for MET16 (Figure 5C).Figure 5.

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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