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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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(A) Reporter expression levels under fully inducing conditions (0 mM phosphate) plotted against relative Pho4 binding affinity. Motifs are indicated by the same two bases used in Figure 2. The data have been fit to a standard binding isotherm, yielding an apparent protein concentration, relative to the mean affinity, of 4 (Methods). (B) Alternative representation of the data in panel A, based on calculating the predicted occupancy of each motif at a protein concentration 4 times the mean Kd. The line is a linear fit of expression versus predicted occupancy.
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gkt210-F3: (A) Reporter expression levels under fully inducing conditions (0 mM phosphate) plotted against relative Pho4 binding affinity. Motifs are indicated by the same two bases used in Figure 2. The data have been fit to a standard binding isotherm, yielding an apparent protein concentration, relative to the mean affinity, of 4 (Methods). (B) Alternative representation of the data in panel A, based on calculating the predicted occupancy of each motif at a protein concentration 4 times the mean Kd. The line is a linear fit of expression versus predicted occupancy.

Mentions: To quantify these effects, we first looked at the correlation between the Pho4 affinity of the binding site and the level of gene expression under maximally induced conditions. As affinity increases, expression asymptotically approaches a maximum, implying that occupancy of the site is becoming saturated (Figure 3A). The curve resembles the classic hyperbolic curve that describes occupancy of a binding site as a function of protein concentration. This is not a coincidence. The relationship between affinity, concentration and binding occupancy that allows us to determine the affinity of a site from the change in occupancy as a function of protein concentration can also be used to determine protein concentration from the change in occupancy as a function of affinity. In this case, we do not have values for the in vivo binding occupancy per se, but we can instead use gene expression as a proxy for binding occupancy. In addition, we do not know the absolute affinities of the binding sites under in vivo conditions, so the concentration that can be determined is not an absolute concentration but a concentration expressed relative to the Kd of the binding sites. Fitting the data in Figure 3A to two parameters, the maximum expression level and the concentration of Pho4, we find a good fit at a Pho4 concentration four times the mean Kd of the binding sites. Using this value of [Pho4] = 4, we can transform affinities into predicted fractional occupancies, yielding a linear relationship between expression and predicted occupancy (R = 0.94; Figure 3B). This is a remarkably strong correlation considering that the occupancy of binding sites in vivo was predicted on the basis of subtle differences in affinity in vitro; we conclude that the specificity in vivo must be very similar to the specificity in vitro (8). Furthermore, the experimental value that was correlated to predicted binding is the expression level of reporter genes. Expression levels need not have been related in some simple fashion to binding, but the strength and linearity of the correlation suggests that median single-cell GFP fluorescence values are, in fact, an accurate proxy for transcription factor binding in this system. The effective Pho4 concentration is also likely to be reasonably well estimated because both the linearity of the fit and the value of the correlation coefficient fall off as Pho4 concentration values vary from the optimum (Supplementary Figure S4). We have less confidence in the absolute Pho4 occupancy values because these values are based on the assumption that expression asymptotically approaches its limit entirely because Pho4 binding asymptotically approaches saturation. In reality, there may be additional factors, such as the saturation of RNA polymerase activity, that affect maximal expression level. However, these effects do not materially affect our interpretation, as the excellent fit that we find between in vitro affinity and expression requires none of the assumptions that are required for the estimation of occupancy.Figure 3.


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)

(A) Reporter expression levels under fully inducing conditions (0 mM phosphate) plotted against relative Pho4 binding affinity. Motifs are indicated by the same two bases used in Figure 2. The data have been fit to a standard binding isotherm, yielding an apparent protein concentration, relative to the mean affinity, of 4 (Methods). (B) Alternative representation of the data in panel A, based on calculating the predicted occupancy of each motif at a protein concentration 4 times the mean Kd. The line is a linear fit of expression versus predicted occupancy.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643608&req=5

gkt210-F3: (A) Reporter expression levels under fully inducing conditions (0 mM phosphate) plotted against relative Pho4 binding affinity. Motifs are indicated by the same two bases used in Figure 2. The data have been fit to a standard binding isotherm, yielding an apparent protein concentration, relative to the mean affinity, of 4 (Methods). (B) Alternative representation of the data in panel A, based on calculating the predicted occupancy of each motif at a protein concentration 4 times the mean Kd. The line is a linear fit of expression versus predicted occupancy.
Mentions: To quantify these effects, we first looked at the correlation between the Pho4 affinity of the binding site and the level of gene expression under maximally induced conditions. As affinity increases, expression asymptotically approaches a maximum, implying that occupancy of the site is becoming saturated (Figure 3A). The curve resembles the classic hyperbolic curve that describes occupancy of a binding site as a function of protein concentration. This is not a coincidence. The relationship between affinity, concentration and binding occupancy that allows us to determine the affinity of a site from the change in occupancy as a function of protein concentration can also be used to determine protein concentration from the change in occupancy as a function of affinity. In this case, we do not have values for the in vivo binding occupancy per se, but we can instead use gene expression as a proxy for binding occupancy. In addition, we do not know the absolute affinities of the binding sites under in vivo conditions, so the concentration that can be determined is not an absolute concentration but a concentration expressed relative to the Kd of the binding sites. Fitting the data in Figure 3A to two parameters, the maximum expression level and the concentration of Pho4, we find a good fit at a Pho4 concentration four times the mean Kd of the binding sites. Using this value of [Pho4] = 4, we can transform affinities into predicted fractional occupancies, yielding a linear relationship between expression and predicted occupancy (R = 0.94; Figure 3B). This is a remarkably strong correlation considering that the occupancy of binding sites in vivo was predicted on the basis of subtle differences in affinity in vitro; we conclude that the specificity in vivo must be very similar to the specificity in vitro (8). Furthermore, the experimental value that was correlated to predicted binding is the expression level of reporter genes. Expression levels need not have been related in some simple fashion to binding, but the strength and linearity of the correlation suggests that median single-cell GFP fluorescence values are, in fact, an accurate proxy for transcription factor binding in this system. The effective Pho4 concentration is also likely to be reasonably well estimated because both the linearity of the fit and the value of the correlation coefficient fall off as Pho4 concentration values vary from the optimum (Supplementary Figure S4). We have less confidence in the absolute Pho4 occupancy values because these values are based on the assumption that expression asymptotically approaches its limit entirely because Pho4 binding asymptotically approaches saturation. In reality, there may be additional factors, such as the saturation of RNA polymerase activity, that affect maximal expression level. However, these effects do not materially affect our interpretation, as the excellent fit that we find between in vitro affinity and expression requires none of the assumptions that are required for the estimation of occupancy.Figure 3.

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