Promoter binding and activity of genomic EWSR1/FLI1 and E2F3 binding regions. (A) Firefly luciferase reporter assays for 10 arbitrarily chosen genes identified by ChIP-seq as EWSR1/FLI1 and E2F3 target genes. Promoter fragments (CDK2: −122/+458; E2F3: −272/+327; RAD51: −186/+164; VRK1: −269/+100; RFC2: −400/+25; ATAD2: −368/+202; RRM2: −463/+191; GEMIN4: −275/+87; MFLI1P: −251/+70; SKP2: −240/348) were cloned into the pGL4.10 vector (Promega) and tested for responsiveness to conditional EWSR1/FLI1 knockdown in A673 ES cells 48 h after doxycycline-induced EWSR1/FLI1 shRNA induction (dark green) or shRNA-induced knockdown of E2F3 (red). As negative controls, promoter activities of an expressed gene that does not show a change in mRNA expression after the EWSR1/FLI1 knockdown (PRKCI: −139/265), and of the empty vector (pGL4.10) are shown. The y-axis represents the promoter activity relative to control conditions. Means and standard deviations of at least three independent experiments, each performed in triplicate, are shown. (B) Fold changes in reporter activity of wild-type and mutant ATAD2 reporter constructs in the presence (light green) and doxycycline-induced absence (dark green) of EWSR1/FLI1 48 h after EWSR1/FLI1 shRNA induction. See Supplemental Figure S3 for ChIP and luciferase assays.
