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Differential binding of the related transcription factors Pho4 and Cbf1 can tune the sensitivity of promoters to different levels of an induction signal.

Aow JS, Xue X, Run JQ, Lim GF, Goh WS, Clarke ND - Nucleic Acids Res. (2013)

Bottom Line: Chromatin immunoprecipitation and computational analyses of natural Pho4 target genes, along with the activities of the reporter constructs, indicates that genes differ in their sensitivity to intermediate induction signals in part because of differences in their affinity for Cbf1.The induction sensitivity of both natural Pho4 target genes and reporter genes was well explained only by a model that assumes a role for Cbf1 in remodeling chromatin.Our analyses highlight the importance of taking into account the activities of related transcription factors in explaining system-wide gene expression data.

View Article: PubMed Central - PubMed

Affiliation: Computational and Systems Biology, Genome Institute of Singapore, 60 Biopolis St., Singapore 138672, Singapore.

ABSTRACT
Transcription factors that belong to the same family typically have similar, but not identical, binding specificities. As such, they can be expected to compete differentially for binding to different variants of their binding sites. Pho4 is a yeast factor whose nuclear concentration is up-regulated in low phosphate, while the related factor, Cbf1, is constitutively expressed. We constructed 16 GFP-reporter genes containing all palindromic variants of the motif NNCACGTGNN, and determined their activities at a range of phosphate concentrations. Pho4 affinity did not explain expression data well except under fully induced conditions. However, reporter activity was quantitatively well explained under all conditions by a model in which Cbf1 itself has modest activating activity, and Pho4 and Cbf1 compete with one another. Chromatin immunoprecipitation and computational analyses of natural Pho4 target genes, along with the activities of the reporter constructs, indicates that genes differ in their sensitivity to intermediate induction signals in part because of differences in their affinity for Cbf1. The induction sensitivity of both natural Pho4 target genes and reporter genes was well explained only by a model that assumes a role for Cbf1 in remodeling chromatin. Our analyses highlight the importance of taking into account the activities of related transcription factors in explaining system-wide gene expression data.

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Related in: MedlinePlus

Heatmaps showing relative occupancy by Pho4 and Cbf1 at all 256 variants of the binding site NNCACGTGNN; rows indicate the 5′ dinucleotide, columns the 3′ dinucleotide. Occupancies for Pho4 and Cbf1 (left and center, respectively) are based on the relative free energies of binding for dinucleotides flanking the CACGTG core (8), with the assumed protein concentration adjusted so the mean occupancy among all sites is 0.5. The fold-difference between Pho4 and Cbf1 occupancies is shown in the panel on the right, with motifs having higher occupancy for Pho4 shown in blue and those with higher occupancy for Cbf1 shown in yellow. The sequence logos for Pho4 and Cbf1 are based on a consensus CACGTG core plus flanking base preferences that are derived from a linear regression of the experimentally measured free energy differences for the 16 dinucleotide free energy values (8). The result is a best-fit estimation for the relative contributions to binding of each of the four bases at each of the two positions.
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gkt210-F1: Heatmaps showing relative occupancy by Pho4 and Cbf1 at all 256 variants of the binding site NNCACGTGNN; rows indicate the 5′ dinucleotide, columns the 3′ dinucleotide. Occupancies for Pho4 and Cbf1 (left and center, respectively) are based on the relative free energies of binding for dinucleotides flanking the CACGTG core (8), with the assumed protein concentration adjusted so the mean occupancy among all sites is 0.5. The fold-difference between Pho4 and Cbf1 occupancies is shown in the panel on the right, with motifs having higher occupancy for Pho4 shown in blue and those with higher occupancy for Cbf1 shown in yellow. The sequence logos for Pho4 and Cbf1 are based on a consensus CACGTG core plus flanking base preferences that are derived from a linear regression of the experimentally measured free energy differences for the 16 dinucleotide free energy values (8). The result is a best-fit estimation for the relative contributions to binding of each of the four bases at each of the two positions.

Mentions: Pho4 and Cbf1 differ subtly in their binding specificities. These differences have been studied most thoroughly in vitro using an assay that rapidly traps protein–DNA complexes in a microfluidic device (8). This technique was used to measure the affinities of Pho4 and Cbf1 to each of the 16 possible dinucleotides flanking one side of the CACGTG core. Assuming that each half of the binding site contributes independently to affinity, these values allow the calculation of relative affinities for all 256 variants of the NNCACGTGNN motif (Figure 1). The richness and presumptive accuracy of these data offer a unique opportunity to ask how well in vitro binding affinity data can explain expression in vivo. We show that they do, in fact, do a remarkably good job explaining gene expression data for 16 reporter constructs over a range of phosphate concentrations. However, to explain the data at phosphate concentrations higher than ∼80 μM, we find that a modest, but significant, contribution to transcriptional activation is required from Cbf1; also required to explain the data is competition for binding between Pho4 and Cbf1. Finally, we show that differences in the affinity for Cbf1 can explain much of the differences among natural Pho4 target genes in terms of their sensitivity to induction at moderate phosphate concentrations.Figure 1.


Differential binding of the related transcription factors Pho4 and Cbf1 can tune the sensitivity of promoters to different levels of an induction signal.

Aow JS, Xue X, Run JQ, Lim GF, Goh WS, Clarke ND - Nucleic Acids Res. (2013)

Heatmaps showing relative occupancy by Pho4 and Cbf1 at all 256 variants of the binding site NNCACGTGNN; rows indicate the 5′ dinucleotide, columns the 3′ dinucleotide. Occupancies for Pho4 and Cbf1 (left and center, respectively) are based on the relative free energies of binding for dinucleotides flanking the CACGTG core (8), with the assumed protein concentration adjusted so the mean occupancy among all sites is 0.5. The fold-difference between Pho4 and Cbf1 occupancies is shown in the panel on the right, with motifs having higher occupancy for Pho4 shown in blue and those with higher occupancy for Cbf1 shown in yellow. The sequence logos for Pho4 and Cbf1 are based on a consensus CACGTG core plus flanking base preferences that are derived from a linear regression of the experimentally measured free energy differences for the 16 dinucleotide free energy values (8). The result is a best-fit estimation for the relative contributions to binding of each of the four bases at each of the two positions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643608&req=5

gkt210-F1: Heatmaps showing relative occupancy by Pho4 and Cbf1 at all 256 variants of the binding site NNCACGTGNN; rows indicate the 5′ dinucleotide, columns the 3′ dinucleotide. Occupancies for Pho4 and Cbf1 (left and center, respectively) are based on the relative free energies of binding for dinucleotides flanking the CACGTG core (8), with the assumed protein concentration adjusted so the mean occupancy among all sites is 0.5. The fold-difference between Pho4 and Cbf1 occupancies is shown in the panel on the right, with motifs having higher occupancy for Pho4 shown in blue and those with higher occupancy for Cbf1 shown in yellow. The sequence logos for Pho4 and Cbf1 are based on a consensus CACGTG core plus flanking base preferences that are derived from a linear regression of the experimentally measured free energy differences for the 16 dinucleotide free energy values (8). The result is a best-fit estimation for the relative contributions to binding of each of the four bases at each of the two positions.
Mentions: Pho4 and Cbf1 differ subtly in their binding specificities. These differences have been studied most thoroughly in vitro using an assay that rapidly traps protein–DNA complexes in a microfluidic device (8). This technique was used to measure the affinities of Pho4 and Cbf1 to each of the 16 possible dinucleotides flanking one side of the CACGTG core. Assuming that each half of the binding site contributes independently to affinity, these values allow the calculation of relative affinities for all 256 variants of the NNCACGTGNN motif (Figure 1). The richness and presumptive accuracy of these data offer a unique opportunity to ask how well in vitro binding affinity data can explain expression in vivo. We show that they do, in fact, do a remarkably good job explaining gene expression data for 16 reporter constructs over a range of phosphate concentrations. However, to explain the data at phosphate concentrations higher than ∼80 μM, we find that a modest, but significant, contribution to transcriptional activation is required from Cbf1; also required to explain the data is competition for binding between Pho4 and Cbf1. Finally, we show that differences in the affinity for Cbf1 can explain much of the differences among natural Pho4 target genes in terms of their sensitivity to induction at moderate phosphate concentrations.Figure 1.

Bottom Line: Chromatin immunoprecipitation and computational analyses of natural Pho4 target genes, along with the activities of the reporter constructs, indicates that genes differ in their sensitivity to intermediate induction signals in part because of differences in their affinity for Cbf1.The induction sensitivity of both natural Pho4 target genes and reporter genes was well explained only by a model that assumes a role for Cbf1 in remodeling chromatin.Our analyses highlight the importance of taking into account the activities of related transcription factors in explaining system-wide gene expression data.

View Article: PubMed Central - PubMed

Affiliation: Computational and Systems Biology, Genome Institute of Singapore, 60 Biopolis St., Singapore 138672, Singapore.

ABSTRACT
Transcription factors that belong to the same family typically have similar, but not identical, binding specificities. As such, they can be expected to compete differentially for binding to different variants of their binding sites. Pho4 is a yeast factor whose nuclear concentration is up-regulated in low phosphate, while the related factor, Cbf1, is constitutively expressed. We constructed 16 GFP-reporter genes containing all palindromic variants of the motif NNCACGTGNN, and determined their activities at a range of phosphate concentrations. Pho4 affinity did not explain expression data well except under fully induced conditions. However, reporter activity was quantitatively well explained under all conditions by a model in which Cbf1 itself has modest activating activity, and Pho4 and Cbf1 compete with one another. Chromatin immunoprecipitation and computational analyses of natural Pho4 target genes, along with the activities of the reporter constructs, indicates that genes differ in their sensitivity to intermediate induction signals in part because of differences in their affinity for Cbf1. The induction sensitivity of both natural Pho4 target genes and reporter genes was well explained only by a model that assumes a role for Cbf1 in remodeling chromatin. Our analyses highlight the importance of taking into account the activities of related transcription factors in explaining system-wide gene expression data.

Show MeSH
Related in: MedlinePlus