RECOMBINANT ACTIVATION DOMAINS OF VIRION PROTEIN-16 AND HUMAN ESTROGEN-RECEPTOR GENERATE TRANSCRIPTIONAL INTERFERENCE IN-VITRO BY DISTINCT MECHANISMS

Overexpression of transcriptional activators in transfection assays ma y inhibit their own activity or interfere with trans-activation by dif ferent sequence-specific transcription factors. In this study we show that this phenomenon of transcriptional interference (squelching) can be mimicked in vitro by adding recombinant activation domains to nucle ar extracts. We demonstrate that the acidic activation domain of virio n protein 16 interferes both with basal transcription from TATA-box pr omoters and promoters activated by various trans-activators in two dif ferent mammalian cell-free transcription systems. This suggests that v irion protein 16 interacts with and thereby sequesters a basal transcr iption factor. In contrast the recombinant activation function 2 (AF-2 ) of human estrogen receptor does not affect basal promoter activity b ut inhibits TATA promoters activated by human progesterone receptor (h PR) or Sp1 as well as the beta-globin and adenovirus major late promot er. By analyzing the effects of AF-2 on DNA binding of hPR and Spl we found that AF-2 inhibits the DNA binding activity of hPR, but not Sp1. Our data suggest that the recombinant AF-2 squelches Spl trans-activa tion by sequestering a common coactivator(s), whereas hPR function mig ht be inhibited due to competition for a common cofactor stabilizing h PR dimers or through the formation of inactive heterodimers between AF -2 and hPR.