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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

Phosphate-concentration dependence of gene expression from promoters containing each of the 16 possible palindromic Pho4/Cbf1 motifs (NNCACGTGNN). The four graphs group sites according to the identity of the distal flanking base; for example, the top graph shows expression for the four promoters that contain variants of CNCACGTGNG. Within each graph, the color denotes the base immediately 5′ to the core hexamer; for example, the blue line in each graph indicates a variant of NCCACGTGGN. Also shown in this panel are the relative affinities of the 16 sites for Pho4 and Cbf1, according to the color scheme in Figure 1.
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gkt210-F2: Phosphate-concentration dependence of gene expression from promoters containing each of the 16 possible palindromic Pho4/Cbf1 motifs (NNCACGTGNN). The four graphs group sites according to the identity of the distal flanking base; for example, the top graph shows expression for the four promoters that contain variants of CNCACGTGNG. Within each graph, the color denotes the base immediately 5′ to the core hexamer; for example, the blue line in each graph indicates a variant of NCCACGTGGN. Also shown in this panel are the relative affinities of the 16 sites for Pho4 and Cbf1, according to the color scheme in Figure 1.

Mentions: The gene VTC4 is a direct regulatory target of Pho4 as defined by ChIP experiments and by a joint statistical analysis of sequence motifs and the effect of constitutive Pho4 expression on gene expression (Methods; Supplementary Figure S2) (5,22,23). We used the VTC4 promoter as a backbone for construction of 16 GFP fusions that differ at a single Pho4/Cbf1 binding site. The wild-type VTC4 promoter contains two perfect CACGTG motifs but, based on data from in vitro affinity measurements (8), one of these sites (caCACGTGaa) is predicted to bind Pho4 ∼30-fold less well than the other (cgCACGTGgc). Nevertheless, to avoid complications in interpretation, we knocked out the weaker site in all of our reporter constructs, using several substitutions in the core motif. The remaining site was replaced by all 16 possible palindromic variants of the sequence nnCACGTGnn. (The promoter sequences are provided as Supplementary Figure S3). The 16 reporter constructs were separately integrated by homologous recombination into a diploid yeast strain, replacing one of the VTC4 alleles with the gene fusion (Methods). Each of the 16 reporter strains was then assayed for GFP expression at eight different phosphate concentrations ranging from 0 to 10 mM (Figure 2).Figure 2.


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)

Phosphate-concentration dependence of gene expression from promoters containing each of the 16 possible palindromic Pho4/Cbf1 motifs (NNCACGTGNN). The four graphs group sites according to the identity of the distal flanking base; for example, the top graph shows expression for the four promoters that contain variants of CNCACGTGNG. Within each graph, the color denotes the base immediately 5′ to the core hexamer; for example, the blue line in each graph indicates a variant of NCCACGTGGN. Also shown in this panel are the relative affinities of the 16 sites for Pho4 and Cbf1, according to the color scheme in Figure 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt210-F2: Phosphate-concentration dependence of gene expression from promoters containing each of the 16 possible palindromic Pho4/Cbf1 motifs (NNCACGTGNN). The four graphs group sites according to the identity of the distal flanking base; for example, the top graph shows expression for the four promoters that contain variants of CNCACGTGNG. Within each graph, the color denotes the base immediately 5′ to the core hexamer; for example, the blue line in each graph indicates a variant of NCCACGTGGN. Also shown in this panel are the relative affinities of the 16 sites for Pho4 and Cbf1, according to the color scheme in Figure 1.
Mentions: The gene VTC4 is a direct regulatory target of Pho4 as defined by ChIP experiments and by a joint statistical analysis of sequence motifs and the effect of constitutive Pho4 expression on gene expression (Methods; Supplementary Figure S2) (5,22,23). We used the VTC4 promoter as a backbone for construction of 16 GFP fusions that differ at a single Pho4/Cbf1 binding site. The wild-type VTC4 promoter contains two perfect CACGTG motifs but, based on data from in vitro affinity measurements (8), one of these sites (caCACGTGaa) is predicted to bind Pho4 ∼30-fold less well than the other (cgCACGTGgc). Nevertheless, to avoid complications in interpretation, we knocked out the weaker site in all of our reporter constructs, using several substitutions in the core motif. The remaining site was replaced by all 16 possible palindromic variants of the sequence nnCACGTGnn. (The promoter sequences are provided as Supplementary Figure S3). The 16 reporter constructs were separately integrated by homologous recombination into a diploid yeast strain, replacing one of the VTC4 alleles with the gene fusion (Methods). Each of the 16 reporter strains was then assayed for GFP expression at eight different phosphate concentrations ranging from 0 to 10 mM (Figure 2).Figure 2.

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