Limits...
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.

Show MeSH

Related in: MedlinePlus

Training and predictions.(A) Training results for 7 constructs. RNA levels and model results are as shown in the key; the model result trace obscures the data in regions where both are superimposed. The regions of the eve locus used to generate the training data are indicated schematically. (B–G) Predictions of gene expression driven by DNA sequences that were not used for training. The sequences used are fully described in Table S4. Black lines are predicted RNA expression and colored lines are quantitative protein profiles of the corresponding endogenous loci. The scale of relative fluorescence levels for RNA is shown at the left of graphs, that for proteins on the right. All protein patterns are taken from the FlyEx database (http://urchin.spbcas.ru/flyex) [7]. An asterisk on a panel indicates the prediction was not made from model 6: D1-2 are from model 2, G1 is from model 7, and G2 is from model 1. See text, Figure S4 and Table S1 for details. (B) 5 mutant eve enhancers, described fully in the main text. (C) Stripe 2 enhancers from 16 different Drosophila species, with abbreviations and panel numbers (see Table S5 for full species name). The enhancer from D. persimilis (per), D. grimshawi (gri), D. mojavensis (moj) and willistoni (wil) was first identified in this study. (D) Other D. melanogaster eve enhancers. (D1) Stripe 5 enhancer. (D2) Stripe 4/6 enhancer. (D3) pseudoobscura- melanogaster stripe 2 chimera(p1-m2). (D4) melanogaster- pseudoobscura stripe 2 chimera(m1-p2). (E) Stripe 2 (S2E; E1–E6) and stripe 3/7 (S37E, E7–E12) enhancers from 6 Sepsid species, with abbreviations (see Table S5). (F) 5 non- eve enhancers from the D. melanogaster genes hb (F1), Kr (F2), run (F3-4), and h (F5). (G) Large 5′ (G1) and 3′ (G2) eve regulatory DNAs that contain multiple enhancers.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3585115&req=5

pgen-1003243-g004: Training and predictions.(A) Training results for 7 constructs. RNA levels and model results are as shown in the key; the model result trace obscures the data in regions where both are superimposed. The regions of the eve locus used to generate the training data are indicated schematically. (B–G) Predictions of gene expression driven by DNA sequences that were not used for training. The sequences used are fully described in Table S4. Black lines are predicted RNA expression and colored lines are quantitative protein profiles of the corresponding endogenous loci. The scale of relative fluorescence levels for RNA is shown at the left of graphs, that for proteins on the right. All protein patterns are taken from the FlyEx database (http://urchin.spbcas.ru/flyex) [7]. An asterisk on a panel indicates the prediction was not made from model 6: D1-2 are from model 2, G1 is from model 7, and G2 is from model 1. See text, Figure S4 and Table S1 for details. (B) 5 mutant eve enhancers, described fully in the main text. (C) Stripe 2 enhancers from 16 different Drosophila species, with abbreviations and panel numbers (see Table S5 for full species name). The enhancer from D. persimilis (per), D. grimshawi (gri), D. mojavensis (moj) and willistoni (wil) was first identified in this study. (D) Other D. melanogaster eve enhancers. (D1) Stripe 5 enhancer. (D2) Stripe 4/6 enhancer. (D3) pseudoobscura- melanogaster stripe 2 chimera(p1-m2). (D4) melanogaster- pseudoobscura stripe 2 chimera(m1-p2). (E) Stripe 2 (S2E; E1–E6) and stripe 3/7 (S37E, E7–E12) enhancers from 6 Sepsid species, with abbreviations (see Table S5). (F) 5 non- eve enhancers from the D. melanogaster genes hb (F1), Kr (F2), run (F3-4), and h (F5). (G) Large 5′ (G1) and 3′ (G2) eve regulatory DNAs that contain multiple enhancers.

Mentions: We fit the model described above to lacZ expression driven by the four fusion constructs shown in Figure 1 together with three additional fragments of the eve promoter, MSE2, MSE3, and 1700 during T6 (Figure 4A); fits were also performed to the four fusion constructs without the additional fragments (Figure S3). Inclusion of the three additional P-element constructs improved the predictive power of the model at the cost of one additional free position effect scaling parameter for each construct. Our TF dataset contains all of the factors essential for eve regulation in a region extending from the 1–2 eve interstripe to a position just posterior of stripe 7 (35% to 92% EL); additional TFs act on eve in the head and tail regions. These data constituted 406 independent observations of transcription rate corresponding to 58 combinations of nine TF concentrations acting on 7 constructs in each time class. The activators are Bcd, Cad, Drosophila-STAT (D-STAT), and Dichaete. The repressors are Kr, Kni, Gt, Tailless (Tll), and Hb. Of these, Hb was subject to coactivation by Bcd [20] or Cad, and hence it also functions as an activator. Independent experimental data (Text S1) allowed us to define binding thresholds for Hb and Bcd unambiguously, but in the case of other TFs these data implied a range of values for PWM thresholds and we allowed the threshold to be a free parameter within this range.


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)

Training and predictions.(A) Training results for 7 constructs. RNA levels and model results are as shown in the key; the model result trace obscures the data in regions where both are superimposed. The regions of the eve locus used to generate the training data are indicated schematically. (B–G) Predictions of gene expression driven by DNA sequences that were not used for training. The sequences used are fully described in Table S4. Black lines are predicted RNA expression and colored lines are quantitative protein profiles of the corresponding endogenous loci. The scale of relative fluorescence levels for RNA is shown at the left of graphs, that for proteins on the right. All protein patterns are taken from the FlyEx database (http://urchin.spbcas.ru/flyex) [7]. An asterisk on a panel indicates the prediction was not made from model 6: D1-2 are from model 2, G1 is from model 7, and G2 is from model 1. See text, Figure S4 and Table S1 for details. (B) 5 mutant eve enhancers, described fully in the main text. (C) Stripe 2 enhancers from 16 different Drosophila species, with abbreviations and panel numbers (see Table S5 for full species name). The enhancer from D. persimilis (per), D. grimshawi (gri), D. mojavensis (moj) and willistoni (wil) was first identified in this study. (D) Other D. melanogaster eve enhancers. (D1) Stripe 5 enhancer. (D2) Stripe 4/6 enhancer. (D3) pseudoobscura- melanogaster stripe 2 chimera(p1-m2). (D4) melanogaster- pseudoobscura stripe 2 chimera(m1-p2). (E) Stripe 2 (S2E; E1–E6) and stripe 3/7 (S37E, E7–E12) enhancers from 6 Sepsid species, with abbreviations (see Table S5). (F) 5 non- eve enhancers from the D. melanogaster genes hb (F1), Kr (F2), run (F3-4), and h (F5). (G) Large 5′ (G1) and 3′ (G2) eve regulatory DNAs that contain multiple enhancers.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003243-g004: Training and predictions.(A) Training results for 7 constructs. RNA levels and model results are as shown in the key; the model result trace obscures the data in regions where both are superimposed. The regions of the eve locus used to generate the training data are indicated schematically. (B–G) Predictions of gene expression driven by DNA sequences that were not used for training. The sequences used are fully described in Table S4. Black lines are predicted RNA expression and colored lines are quantitative protein profiles of the corresponding endogenous loci. The scale of relative fluorescence levels for RNA is shown at the left of graphs, that for proteins on the right. All protein patterns are taken from the FlyEx database (http://urchin.spbcas.ru/flyex) [7]. An asterisk on a panel indicates the prediction was not made from model 6: D1-2 are from model 2, G1 is from model 7, and G2 is from model 1. See text, Figure S4 and Table S1 for details. (B) 5 mutant eve enhancers, described fully in the main text. (C) Stripe 2 enhancers from 16 different Drosophila species, with abbreviations and panel numbers (see Table S5 for full species name). The enhancer from D. persimilis (per), D. grimshawi (gri), D. mojavensis (moj) and willistoni (wil) was first identified in this study. (D) Other D. melanogaster eve enhancers. (D1) Stripe 5 enhancer. (D2) Stripe 4/6 enhancer. (D3) pseudoobscura- melanogaster stripe 2 chimera(p1-m2). (D4) melanogaster- pseudoobscura stripe 2 chimera(m1-p2). (E) Stripe 2 (S2E; E1–E6) and stripe 3/7 (S37E, E7–E12) enhancers from 6 Sepsid species, with abbreviations (see Table S5). (F) 5 non- eve enhancers from the D. melanogaster genes hb (F1), Kr (F2), run (F3-4), and h (F5). (G) Large 5′ (G1) and 3′ (G2) eve regulatory DNAs that contain multiple enhancers.
Mentions: We fit the model described above to lacZ expression driven by the four fusion constructs shown in Figure 1 together with three additional fragments of the eve promoter, MSE2, MSE3, and 1700 during T6 (Figure 4A); fits were also performed to the four fusion constructs without the additional fragments (Figure S3). Inclusion of the three additional P-element constructs improved the predictive power of the model at the cost of one additional free position effect scaling parameter for each construct. Our TF dataset contains all of the factors essential for eve regulation in a region extending from the 1–2 eve interstripe to a position just posterior of stripe 7 (35% to 92% EL); additional TFs act on eve in the head and tail regions. These data constituted 406 independent observations of transcription rate corresponding to 58 combinations of nine TF concentrations acting on 7 constructs in each time class. The activators are Bcd, Cad, Drosophila-STAT (D-STAT), and Dichaete. The repressors are Kr, Kni, Gt, Tailless (Tll), and Hb. Of these, Hb was subject to coactivation by Bcd [20] or Cad, and hence it also functions as an activator. Independent experimental data (Text S1) allowed us to define binding thresholds for Hb and Bcd unambiguously, but in the case of other TFs these data implied a range of values for PWM thresholds and we allowed the threshold to be a free parameter within this range.

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