Fig. 6

A fragment from CoolUp gene pastrel with increased enhancer activity at low temperature. (A) Increased DNA accessibility at low temperature in region (grey shading) in the 5′ region of pst, a gene with higher transcript levels at low temperature. The genomic pst region is shown schematically (top), as well as browser tracks obtained from S2R+ cells at the indicated temperatures (average of three biological replicates) by either ATAC-Seq data (middle) or 3′ RNA-Seq (bottom). (B) Quantification of pst transcript levels at the indicated temperatures in S2R+ cells (top) and adult male flies (bottom). Results from two independent analyses, by microarray and 3′ RNA-Seq, respectively, are displayed in case of S2R+ cells. The data for flies was obtained by 3′ RNA-Seq. Average of three biological replicates and s.d. are shown, relative to expression at 25 °C, which was set to 1. (C) Quantification of EGFP-Pst protein expression levels at the indicated temperatures by flow cytometry. Culture aliquots of S2R + _g-EGFP-pst cells were shifted to the indicated temperatures and analyzed at the indicated times after the shift. Average of three biological replicates and s.d. are shown, relative to expression at 25 °C, which was set to 1. (D) Temperature dependence of the enhancer activity of the pst_E1 fragment (shaded region in panel A) analyzed after RMCE with SR9rg cells. For comparison the fragments Hsp23_E2, sqh_E2 and 20 × UAS were analyzed in parallel. After RMCE, cells were shifted eventually to the indicated temperatures and 48 h later analyzed by flow cytometry