Wu S Y, Thomas M C, Hou S Y, Likhite V, Chiang C M
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
J Biol Chem. 1999 Aug 13;274(33):23480-90. doi: 10.1074/jbc.274.33.23480.
TFIID is a general transcription factor required for the assembly of the transcription machinery on most eukaryotic promoters transcribed by RNA polymerase II. Although the TATA-binding subunit (TBP) of TFIID is able to support core promoter and activator-dependent transcription under some circumstances, the roles of TBP-associated factors (TAF(II)s) in TFIID-mediated activation remain unclear. To define the evolutionarily conserved function of TFIID and to elucidate the roles of TAF(II)s in gene activation, we have cloned the mouse TAF(II)55 subunit of TFIID and further isolated mouse TFIID from a murine FM3A-derived cell line that constitutively expresses FLAG-tagged mouse TAF(II)55. Both mouse and human TFIIDs are capable of mediating transcriptional activation by Gal4 fusions containing different activation domains in a highly purified human cell-free transcription system devoid of TFIIA and Mediator. Although TAF(II)-independent activation by Gal4-VP16 can also be observed in this highly purified human transcription system with either mouse or yeast TBP, TAF(II)s are strictly required for estrogen receptor-mediated activation independently of the core promoter sequence. In addition, TAF(II)s are necessary for transcription from a preassembled chromatin template. These findings clearly demonstrate an essential role of TAF(II)s as a transcriptional coactivator for estrogen receptor and in chromatin transcription.
TFIID是一种通用转录因子，对于在大多数由RNA聚合酶II转录的真核启动子上组装转录机制而言是必需的。尽管TFIID的TATA结合亚基（TBP）在某些情况下能够支持核心启动子和激活因子依赖性转录，但TBP相关因子（TAF(II)s）在TFIID介导的激活中的作用仍不清楚。为了确定TFIID在进化上保守的功能并阐明TAF(II)s在基因激活中的作用，我们克隆了TFIID的小鼠TAF(II)55亚基，并进一步从组成型表达FLAG标签的小鼠TAF(II)55的鼠源FM3A衍生细胞系中分离出小鼠TFIID。在不含TFIIA和中介体的高度纯化的人无细胞转录系统中，小鼠和人TFIID都能够通过含有不同激活域的Gal4融合蛋白介导转录激活。尽管在这个高度纯化的人转录系统中，使用小鼠或酵母TBP也能观察到Gal4-VP16的不依赖TAF(II)的激活，但雌激素受体介导的激活严格需要TAF(II)s，且不依赖于核心启动子序列。此外，TAF(II)s对于从预组装的染色质模板进行转录是必需的。这些发现清楚地证明了TAF(II)s作为雌激素受体转录共激活因子以及在染色质转录中的重要作用。
