Recapitulation of the DES-dependent chromatin-independent transcriptional repression by ETO in vitro. (A) Top panel, scheme for in vitro transcription from chromatin templates with ETO added at various time points. The experiment is essentially the same as that shown in Fig. 5G except that purified TFIID, equivalent to 5 ng of TBP, was included at the PIC assembly step to facilitate transcription by Gal4-HEB(1-548) from chromatin templates. Bottom panel, effects of ETO added at different indicated steps on transcriptional activation by Gal4-HEB(1-548). (B) Top panel, scheme for in vitro transcription from naked DNA templates. Bottom panel, effects of ETO on Gal4-HEB(1-548)-dependent activation and basal transcription. (C) Schematic representation of ETO and ETO derivatives. (D) Effects of ETO and ETO derivatives on Gal4-HEB(1-373)-dependent transcription from naked DNA templates. WT, wild-type ETO; Δ403-post-PIC, ETOΔ403 was added after PIC assembly (i.e., with nucleoside triphosphates). (E) Effects of ETO and ETO derivatives on Gal4-HEB-AD1- or Gal4-HEB(1-373)-dependent transcription from naked DNA templates. (F) Effects of ETO, ETOΔ403, AML1-ETO, ETO-2, or MTGR1 on Gal4-HEB(1-373)-dependent transcription from naked DNA templates. Right panel, Coomassie blue staining of purified ETO-2, MTGR1, and AML1-ETO proteins following sodium dodecyl sulfate-polyacrylamide gel electrophoresis.