Figure EV1. DNA affinity purifications uncover differentially bound proteins at functionally distinct promoters.

1. Heat map of ~17,000 Drosophila melanogaster protein‐coding gene promoters displaying % match to position weight matrix (PWM) score. Promoters were clustered with k‐means clustering. Nine clusters emerged which display developmental (clusters 1–3) and housekeeping (clusters 5–7) motifs. Promoters in cluster 4 are enriched in Ohler8 and E‐box motifs and can respond to both developmental and housekeeping coactivators as defined by Haberle, V. et al, 2019. Cluster 9 had no strong matches to any motif PWM.
2. Pie chart of all expressed Drosophila melanogaster protein‐coding gene promoters (~170,000) grouped based on motif content (left), and all expressed protein‐coding genes from Drosophila S2 cells (~10,000). Only the main motif groups studied in this paper that are classified as housekeeping or developmental are shown. Group labeled as “other” contains promoters with motifs such as Ohler 8 and E‐box or not motifs which could not be assigned as developmental or housekeeping.
3. Luciferase activity assay measuring the basal or activated state of tested core promoter fragments. To measure basal activity, 121‐bp‐long promoter fragments cloned upstream of a luciferase gene (P). To measure the activated state of the core promoters we cloned the Drosophila Zdfh1 enhancer upstream of the promoter fragments (E + P). Plasmids were transfected into Drosophila S2 cells and activity was measured after 48 h. Firefly luciferase values were normalized to co‐transfected Renilla luciferase values to control for transfection efficiency. Error bars represent standard deviation across four biological replicates.
4. Rank plot of protein binding enrichment on TATA and DRE promoters over the control DNA pool from the DNA‐purification mass spectrometry assay. Highlighted proteins are the Pol II PIC components and the DRE binding factor DREF.
5. DNA‐purification assay with a pool of 25 TATA‐box promoters, and two individual TATA‐box promoters in which the TATA‐box was mutated (left panel). The assay was performed with a nuclear extract expressing TBP‐FLAG that was tracked with a western blot. DNA‐purification of a pool of 20 DRE promoters and three individual DRE promoters in which the DRE motif was mutated. The assay was performed with a nuclear extract expressing DREF‐FLAG and followed with a western blot (right panel). Note that DREF binding is reduced to background levels while TBP is still slightly enriched compared with negative controls, consistent with TBP binding to non‐TATA‐box developmental core promoters (Fig 2B and E).