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A model of spatially restricted transcription in opposing gradients of activators and repressors.

White MA, Parker DS, Barolo S, Cohen BA - Mol. Syst. Biol. (2012)

Bottom Line: This model quantitatively predicts the boundaries of gene expression within OARGs.When trained on experimental data, our model accounts for the counterintuitive observation that increasing the affinity of binding sites in enhancers of Hedgehog target genes produces more restricted transcription within Hedgehog gradients in Drosophila.Because our model is general, it may explain the role of low-affinity binding sites in many contexts, including mammalian Hedgehog gradients.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St Louis, MO 63108, USA.

ABSTRACT
Morphogens control patterns of transcription in development, often by establishing concentration gradients of a single transcriptional activator. However, many morphogens, including Hedgehog, create opposing activator and repressor gradients (OARGs). In contrast to single activator gradients, it is not well understood how OARGs control transcriptional patterns. We present a general thermodynamic model that explains how spatial patterns of gene expression are established within OARGs. The model predicts that differences in enhancer binding site affinities for morphogen-responsive transcription factors (TFs) produce discrete transcriptional boundaries, but only when either activators or repressors bind cooperatively. This model quantitatively predicts the boundaries of gene expression within OARGs. When trained on experimental data, our model accounts for the counterintuitive observation that increasing the affinity of binding sites in enhancers of Hedgehog target genes produces more restricted transcription within Hedgehog gradients in Drosophila. Because our model is general, it may explain the role of low-affinity binding sites in many contexts, including mammalian Hedgehog gradients.

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High-affinity Gli sites in the wg enhancer repress expression within Hh gradients of embryonic parasegments. (A) Four low-affinity Gli sites in the wg embryonic ectoderm enhancer (Von Ohlen and Hooper, 1997) are conserved in 12 Drosophila species. The Gli binding site is shown in bold, and mismatches to the consensus sequence (gaccaccca) are shown in red. Numbers in parentheses indicate the measured affinity of the site relative to the consensus sequence (see Materials and methods). Species abbreviations: mel, D. melanogaster; sim, D. simulans; sec, D. sechellia; yak, D. yakuba; ere, D. erecta; ana, D. ananasae; pse, D. pseudoobscura; per, D. persimilis; wil, D. willistoni; vir, D. virilis; moj, D. mojavensis; gri, D. grimshawi. (B) GFP reporter gene expression in embryonic parasegments driven by an Hh-responsive wg enhancer. Magenta indicates Hh-lacZ (see Materials and methods). The white box indicates the ventral half of parasegments 6 and 7 that were quantified. (C) Increasing the affinity of Gli sites produces repression. Normalized GFP expression from low-affinity (dashed blue line) and high-affinity (dashed red line) versions of the wg enhancer shows mean Gli-mediated activation (positive y axis) or repression (negative y axis) along the Hh gradient (x axis) of parasegments 6 and 7. Vertical bars indicate standard error. GFP expression is defined relative to GFP expression from a basal, Gli-independent enhancer in which the Gli sites have been abolished. The cooperative repression model (solid lines) trained on these data accurately identifies regions where low-affinity sites produce activation and high-affinity sites produce repression. Wedges indicate the orientation of the Hedgehog gradient and opposing gradients of Gli activators and repressors. Reproducibility of wg reporter measurements in three different transgenic lines is shown in Supplementary Figure 3. Source data is available for this figure in the Supplementary Information.
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f4: High-affinity Gli sites in the wg enhancer repress expression within Hh gradients of embryonic parasegments. (A) Four low-affinity Gli sites in the wg embryonic ectoderm enhancer (Von Ohlen and Hooper, 1997) are conserved in 12 Drosophila species. The Gli binding site is shown in bold, and mismatches to the consensus sequence (gaccaccca) are shown in red. Numbers in parentheses indicate the measured affinity of the site relative to the consensus sequence (see Materials and methods). Species abbreviations: mel, D. melanogaster; sim, D. simulans; sec, D. sechellia; yak, D. yakuba; ere, D. erecta; ana, D. ananasae; pse, D. pseudoobscura; per, D. persimilis; wil, D. willistoni; vir, D. virilis; moj, D. mojavensis; gri, D. grimshawi. (B) GFP reporter gene expression in embryonic parasegments driven by an Hh-responsive wg enhancer. Magenta indicates Hh-lacZ (see Materials and methods). The white box indicates the ventral half of parasegments 6 and 7 that were quantified. (C) Increasing the affinity of Gli sites produces repression. Normalized GFP expression from low-affinity (dashed blue line) and high-affinity (dashed red line) versions of the wg enhancer shows mean Gli-mediated activation (positive y axis) or repression (negative y axis) along the Hh gradient (x axis) of parasegments 6 and 7. Vertical bars indicate standard error. GFP expression is defined relative to GFP expression from a basal, Gli-independent enhancer in which the Gli sites have been abolished. The cooperative repression model (solid lines) trained on these data accurately identifies regions where low-affinity sites produce activation and high-affinity sites produce repression. Wedges indicate the orientation of the Hedgehog gradient and opposing gradients of Gli activators and repressors. Reproducibility of wg reporter measurements in three different transgenic lines is shown in Supplementary Figure 3. Source data is available for this figure in the Supplementary Information.

Mentions: To determine whether this behavior occurs in response to other Gli OARGs, we examined the role of Gli binding site affinity in another Hh target gene, wingless (wg), which is expressed in a different tissue and developmental time point in Drosophila. Like dpp in the wing disc, the expression of wg in the Hh gradients of embryonic parasegments is driven by conserved, low-affinity Gli sites (Figure 4A; Von Ohlen and Hooper, 1997). We tested the role of Gli binding site affinity by using low- and high-affinity versions of the wg enhancer to drive the expression of a GFP reporter gene in the parasegments of Drosophila embryos (Figure 4B). Low- and high-affinity versions of the wg enhancer were compared to a ‘basal', Gli-independent wg enhancer in which the Gli sites were abolished. This basal enhancer revealed the effects of other factors acting on the wg enhancer, and activation and repression by Gli were defined relative to the basal levels of expression across each parasegment. Reporter gene expression above basal expression indicated Gli-mediated activation, while expression below basal levels indicated Gli-mediated repression.


A model of spatially restricted transcription in opposing gradients of activators and repressors.

White MA, Parker DS, Barolo S, Cohen BA - Mol. Syst. Biol. (2012)

High-affinity Gli sites in the wg enhancer repress expression within Hh gradients of embryonic parasegments. (A) Four low-affinity Gli sites in the wg embryonic ectoderm enhancer (Von Ohlen and Hooper, 1997) are conserved in 12 Drosophila species. The Gli binding site is shown in bold, and mismatches to the consensus sequence (gaccaccca) are shown in red. Numbers in parentheses indicate the measured affinity of the site relative to the consensus sequence (see Materials and methods). Species abbreviations: mel, D. melanogaster; sim, D. simulans; sec, D. sechellia; yak, D. yakuba; ere, D. erecta; ana, D. ananasae; pse, D. pseudoobscura; per, D. persimilis; wil, D. willistoni; vir, D. virilis; moj, D. mojavensis; gri, D. grimshawi. (B) GFP reporter gene expression in embryonic parasegments driven by an Hh-responsive wg enhancer. Magenta indicates Hh-lacZ (see Materials and methods). The white box indicates the ventral half of parasegments 6 and 7 that were quantified. (C) Increasing the affinity of Gli sites produces repression. Normalized GFP expression from low-affinity (dashed blue line) and high-affinity (dashed red line) versions of the wg enhancer shows mean Gli-mediated activation (positive y axis) or repression (negative y axis) along the Hh gradient (x axis) of parasegments 6 and 7. Vertical bars indicate standard error. GFP expression is defined relative to GFP expression from a basal, Gli-independent enhancer in which the Gli sites have been abolished. The cooperative repression model (solid lines) trained on these data accurately identifies regions where low-affinity sites produce activation and high-affinity sites produce repression. Wedges indicate the orientation of the Hedgehog gradient and opposing gradients of Gli activators and repressors. Reproducibility of wg reporter measurements in three different transgenic lines is shown in Supplementary Figure 3. Source data is available for this figure in the Supplementary Information.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3472688&req=5

f4: High-affinity Gli sites in the wg enhancer repress expression within Hh gradients of embryonic parasegments. (A) Four low-affinity Gli sites in the wg embryonic ectoderm enhancer (Von Ohlen and Hooper, 1997) are conserved in 12 Drosophila species. The Gli binding site is shown in bold, and mismatches to the consensus sequence (gaccaccca) are shown in red. Numbers in parentheses indicate the measured affinity of the site relative to the consensus sequence (see Materials and methods). Species abbreviations: mel, D. melanogaster; sim, D. simulans; sec, D. sechellia; yak, D. yakuba; ere, D. erecta; ana, D. ananasae; pse, D. pseudoobscura; per, D. persimilis; wil, D. willistoni; vir, D. virilis; moj, D. mojavensis; gri, D. grimshawi. (B) GFP reporter gene expression in embryonic parasegments driven by an Hh-responsive wg enhancer. Magenta indicates Hh-lacZ (see Materials and methods). The white box indicates the ventral half of parasegments 6 and 7 that were quantified. (C) Increasing the affinity of Gli sites produces repression. Normalized GFP expression from low-affinity (dashed blue line) and high-affinity (dashed red line) versions of the wg enhancer shows mean Gli-mediated activation (positive y axis) or repression (negative y axis) along the Hh gradient (x axis) of parasegments 6 and 7. Vertical bars indicate standard error. GFP expression is defined relative to GFP expression from a basal, Gli-independent enhancer in which the Gli sites have been abolished. The cooperative repression model (solid lines) trained on these data accurately identifies regions where low-affinity sites produce activation and high-affinity sites produce repression. Wedges indicate the orientation of the Hedgehog gradient and opposing gradients of Gli activators and repressors. Reproducibility of wg reporter measurements in three different transgenic lines is shown in Supplementary Figure 3. Source data is available for this figure in the Supplementary Information.
Mentions: To determine whether this behavior occurs in response to other Gli OARGs, we examined the role of Gli binding site affinity in another Hh target gene, wingless (wg), which is expressed in a different tissue and developmental time point in Drosophila. Like dpp in the wing disc, the expression of wg in the Hh gradients of embryonic parasegments is driven by conserved, low-affinity Gli sites (Figure 4A; Von Ohlen and Hooper, 1997). We tested the role of Gli binding site affinity by using low- and high-affinity versions of the wg enhancer to drive the expression of a GFP reporter gene in the parasegments of Drosophila embryos (Figure 4B). Low- and high-affinity versions of the wg enhancer were compared to a ‘basal', Gli-independent wg enhancer in which the Gli sites were abolished. This basal enhancer revealed the effects of other factors acting on the wg enhancer, and activation and repression by Gli were defined relative to the basal levels of expression across each parasegment. Reporter gene expression above basal expression indicated Gli-mediated activation, while expression below basal levels indicated Gli-mediated repression.

Bottom Line: This model quantitatively predicts the boundaries of gene expression within OARGs.When trained on experimental data, our model accounts for the counterintuitive observation that increasing the affinity of binding sites in enhancers of Hedgehog target genes produces more restricted transcription within Hedgehog gradients in Drosophila.Because our model is general, it may explain the role of low-affinity binding sites in many contexts, including mammalian Hedgehog gradients.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St Louis, MO 63108, USA.

ABSTRACT
Morphogens control patterns of transcription in development, often by establishing concentration gradients of a single transcriptional activator. However, many morphogens, including Hedgehog, create opposing activator and repressor gradients (OARGs). In contrast to single activator gradients, it is not well understood how OARGs control transcriptional patterns. We present a general thermodynamic model that explains how spatial patterns of gene expression are established within OARGs. The model predicts that differences in enhancer binding site affinities for morphogen-responsive transcription factors (TFs) produce discrete transcriptional boundaries, but only when either activators or repressors bind cooperatively. This model quantitatively predicts the boundaries of gene expression within OARGs. When trained on experimental data, our model accounts for the counterintuitive observation that increasing the affinity of binding sites in enhancers of Hedgehog target genes produces more restricted transcription within Hedgehog gradients in Drosophila. Because our model is general, it may explain the role of low-affinity binding sites in many contexts, including mammalian Hedgehog gradients.

Show MeSH
Related in: MedlinePlus