Ligand-dependent activation of transcription in vitro by retinoic acid receptor alpha retinoid X receptor alpha heterodimers that mimics transactivation by retinoids in vivo

All-trans and 9-cis retinoic acids (RA) signals are transduced by retinoic acid receptor/retinoid X receptor (RAR/RXR) heterodimers that act as functi onal units controlling the transcription of RA-responsive genes. With the a im of elucidating the underlying molecular mechanisms, we have developed an in vitro transcription system using a chromatin template made up of a mini mal promoter and a direct repeat with 5-spacing-based RA response element. RAR alpha and RXR alpha were expressed in and purified from baculovirus-inf ected Sf9 cells, and transcription was carried out by using naked DNA or ch romatin templates. Transcription from naked templates was not affected by t he presence of RA and/or RAR/RXR heterodimers. in contrast, very little tra nscription occurred from chromatin templates in the absence of RA or RAR/RX R heterodimers whereas their addition resulted in a dosage-dependent stimul ation of transcription that never exceeded that occurring on naked DNA temp lates. Most importantly, the addition of synthetic agonistic or antagonisti c retinoids to the chromatin transcription system mimicked their stimulator y or inhibitory action irt vivo, and activation by a RXR-specific retinoid was subordinated to the binding of an agonist ligand to the RAR partner. Mo reover, the addition of the p300 coactivator generated a synergistic enhanc ement of transcription. Thus, the dissection of this transcription system u ltimately should lead to the elucidation of the molecular mechanisms by whi ch RAR/RXR heterodimers control transcription in a ligand-dependent manner.