Isolation of mouse TFIID and functional characterization of TBP and TFIID in mediating estrogen receptor and chromatin transcription

TFIID is a general transcription factor required for the assembly of the tr anscription machinery on most eukaryotic promoters transcribed by RNA polym erase II. Although the TATA-binding subunit (TBP) of TFIID is able to suppo rt core promoter and activator-dependent transcription under some circumsta nces, the roles of TBP-associated factors (TAF(II)s) in TFIID-mediated acti vation remain unclear. To define the evolutionarily conserved function of T FIID and to elucidate the roles of TAF(II)s in gene activation, we have clo ned the mouse TAF(II)55 subunit of TFIID and further isolated mouse TFIID f rom a murine FM3A-derived cell line that constitutively expresses FLAG-tagg ed mouse TAF(II)55. Both mouse and human TFIIDs are capable of mediating tr anscriptional activation by Ga14 fusions containing different activation do mains in a highly purified human cell-free transcription system devoid of T FIIA and Mediator. Although TAF(II)-independent activation by Ga14-VP16 can also be observed in this highly purified human transcription system with e ither mouse or yeast TBP, TAF(II)s are strictly required for estrogen recep tor-mediated activation independently of the core promoter sequence. In add ition, TAF(II)s are necessary for transcription from a preassembled chromat in template. These findings clearly demonstrate an essential role of TAF(II )s as a transcriptional coactivator for estrogen receptor and in chromatin transcription.