Figure 1(A) The promoter region was divided into 7 building blocks: block 1 with 239 bp of a potential -1 nucleosomal sequence; block 2 with 73 bp sequence representing the ecdysone receptor binding region; block 3-6 with 131 bp sequence representing the native and perturbative core promoter regions from different architectures; block 7 with 240 bp of a potential +1 nucleosomal sequence. (B) Synthetic promoter design - building blocks. The promoter region (sketch in lower panel) was divided into 7 building blocks: block 1 with 239 bp sequence representing a potential -1 nucleosome; block 2 with 73 bp sequence representing the ecdysone receptor binding region; block 3-6 with 131 bp sequence representing the native and perturbative core promoter regions from different architectures; block 7 with 240 bp sequence representing a potential +1 nucleosome. ( (C) Control co-transfected vector (backbone not represented) used for data normalization (Material and Methods), and consisting in a pTran promoter driving the expression of the Renilla Luciferase gene. (D) Simplified dual luciferase assay experimental workflow. To measure promoter activity quantitatively on a large scale with high reproducibility we integrated the golden gate cloning strategy (BsaI cloning) with a high-throughput experimental pipeline using automated robot systems for colony picking, reporter plasmids isolation, transient co-transfection and dual luciferase assay (Figure EV1 and Materials and Methods for details). ( (E) Normalized expression levels of the native core promoters. Their activities spanned over a broad range of three orders of magnitude (promoter constructs contained block 1.11 and block 7.11 combination as nucleosomal sequences). Each color represents a different class of core promoter architecture. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements. (including new cell transfection procedures and measurements of promoter activities). (F) Confocal fluorescence sections of living D. mel. embryos (after ~40min at stage5 during embryonic development) expressing an optimized reporter mNeonGreen protein, and carrying hunchback anterior enhancer - tested core promoter - mNeon. The fluorescence signal of mNeonGreen can be seen (in false colors) in the nuclei at the embryo peripheries. The promoters tested correspond to motif knockouts or motif substitution with consensus sequences from the constitutive MED4 (on the left) and the developmental pain (on the right) promoters. The ko motifs or the type of mutations are indicated in white, together with the normalized expression levels (in bracket) measured with our Luciferase assay pipeline. Whereas MED4 promoters drive strong expression along the entire anterior (A) - posterior (P) axis of the embryo, pain embryos show weaker expression, consistently with the expression levels measured in the Luciferase assay. Noteworthy, in contrast to the homogenous AP expression with the constitutive MED4 gene, the A-P expressions patterns for developmental pain resemble the known AP gradient of expression typically observed for the Hb enhancer. The white arrows indicate the fluorescence signals of the nuclei in the anterior part of the embryos. (G - H) Quantification of the expression patterns in developing embryos, projected along the A-P axis for MED4 (G) and for pain (H) for the different promoter variants, respectively. The errors bars are standard deviations from 3-4 biological replicates measurements (different embryos). The fluorescence background measured in a wild-type embryo is shown as yellow dotted lines. The fluorescence patterns for pain recapitulate the typical hb_ant enhancer activity, characterized by a gradient of reporter expression (black arrow in H) with a sharp drop at around A-P=50%, which was expected for a developmental gene. An exemplary AP profile for the hb_ant enhancer is shown as grey empty triangles (the background was adjusted at about 3000 a.u for better comparison). In contrast, the constructs containing the constitutive MED4 gene promoter lead to a stronger and more homogeneous expression with an only slightly enhanced expression level at the anterior tip (black arrow in G). (I) Scatter plot of expression levels obtained in D. mel. S2 cells by our Luciferase assay pipeline vs mNeonGreen reporter expression in living D. mel. embryos, revealing a high correlation (Pearson coefficient 0.91) between the two datasets. Error bars represent standard deviations of 3-4 biological replicate measurements.