Regulation of TAFI Activity by TAF7

通用转录因子TFIID由TATA结合蛋白(TBP)和一系列TBP相关因子(TAFs)组成，TBP相关因子共同参与转录预起始复合体的组装。其中一种TAFs具有转录MHC I类基因所必需的乙酰转移酶(AT)活性：抑制AT活性会抑制转录。为了确定可能调节TAFI AT活性的潜在细胞因子，筛选了一个含有TAFI片段(848-1279氨基酸)的酵母双杂交文库，该片段跨越其AT结构域和RAP74结合域的一部分。TFIID组分TAF7被分离出来，并发现主要与RAP74结合结构域相互作用。TAF7与TAFI结合可抑制其AT活性。重要的是，在体外转录实验中加入重组TAF7可以抑制TAFI依赖的MHC I类转录。因此，TAF7能够通过调节其AT活性来调节TAFI的功能。为了研究TAF7在转录抑制TAF1 AT活性中的作用，我们建立了以MHC I类启动子为模板的体外预起始复合体(PIC)组装实验。在本实验中，PIC的转录起始于体内观察到的位点，依赖于所有4个dNTP，并被转录抑制物抑制。此外，我们发现TAF7与PIC组装。目前正在研究的是TAF7-对TAF1AT活性的抑制被解除，允许转录进行的机制。

The general transcription factor, TFIID, consists of the TATA binding protein (TBP) associated with a series of TBP-associated factors (TAFs) that together participate in the assembly of the transcription preinitiation complex. One of the TAFs, has acetyl transferase (AT) activity that is necessary for transcription of MHC class I genes: inhibition of the AT activity represses transcription. To identify potential cellular factors that might regulate the AT activity of TAFI, a yeast two-hybrid library was screened with a TAFI segment (848-1279 aa) that spanned part of its AT domain and its RAP74 binding domain. The TFIID component, TAF7, was isolated and found to interact predominantly with the RAP74-binding domain. TAF7 binding to TAFI inhibits its AT activity. Importantly, addition of recombinant TAF7 to in vitro transcription assays inhibits TAFI-dependent MHC class I transcription. Thus, TAF7 is capable of regulating TAFI function by modulating its AT activity. To investigate the role in transcription of TAF7 repression of TAF1 AT activity, we have developed an in vitro preinitiation complex (PIC) assembly assay, using the MHC class I promoter as the template. In this assay, transcription from the PIC initiates at the sites observed in vivo, depends on all 4 dNTP's and is inhibited by inhibitors of transcription. Furthermore, we find that TAF7 assembles with the PIC. Currently under investigation is the mechanism by which TAF7-repression of the TAF1 AT activity is relieved, allowing transcription to proceed.