CHROMATIN, TAFS, AND A NOVEL MULTIPROTEIN COACTIVATOR ARE REQUIRED FOR SYNERGISTIC ACTIVATION BY SP1 AND SREBP-1A IN-VITRO

The promoter selectivity factor Sp1 often cooperates with other enhanc er-binding proteins to activate transcription. To study the molecular underpinnings of these regulatory events, we have reconstituted in vit ro the synergy observed in vivo between Sp1 and the sterol-regulated f actor SREBP-1a at the low density lipoprotein receptor (LDLR) promoter . Using a highly purified human transcription system, we found that ch romatin, TAFs, and a novel SREBP-binding coactivator activity, which i ncludes CBP, are all required to mediate full synergistic activation b y Sp1 and SREBP-1a. The SREBP-binding domain of CBP inhibits activatio n by SREBP-1a and Sp1 in a dominant-negative fashion that is both chro matin- and activator-specific. Whereas recombinant CBP alone is not su fficient to mediate activation, a human cellular fraction containing C BP can support high levels of chromatin-dependent synergistic activati on. Purification of this activity to near homogeneity resulted in the identification of a multiprotein coactivator, including CBP, that sele ctively binds to the SREBP-1a activation domain and is capable of medi ating high levels of synergistic activation by SREBP/Sp1 on chromatin templates. The development of a reconstituted chromatin transcription system has allowed us to isolate a novel coactivator that is recruited by the SREBP-1a activation domain and that functions in concert with TFIID to coordinate the action of multiple activators at complex promo ters in the context of chromatin.