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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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Sensitivity of natural promoters to intermediate phosphate concentrations is related to Cbf1 affinity. (A) Normalized gene expression levels at different phosphate concentrations. Expression was determined by microarray analysis at 0 μM, 10 μM, 100 μM, 1 mM and 10 mM [Pi], and was normalized between a value of 1 (average for 1 and 10 mM, as these were similarly low) and 100 (0 μM). As indicated in the legend, the colored circles represent the expression level in 10 μM phosphate, while triangles shows expression in 100 μM phosphate. The line midway between the two is the log-average of these values; we use this value as a measure of the sensitivity of a gene to intermediate phosphate. Blue and red genes are the same as those classified by Kim and O’Shea (25) as having low and high thresholds for induction, and are colored in the same way. [Some gene names are different, reflecting changes in the nomenclature used in the Saccharomyces Genome Database (SGD) (20)]. VTC4 (gray) has been included in this analysis, as its promoter was the basis for the reporter experiments described here. (B) ChIP of Pho4 (closed squares) and Cbf1 (open squares) in high and low phosphate. Values and error bars are the average and standard error of enrichment values for two sets of experiments using Myc-tagged and HA-tagged proteins (Methods). Colors are as in panel A. (C) Correlation of induction sensitivity with the change in transcription factor binding. The fold-difference in ChIP enrichment values are shown for both for Pho4 (closed squares; 0/10 mM phosphate) and Cbf1 (open squares; 10/0 mM). Note that the Pho4 and Cbf1 ChIP enrichment ratios can be compared for a given gene because the two values share the same y-axis values. (D) Correlation between the experimentally determined phosphate sensitivity of expression and two in silico predictions. One (X’s) is the model used for the reporter constructs in Figure 4, which takes into account competition between Cbf1 and Pho4 and ascribes a low level of transcriptional activity to bound Cbf1. The second model (circles) includes the same terms, but also models chromatin changes as reported for the reporter gene sensitivity values in Figure 5. The dashed and solid lines indicate fits to these predictions, respectively. The parameters used, and an analysis of the sensitivity to those parameters, are shown in Supplementary Figure S11. (E) Correlation coefficients for the chromatin remodeling model shown in panel D, along with the effect of leaving out certain terms. The error bars for the top histogram bar indicates the 95% confidence interval for the correlation as defined by bootstrap resampling. Leaving out any term that is required for modeling the cooperative effects of Cbf1 chromatin remodeling results in an insignificant correlation. The terms are labeled a–d: (a) are there two states for the promoter, ‘open’ and ‘closed’?; (b) is there tighter binding of Pho4 and Cbf1 to the ‘open’ state?; (c) does Cbf1 binding shift the equilibrium toward the open state?; (d) is Cbf1 included in the model at all?
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gkt210-F6: Sensitivity of natural promoters to intermediate phosphate concentrations is related to Cbf1 affinity. (A) Normalized gene expression levels at different phosphate concentrations. Expression was determined by microarray analysis at 0 μM, 10 μM, 100 μM, 1 mM and 10 mM [Pi], and was normalized between a value of 1 (average for 1 and 10 mM, as these were similarly low) and 100 (0 μM). As indicated in the legend, the colored circles represent the expression level in 10 μM phosphate, while triangles shows expression in 100 μM phosphate. The line midway between the two is the log-average of these values; we use this value as a measure of the sensitivity of a gene to intermediate phosphate. Blue and red genes are the same as those classified by Kim and O’Shea (25) as having low and high thresholds for induction, and are colored in the same way. [Some gene names are different, reflecting changes in the nomenclature used in the Saccharomyces Genome Database (SGD) (20)]. VTC4 (gray) has been included in this analysis, as its promoter was the basis for the reporter experiments described here. (B) ChIP of Pho4 (closed squares) and Cbf1 (open squares) in high and low phosphate. Values and error bars are the average and standard error of enrichment values for two sets of experiments using Myc-tagged and HA-tagged proteins (Methods). Colors are as in panel A. (C) Correlation of induction sensitivity with the change in transcription factor binding. The fold-difference in ChIP enrichment values are shown for both for Pho4 (closed squares; 0/10 mM phosphate) and Cbf1 (open squares; 10/0 mM). Note that the Pho4 and Cbf1 ChIP enrichment ratios can be compared for a given gene because the two values share the same y-axis values. (D) Correlation between the experimentally determined phosphate sensitivity of expression and two in silico predictions. One (X’s) is the model used for the reporter constructs in Figure 4, which takes into account competition between Cbf1 and Pho4 and ascribes a low level of transcriptional activity to bound Cbf1. The second model (circles) includes the same terms, but also models chromatin changes as reported for the reporter gene sensitivity values in Figure 5. The dashed and solid lines indicate fits to these predictions, respectively. The parameters used, and an analysis of the sensitivity to those parameters, are shown in Supplementary Figure S11. (E) Correlation coefficients for the chromatin remodeling model shown in panel D, along with the effect of leaving out certain terms. The error bars for the top histogram bar indicates the 95% confidence interval for the correlation as defined by bootstrap resampling. Leaving out any term that is required for modeling the cooperative effects of Cbf1 chromatin remodeling results in an insignificant correlation. The terms are labeled a–d: (a) are there two states for the promoter, ‘open’ and ‘closed’?; (b) is there tighter binding of Pho4 and Cbf1 to the ‘open’ state?; (c) does Cbf1 binding shift the equilibrium toward the open state?; (d) is Cbf1 included in the model at all?

Mentions: It is well established that there are differences in the sensitivity to induction among Pho4-target genes. Lam et al. (24), using GFP fusions for seven Pho4 target genes, classified the genes into two groups on this basis. We validated this observation using a microarray gene expression data obtained at five phosphate concentrations (Figure 6A). Although the microarray and GFP reporter assays are measuring rather different things, the baseline expression in high phosphate for the seven genes that were assayed in common are well correlated (R = 0.95), as is the extent of their induction upon shifting to 0 mM phosphate (R = 0.95) (Supplementary Figure S9). We prefer to use the term expression sensitivity rather than threshold because induction is not an all or none phenomenon, but the concepts are related, and our data can readily be interpreted to produce the same two groups defined by GFP assays (Figure 6A). In addition to the seven genes studied previously, we included in our analysis VTC4, a Pho4 target gene that shows an induction sensitivity intermediate between the two classes defined earlier (Figure 6A).Figure 6.


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)

Sensitivity of natural promoters to intermediate phosphate concentrations is related to Cbf1 affinity. (A) Normalized gene expression levels at different phosphate concentrations. Expression was determined by microarray analysis at 0 μM, 10 μM, 100 μM, 1 mM and 10 mM [Pi], and was normalized between a value of 1 (average for 1 and 10 mM, as these were similarly low) and 100 (0 μM). As indicated in the legend, the colored circles represent the expression level in 10 μM phosphate, while triangles shows expression in 100 μM phosphate. The line midway between the two is the log-average of these values; we use this value as a measure of the sensitivity of a gene to intermediate phosphate. Blue and red genes are the same as those classified by Kim and O’Shea (25) as having low and high thresholds for induction, and are colored in the same way. [Some gene names are different, reflecting changes in the nomenclature used in the Saccharomyces Genome Database (SGD) (20)]. VTC4 (gray) has been included in this analysis, as its promoter was the basis for the reporter experiments described here. (B) ChIP of Pho4 (closed squares) and Cbf1 (open squares) in high and low phosphate. Values and error bars are the average and standard error of enrichment values for two sets of experiments using Myc-tagged and HA-tagged proteins (Methods). Colors are as in panel A. (C) Correlation of induction sensitivity with the change in transcription factor binding. The fold-difference in ChIP enrichment values are shown for both for Pho4 (closed squares; 0/10 mM phosphate) and Cbf1 (open squares; 10/0 mM). Note that the Pho4 and Cbf1 ChIP enrichment ratios can be compared for a given gene because the two values share the same y-axis values. (D) Correlation between the experimentally determined phosphate sensitivity of expression and two in silico predictions. One (X’s) is the model used for the reporter constructs in Figure 4, which takes into account competition between Cbf1 and Pho4 and ascribes a low level of transcriptional activity to bound Cbf1. The second model (circles) includes the same terms, but also models chromatin changes as reported for the reporter gene sensitivity values in Figure 5. The dashed and solid lines indicate fits to these predictions, respectively. The parameters used, and an analysis of the sensitivity to those parameters, are shown in Supplementary Figure S11. (E) Correlation coefficients for the chromatin remodeling model shown in panel D, along with the effect of leaving out certain terms. The error bars for the top histogram bar indicates the 95% confidence interval for the correlation as defined by bootstrap resampling. Leaving out any term that is required for modeling the cooperative effects of Cbf1 chromatin remodeling results in an insignificant correlation. The terms are labeled a–d: (a) are there two states for the promoter, ‘open’ and ‘closed’?; (b) is there tighter binding of Pho4 and Cbf1 to the ‘open’ state?; (c) does Cbf1 binding shift the equilibrium toward the open state?; (d) is Cbf1 included in the model at all?
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Related In: Results  -  Collection

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gkt210-F6: Sensitivity of natural promoters to intermediate phosphate concentrations is related to Cbf1 affinity. (A) Normalized gene expression levels at different phosphate concentrations. Expression was determined by microarray analysis at 0 μM, 10 μM, 100 μM, 1 mM and 10 mM [Pi], and was normalized between a value of 1 (average for 1 and 10 mM, as these were similarly low) and 100 (0 μM). As indicated in the legend, the colored circles represent the expression level in 10 μM phosphate, while triangles shows expression in 100 μM phosphate. The line midway between the two is the log-average of these values; we use this value as a measure of the sensitivity of a gene to intermediate phosphate. Blue and red genes are the same as those classified by Kim and O’Shea (25) as having low and high thresholds for induction, and are colored in the same way. [Some gene names are different, reflecting changes in the nomenclature used in the Saccharomyces Genome Database (SGD) (20)]. VTC4 (gray) has been included in this analysis, as its promoter was the basis for the reporter experiments described here. (B) ChIP of Pho4 (closed squares) and Cbf1 (open squares) in high and low phosphate. Values and error bars are the average and standard error of enrichment values for two sets of experiments using Myc-tagged and HA-tagged proteins (Methods). Colors are as in panel A. (C) Correlation of induction sensitivity with the change in transcription factor binding. The fold-difference in ChIP enrichment values are shown for both for Pho4 (closed squares; 0/10 mM phosphate) and Cbf1 (open squares; 10/0 mM). Note that the Pho4 and Cbf1 ChIP enrichment ratios can be compared for a given gene because the two values share the same y-axis values. (D) Correlation between the experimentally determined phosphate sensitivity of expression and two in silico predictions. One (X’s) is the model used for the reporter constructs in Figure 4, which takes into account competition between Cbf1 and Pho4 and ascribes a low level of transcriptional activity to bound Cbf1. The second model (circles) includes the same terms, but also models chromatin changes as reported for the reporter gene sensitivity values in Figure 5. The dashed and solid lines indicate fits to these predictions, respectively. The parameters used, and an analysis of the sensitivity to those parameters, are shown in Supplementary Figure S11. (E) Correlation coefficients for the chromatin remodeling model shown in panel D, along with the effect of leaving out certain terms. The error bars for the top histogram bar indicates the 95% confidence interval for the correlation as defined by bootstrap resampling. Leaving out any term that is required for modeling the cooperative effects of Cbf1 chromatin remodeling results in an insignificant correlation. The terms are labeled a–d: (a) are there two states for the promoter, ‘open’ and ‘closed’?; (b) is there tighter binding of Pho4 and Cbf1 to the ‘open’ state?; (c) does Cbf1 binding shift the equilibrium toward the open state?; (d) is Cbf1 included in the model at all?
Mentions: It is well established that there are differences in the sensitivity to induction among Pho4-target genes. Lam et al. (24), using GFP fusions for seven Pho4 target genes, classified the genes into two groups on this basis. We validated this observation using a microarray gene expression data obtained at five phosphate concentrations (Figure 6A). Although the microarray and GFP reporter assays are measuring rather different things, the baseline expression in high phosphate for the seven genes that were assayed in common are well correlated (R = 0.95), as is the extent of their induction upon shifting to 0 mM phosphate (R = 0.95) (Supplementary Figure S9). We prefer to use the term expression sensitivity rather than threshold because induction is not an all or none phenomenon, but the concepts are related, and our data can readily be interpreted to produce the same two groups defined by GFP assays (Figure 6A). In addition to the seven genes studied previously, we included in our analysis VTC4, a Pho4 target gene that shows an induction sensitivity intermediate between the two classes defined earlier (Figure 6A).Figure 6.

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