HOX11-dependent repression may be mediated by interactions with components of the basal general transcription machinery. (A) Schematic diagram of the SV40 promoter in control and mutant reporter plasmids: enhancer region (En); GC BOX (GC); TATA-like motif (TATA). (B) Deletion (Δ) of cis-regulatory elements, including the enhancer, GC box, and TATA-like motif from the SV40 promoter, failed to abolish HOX11-mediated repression. Relative CAT activities from cotransfection of pFLAG-HOX11 and parental or mutant SV40 reporter plasmids into C2C12 cells (▪) and HeLa cells (□). Transfection assays were repeated at least 3 times. Variability between assays was not more than 15%. (C) HOX11 represses in vitro transcription driven by purified RNA polymerase II holoenzyme. In vitro transcription assays were performed using 0.3 μg reporter plasmid containing the adenovirus major late promoter driving a G-less cassette (G-less) and 5 tandem copies of the Gal4 DNA-binding site.21 Reaction conditions consisted of 5 μg purified polymerase II holoenzyme and 100 ng purified S-tagged HOX11 or PET control protein. Transcription was measured in the absence or presence of purified Gal4-VP16 proteins (50 ng). Radiolabeled RNA was resolved on a 4.5% acrylamide-6M urea denaturing gel and visualized by autoradiography.
