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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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Fusion constructs.(A) The 7 striped expression pattern of eve, visualized with antibody staining. This and other embryos are oriented dorsal up and anterior to the left. The white rectangle located in the middle of the embryo indicates a 10% strip ranging from 35 to 92% embryo length (EL). (B) Schematic view of the eve gene. The transcript (black box) and early acting enhancers are shown. The distance of the 5′ end of each enhancer from the TSS is specified. The colored boxes and adjacent thick lines indicate the two segments of DNA used to create various reporter constructs. (C) (left) Fluorescence in situ hybridization for lacZ mRNA. (right) Segmented image with nuclear mask. Image segmentation was carried out as described [16]. Intense and punctate fluorescent spots in the nuclei are nascent transcripts. (D) Quantitative expression data for Eve protein and 4 fusion constructs, obtained from the area shown in the white rectangle in B. Embryos were classified temporally as belonging to one of eight time classes (T1–T8) in cleavage cycle 14A (C14A), each about 6.5 min long, as described [6]. T1, T6 and T8 data are shown here, with time after egg deposition (AED). The numbers in parentheses are the number of embryos used to generate the averaged expression profiles of each time class. Arrows indicate regions of major alteration in gene expression after spacer removal. (E) lacZ mRNA expression from individual embryos. 4 fusion constructs and their gene expression at T6 are shown.
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pgen-1003243-g001: Fusion constructs.(A) The 7 striped expression pattern of eve, visualized with antibody staining. This and other embryos are oriented dorsal up and anterior to the left. The white rectangle located in the middle of the embryo indicates a 10% strip ranging from 35 to 92% embryo length (EL). (B) Schematic view of the eve gene. The transcript (black box) and early acting enhancers are shown. The distance of the 5′ end of each enhancer from the TSS is specified. The colored boxes and adjacent thick lines indicate the two segments of DNA used to create various reporter constructs. (C) (left) Fluorescence in situ hybridization for lacZ mRNA. (right) Segmented image with nuclear mask. Image segmentation was carried out as described [16]. Intense and punctate fluorescent spots in the nuclei are nascent transcripts. (D) Quantitative expression data for Eve protein and 4 fusion constructs, obtained from the area shown in the white rectangle in B. Embryos were classified temporally as belonging to one of eight time classes (T1–T8) in cleavage cycle 14A (C14A), each about 6.5 min long, as described [6]. T1, T6 and T8 data are shown here, with time after egg deposition (AED). The numbers in parentheses are the number of embryos used to generate the averaged expression profiles of each time class. Arrows indicate regions of major alteration in gene expression after spacer removal. (E) lacZ mRNA expression from individual embryos. 4 fusion constructs and their gene expression at T6 are shown.

Mentions: We sought a small collection of regulatory DNAs which, by driving reporter expression of lacZ RNA, would provide the maximum amount of information on the rules of transcriptional control. eve is a logical source for such regulatory DNA because it is known that the 7 narrow stripes of gene expression (Figure 1A), each about 3 nuclei wide, form by the repressive action of gap gene encoded TFs such as Hunchback (Hb), Kruppel (Kr), Knirps (Kni) and Giant (Gt), expressed in domains 10–15 nuclei wide [6]. eve stripes 2 and 3 are particularly informative. It has been shown that stripe 2 is repressed by Kr, but stripe 3 evades repression by peak levels of Kr [17]. Hb, on the other hand, represses stripe 3 while it activates stripe 2 expression [18], [19]. These observations provide stringent mechanistic constraints on transcriptional regulation which can be made even more stringent by considering fusions of minimal enhancers expressing the two stripes.


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)

Fusion constructs.(A) The 7 striped expression pattern of eve, visualized with antibody staining. This and other embryos are oriented dorsal up and anterior to the left. The white rectangle located in the middle of the embryo indicates a 10% strip ranging from 35 to 92% embryo length (EL). (B) Schematic view of the eve gene. The transcript (black box) and early acting enhancers are shown. The distance of the 5′ end of each enhancer from the TSS is specified. The colored boxes and adjacent thick lines indicate the two segments of DNA used to create various reporter constructs. (C) (left) Fluorescence in situ hybridization for lacZ mRNA. (right) Segmented image with nuclear mask. Image segmentation was carried out as described [16]. Intense and punctate fluorescent spots in the nuclei are nascent transcripts. (D) Quantitative expression data for Eve protein and 4 fusion constructs, obtained from the area shown in the white rectangle in B. Embryos were classified temporally as belonging to one of eight time classes (T1–T8) in cleavage cycle 14A (C14A), each about 6.5 min long, as described [6]. T1, T6 and T8 data are shown here, with time after egg deposition (AED). The numbers in parentheses are the number of embryos used to generate the averaged expression profiles of each time class. Arrows indicate regions of major alteration in gene expression after spacer removal. (E) lacZ mRNA expression from individual embryos. 4 fusion constructs and their gene expression at T6 are shown.
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pgen-1003243-g001: Fusion constructs.(A) The 7 striped expression pattern of eve, visualized with antibody staining. This and other embryos are oriented dorsal up and anterior to the left. The white rectangle located in the middle of the embryo indicates a 10% strip ranging from 35 to 92% embryo length (EL). (B) Schematic view of the eve gene. The transcript (black box) and early acting enhancers are shown. The distance of the 5′ end of each enhancer from the TSS is specified. The colored boxes and adjacent thick lines indicate the two segments of DNA used to create various reporter constructs. (C) (left) Fluorescence in situ hybridization for lacZ mRNA. (right) Segmented image with nuclear mask. Image segmentation was carried out as described [16]. Intense and punctate fluorescent spots in the nuclei are nascent transcripts. (D) Quantitative expression data for Eve protein and 4 fusion constructs, obtained from the area shown in the white rectangle in B. Embryos were classified temporally as belonging to one of eight time classes (T1–T8) in cleavage cycle 14A (C14A), each about 6.5 min long, as described [6]. T1, T6 and T8 data are shown here, with time after egg deposition (AED). The numbers in parentheses are the number of embryos used to generate the averaged expression profiles of each time class. Arrows indicate regions of major alteration in gene expression after spacer removal. (E) lacZ mRNA expression from individual embryos. 4 fusion constructs and their gene expression at T6 are shown.
Mentions: We sought a small collection of regulatory DNAs which, by driving reporter expression of lacZ RNA, would provide the maximum amount of information on the rules of transcriptional control. eve is a logical source for such regulatory DNA because it is known that the 7 narrow stripes of gene expression (Figure 1A), each about 3 nuclei wide, form by the repressive action of gap gene encoded TFs such as Hunchback (Hb), Kruppel (Kr), Knirps (Kni) and Giant (Gt), expressed in domains 10–15 nuclei wide [6]. eve stripes 2 and 3 are particularly informative. It has been shown that stripe 2 is repressed by Kr, but stripe 3 evades repression by peak levels of Kr [17]. Hb, on the other hand, represses stripe 3 while it activates stripe 2 expression [18], [19]. These observations provide stringent mechanistic constraints on transcriptional regulation which can be made even more stringent by considering fusions of minimal enhancers expressing the two stripes.

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