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Rearrangements of 2.5 kilobases of noncoding DNA from the Drosophila even-skipped locus define predictive rules of genomic cis-regulatory logic.

Kim AR, Martinez C, Ionides J, Ramos AF, Ludwig MZ, Ogawa N, Sharp DH, Reinitz J - PLoS Genet. (2013)

Bottom Line: The most radical effects are generated by juxtaposing the minimal stripe enhancers MSE2 and MSE3 for stripes 2 and 3 with and without small "spacer" segments less than 360 bp in length.The model was highly constrained by the training data, which it described within the limits of experimental error.The model, so constrained, was able to correctly predict expression patterns driven by enhancers for other Drosophila genes; even-skipped enhancers not included in the training set; stripe 2, 3, and 7 enhancers from various Drosophilid and Sepsid species; and long segments of even-skipped regulatory DNA that contain multiple enhancers.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecology and Evolution, Chicago Center for Systems Biology, University of Chicago, Chicago, Illinois, USA.

ABSTRACT
Rearrangements of about 2.5 kilobases of regulatory DNA located 5' of the transcription start site of the Drosophila even-skipped locus generate large-scale changes in the expression of even-skipped stripes 2, 3, and 7. The most radical effects are generated by juxtaposing the minimal stripe enhancers MSE2 and MSE3 for stripes 2 and 3 with and without small "spacer" segments less than 360 bp in length. We placed these fusion constructs in a targeted transformation site and obtained quantitative expression data for these transformants together with their controlling transcription factors at cellular resolution. These data demonstrated that the rearrangements can alter expression levels in stripe 2 and the 2-3 interstripe by a factor of more than 10. We reasoned that this behavior would place tight constraints on possible rules of genomic cis-regulatory logic. To find these constraints, we confronted our new expression data together with previously obtained data on other constructs with a computational model. The model contained representations of thermodynamic protein-DNA interactions including steric interference and cooperative binding, short-range repression, direct repression, activation, and coactivation. The model was highly constrained by the training data, which it described within the limits of experimental error. The model, so constrained, was able to correctly predict expression patterns driven by enhancers for other Drosophila genes; even-skipped enhancers not included in the training set; stripe 2, 3, and 7 enhancers from various Drosophilid and Sepsid species; and long segments of even-skipped regulatory DNA that contain multiple enhancers. The model further demonstrated that elevated expression driven by a fusion of MSE2 and MSE3 was a consequence of the recruitment of a portion of MSE3 to become a functional component of MSE2, demonstrating that cis-regulatory "elements" are not elementary objects.

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Regulatory analysis of M3_2 and M32.(A) The expression profiles driven by M3_2 and M32 are subdivided into four distinct zones I to IV for analysis as shown. Two additional zones V and VI involving expression changes between M2_3 and M23 are shown in Figure S7. (B) Illustration of a catalyzed reaction with free energy change  and activation energy barrier . Catalysis by activators reduces the barrier by . A scale bar of two heatmaps used in (C), (E), and (F) is shown. The  heatmap applies to the vertical bars on the right hand side of these panels and the  heatmap applies to the square panels in (C), (E), and (F). ; compare with Equations 8 and 9 in Figure 3. (C) Distribution of activation energy barrier changes at single binding site resolution for M3_2 as a function of A-P position on the embryo and number of basepairs 5′ to the M3_2 TSS. The positions of MSE2 and MSE3 are schematically shown at the top.  for each activator binding site is shown in the central panel according to the key in (B) and the summed activation  in the right hand bar. Peaks of activation corresponding to stripes 2, 3, and 7 are indicated. (D) Expression levels of RNA expression driven by M3_2 together with regulating TFs at cellular resolution, as shown in the key. In the key, standard abbreviations are used except that Dst indicates D-STAT and Dic indicates Dichaete. (E) and (F) show a regulatory dissection of expression changes induced by removal of the “spacer” with activation represented as in (C). Selected binding sites for M3_2 and M32 are shown at the top of (E) and (F) respectively, with TF specificity indicated by color as shown in the key for (D). The full set of binding sites is shown in Figure S6. The black arrows show binding sites involved in coactivation; the red arrow in (F) indicates the major coactivation interaction in M32. Circled areas indicate groups of binding sites critical for expression changes in different zones as described in the text.
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pgen-1003243-g005: Regulatory analysis of M3_2 and M32.(A) The expression profiles driven by M3_2 and M32 are subdivided into four distinct zones I to IV for analysis as shown. Two additional zones V and VI involving expression changes between M2_3 and M23 are shown in Figure S7. (B) Illustration of a catalyzed reaction with free energy change and activation energy barrier . Catalysis by activators reduces the barrier by . A scale bar of two heatmaps used in (C), (E), and (F) is shown. The heatmap applies to the vertical bars on the right hand side of these panels and the heatmap applies to the square panels in (C), (E), and (F). ; compare with Equations 8 and 9 in Figure 3. (C) Distribution of activation energy barrier changes at single binding site resolution for M3_2 as a function of A-P position on the embryo and number of basepairs 5′ to the M3_2 TSS. The positions of MSE2 and MSE3 are schematically shown at the top. for each activator binding site is shown in the central panel according to the key in (B) and the summed activation in the right hand bar. Peaks of activation corresponding to stripes 2, 3, and 7 are indicated. (D) Expression levels of RNA expression driven by M3_2 together with regulating TFs at cellular resolution, as shown in the key. In the key, standard abbreviations are used except that Dst indicates D-STAT and Dic indicates Dichaete. (E) and (F) show a regulatory dissection of expression changes induced by removal of the “spacer” with activation represented as in (C). Selected binding sites for M3_2 and M32 are shown at the top of (E) and (F) respectively, with TF specificity indicated by color as shown in the key for (D). The full set of binding sites is shown in Figure S6. The black arrows show binding sites involved in coactivation; the red arrow in (F) indicates the major coactivation interaction in M32. Circled areas indicate groups of binding sites critical for expression changes in different zones as described in the text.

Mentions: The fusions introduce six types of quantitative alterations in expression, each of which occurs in a small spatial region containing 2–3 nuclei, which we call a “zone” (Figure 5A). With respect to the M32 fusion compared to M3_2, in zone I stripe 2 expression is increased by a factor of almost four; in zone II the 2–3 interstripe is derepressed; in zone III stripe 3 expression is reduced; and in zone IV stripe 7 expression is increased. With respect to the M23 fusion compared to M2_3, in zone V stripe 2 expression is reduced and in zone VI stripe 3 expression is slightly increased (Figure S7A). We analyzed the causes of these effects by plotting the contributions to the activation (Figure 5B) as a function of position on the A-P axis and the regulatory sequence (Figure 5C), where each position on the A-P axis defines a unique set of TF concentrations as shown in Figure 5D. Annotating these diagrams with the identity of key binding sites and comparing activation in M32 and M3_2 indicates which TFs and binding sites lead to the effects observed Figure 5E–5F). These diagrams show that the major source of activation is from coactivated Hb bound at the hb-3 site by Bcd bound at the bcd-1,bcd-* and bcd-2 sites (Figure 5E–5F and ). With respect to zone I, we found that the increase of gene expression is almost entirely the result of coactivation of two sites of bound Hb by Bcd. It occurs because of the deletion of the “spacer” DNA between MSE3 and MSE2, which reduces the distance between the two Bcd sites in MSE2 and the two Hb sites in MSE3 from more than 400 bp to about 150 bp, permitting coactivation (Figure 5E–5F, lower black arrows; Figure 5F, white arrow).


Rearrangements of 2.5 kilobases of noncoding DNA from the Drosophila even-skipped locus define predictive rules of genomic cis-regulatory logic.

Kim AR, Martinez C, Ionides J, Ramos AF, Ludwig MZ, Ogawa N, Sharp DH, Reinitz J - PLoS Genet. (2013)

Regulatory analysis of M3_2 and M32.(A) The expression profiles driven by M3_2 and M32 are subdivided into four distinct zones I to IV for analysis as shown. Two additional zones V and VI involving expression changes between M2_3 and M23 are shown in Figure S7. (B) Illustration of a catalyzed reaction with free energy change  and activation energy barrier . Catalysis by activators reduces the barrier by . A scale bar of two heatmaps used in (C), (E), and (F) is shown. The  heatmap applies to the vertical bars on the right hand side of these panels and the  heatmap applies to the square panels in (C), (E), and (F). ; compare with Equations 8 and 9 in Figure 3. (C) Distribution of activation energy barrier changes at single binding site resolution for M3_2 as a function of A-P position on the embryo and number of basepairs 5′ to the M3_2 TSS. The positions of MSE2 and MSE3 are schematically shown at the top.  for each activator binding site is shown in the central panel according to the key in (B) and the summed activation  in the right hand bar. Peaks of activation corresponding to stripes 2, 3, and 7 are indicated. (D) Expression levels of RNA expression driven by M3_2 together with regulating TFs at cellular resolution, as shown in the key. In the key, standard abbreviations are used except that Dst indicates D-STAT and Dic indicates Dichaete. (E) and (F) show a regulatory dissection of expression changes induced by removal of the “spacer” with activation represented as in (C). Selected binding sites for M3_2 and M32 are shown at the top of (E) and (F) respectively, with TF specificity indicated by color as shown in the key for (D). The full set of binding sites is shown in Figure S6. The black arrows show binding sites involved in coactivation; the red arrow in (F) indicates the major coactivation interaction in M32. Circled areas indicate groups of binding sites critical for expression changes in different zones as described in the text.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585115&req=5

pgen-1003243-g005: Regulatory analysis of M3_2 and M32.(A) The expression profiles driven by M3_2 and M32 are subdivided into four distinct zones I to IV for analysis as shown. Two additional zones V and VI involving expression changes between M2_3 and M23 are shown in Figure S7. (B) Illustration of a catalyzed reaction with free energy change and activation energy barrier . Catalysis by activators reduces the barrier by . A scale bar of two heatmaps used in (C), (E), and (F) is shown. The heatmap applies to the vertical bars on the right hand side of these panels and the heatmap applies to the square panels in (C), (E), and (F). ; compare with Equations 8 and 9 in Figure 3. (C) Distribution of activation energy barrier changes at single binding site resolution for M3_2 as a function of A-P position on the embryo and number of basepairs 5′ to the M3_2 TSS. The positions of MSE2 and MSE3 are schematically shown at the top. for each activator binding site is shown in the central panel according to the key in (B) and the summed activation in the right hand bar. Peaks of activation corresponding to stripes 2, 3, and 7 are indicated. (D) Expression levels of RNA expression driven by M3_2 together with regulating TFs at cellular resolution, as shown in the key. In the key, standard abbreviations are used except that Dst indicates D-STAT and Dic indicates Dichaete. (E) and (F) show a regulatory dissection of expression changes induced by removal of the “spacer” with activation represented as in (C). Selected binding sites for M3_2 and M32 are shown at the top of (E) and (F) respectively, with TF specificity indicated by color as shown in the key for (D). The full set of binding sites is shown in Figure S6. The black arrows show binding sites involved in coactivation; the red arrow in (F) indicates the major coactivation interaction in M32. Circled areas indicate groups of binding sites critical for expression changes in different zones as described in the text.
Mentions: The fusions introduce six types of quantitative alterations in expression, each of which occurs in a small spatial region containing 2–3 nuclei, which we call a “zone” (Figure 5A). With respect to the M32 fusion compared to M3_2, in zone I stripe 2 expression is increased by a factor of almost four; in zone II the 2–3 interstripe is derepressed; in zone III stripe 3 expression is reduced; and in zone IV stripe 7 expression is increased. With respect to the M23 fusion compared to M2_3, in zone V stripe 2 expression is reduced and in zone VI stripe 3 expression is slightly increased (Figure S7A). We analyzed the causes of these effects by plotting the contributions to the activation (Figure 5B) as a function of position on the A-P axis and the regulatory sequence (Figure 5C), where each position on the A-P axis defines a unique set of TF concentrations as shown in Figure 5D. Annotating these diagrams with the identity of key binding sites and comparing activation in M32 and M3_2 indicates which TFs and binding sites lead to the effects observed Figure 5E–5F). These diagrams show that the major source of activation is from coactivated Hb bound at the hb-3 site by Bcd bound at the bcd-1,bcd-* and bcd-2 sites (Figure 5E–5F and ). With respect to zone I, we found that the increase of gene expression is almost entirely the result of coactivation of two sites of bound Hb by Bcd. It occurs because of the deletion of the “spacer” DNA between MSE3 and MSE2, which reduces the distance between the two Bcd sites in MSE2 and the two Hb sites in MSE3 from more than 400 bp to about 150 bp, permitting coactivation (Figure 5E–5F, lower black arrows; Figure 5F, white arrow).

Bottom Line: The most radical effects are generated by juxtaposing the minimal stripe enhancers MSE2 and MSE3 for stripes 2 and 3 with and without small "spacer" segments less than 360 bp in length.The model was highly constrained by the training data, which it described within the limits of experimental error.The model, so constrained, was able to correctly predict expression patterns driven by enhancers for other Drosophila genes; even-skipped enhancers not included in the training set; stripe 2, 3, and 7 enhancers from various Drosophilid and Sepsid species; and long segments of even-skipped regulatory DNA that contain multiple enhancers.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecology and Evolution, Chicago Center for Systems Biology, University of Chicago, Chicago, Illinois, USA.

ABSTRACT
Rearrangements of about 2.5 kilobases of regulatory DNA located 5' of the transcription start site of the Drosophila even-skipped locus generate large-scale changes in the expression of even-skipped stripes 2, 3, and 7. The most radical effects are generated by juxtaposing the minimal stripe enhancers MSE2 and MSE3 for stripes 2 and 3 with and without small "spacer" segments less than 360 bp in length. We placed these fusion constructs in a targeted transformation site and obtained quantitative expression data for these transformants together with their controlling transcription factors at cellular resolution. These data demonstrated that the rearrangements can alter expression levels in stripe 2 and the 2-3 interstripe by a factor of more than 10. We reasoned that this behavior would place tight constraints on possible rules of genomic cis-regulatory logic. To find these constraints, we confronted our new expression data together with previously obtained data on other constructs with a computational model. The model contained representations of thermodynamic protein-DNA interactions including steric interference and cooperative binding, short-range repression, direct repression, activation, and coactivation. The model was highly constrained by the training data, which it described within the limits of experimental error. The model, so constrained, was able to correctly predict expression patterns driven by enhancers for other Drosophila genes; even-skipped enhancers not included in the training set; stripe 2, 3, and 7 enhancers from various Drosophilid and Sepsid species; and long segments of even-skipped regulatory DNA that contain multiple enhancers. The model further demonstrated that elevated expression driven by a fusion of MSE2 and MSE3 was a consequence of the recruitment of a portion of MSE3 to become a functional component of MSE2, demonstrating that cis-regulatory "elements" are not elementary objects.

Show MeSH
Related in: MedlinePlus