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

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.

Figure 1. 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 . 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 . 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.

Figure 2. Model equations: TF binding to DNA.

The model equations for binding site prediction (Equation 1 and 2), cooperative and competitive binding (Equation 3) and fractional occupancy calculation (Equation 4) are shown together in a flow diagram with cartoons of each mechanism on the left and an example application in blue with 5 TF binding sites. Subgrouping process partitioning the binding sites into independent binding groups allows faster computation without losing accuracy. In the example, we set the range of quenching to 20 bp.

Figure 3. Model equations: protein–protein interactions.

The model equations for coactivation (Equation 5), short-range quenching (Equation 6), direct repression (Equation 7), adaptor factor recruitment (Equation 8 and 9) and activation synergy (Equation 10) are shown together in a flow diagram with cartoons of each mechanism on the left and an example application in blue with 5 TF binding sites.

Figure 4. 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) . 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.

Figure 5. 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.