Fig. 7

Characterization of the CoolUp enhancer from pastrel. (A) The CoolUp enhancer activity of the CoolOpen region in the pst 5′ region identified by ATAC-Seq (pst_E1, light grey shading) was further analyzed with terminal and internal deletion series. Comparison of E1n and E1o revealed an internal region essential for enhancer activity (dark grey shading). The internal deletions d1–8 eliminate predicted transcription factor binding sites. The consecutive 5 bp deletions d9–18 scan the d4-d5 region. (B-D) Comparison of enhancer activity of E1 and derived fragments at the indicated temperatures as detected after RMCE with SR9rg cells. Bar diagrams display the median GFP signal intensity in the scatter plot window with cells expressing green but not red fluorescence. Analysis of the fragments E1, E1n and E1o (B), E1n derivatives carrying one of the deletions d1–8 (C), or one of the deletions d9–18 (D). Average of duplicates +/− s.d. shown in (B), and values from a single experiment in (D). (E) Characterization of the role of transcription factors (TFs) with predicted binding sites in E1n. The indicated TFs were depleted in SR9rg > pst_E1n (mRuby⁻, GFP⁺) cells, followed by a shift of culture aliquots to the indicated temperature and subsequent flow cytometric analysis. Bar diagram represents median GFP intensity of the cell population lacking red fluorescence. In most cases, two independent dsRNA preparations generated from distinct amplicons (xy_1 and xy_2) were used for depletion. Untreated S2R+ cells and SR9rg > pst_E1n (mRuby⁻, GFP⁺) cells treated with lacZ dsRNA were used as negative and positive control, respectively. (F) Schematic summary model for the control of pst_E1 CoolUp enhancer activity. The JAK/STAT signaling pathway (with the transmembrane receptor Dome and the downstream TF STAT92E) acts positively. The TFs Pnt and Ets97D act as competing activator and repressor, respectively, presumably downstream of the Pvr receptor tyrosine kinase