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。分离得到TFIID组分TAF7，并发现其抑制TAF1AT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。PIC的组装是由通用转录因子(GTF)TFIID完成的。在本实验中，I类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。我们的研究集中在TFIID组件TAF7在调节从PIC组装到起始和延伸的转变中的作用。我们发现，在PIC的形成过程中，TAF7仍然与TFIID相关，但在转录起始和延伸时会释放出来。TAF7的释放是TAF1自动磷酸化的结果。令人惊讶的是，伴随着TAF1的释放，TAF7与转录延伸因子P-TEFb结合。TAF7与P-TEFb的结合伴随着P-TEFb的激酶亚基TAF7和CDK9的磷酸化。重要的是，在转录延伸过程中，TAF7仍然与P-TEFb和转录延伸复合体相关。基于这些发现，我们提出了一个TAF7作为检查点调节因子的模型：抑制转录启动，直到PIC组装完成，然后激活延伸。

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 inhibit TAF1 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. The assembly of the PIC is nucleated by the general transcription factor (GTF), TFIID. In this assay, transcription from the class I PIC initiates at the sites observed in vivo, depends on all 4 dNTP's and is inhibited by inhibitors of transcription. Our study has focused on the role of the TFIID component, TAF7, in regulating the transition from PIC assembly to initiation and elongation. We found that TAF7 remains associated with TFIID during the formation of the PIC, but is released upon transcription initiation and elongation. The release of TAF7 results from autophosphorylation of TAF1. Surprisingly, concomitant with release from TAF1, TAF7 binds to the transcription elongation factor, P-TEFb. Binding of TAF7 to P-TEFb is accompanied by the phosphorylation of both TAF7 and CDK9, the kinase subunit of P-TEFb. Importantly, TAF7 remains associated with P-TEFb and the transcription elongation complex during transcription elongation. Based on these findings, we propose a model in which TAF7 functions as a check-point regulator: inhibiting transcription initiation until PIC assembly is complete and then activating elongation.