Regulation of TAFI Activity by TAF7

一般转录因子TFIID由TATA结合蛋白（TBP）和一系列TBP相关因子（TAFs）组成，这些因子共同参与转录起始前复合物的组装。其中一种TAFs具有乙酰转移酶（AT）活性，这是MHC I类基因转录所必需的：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，我们筛选了一个酵母双杂交文库，其中TAFI片段（848-1279 aa）跨越了部分AT结构域和RAP74结合结构域。分离得到TFIID组分TAF7，发现其抑制TAF1 AT活性。重要的是，在体外转录分析中加入重组TAF7可抑制tafi依赖性MHC I类转录。因此，TAF7能够通过调节其AT活性来调节TAFI功能。为了研究TAF7抑制TAF1 AT活性在转录中的作用，我们开发了一种体外预起始复合物（PIC）组装试验，使用MHC I类启动子作为模板。PIC的组装由通用转录因子（GTF） TFIID成核。在这项实验中，I类PIC的转录在体内观察到的位点开始，依赖于所有4个dNTP，并被转录抑制剂抑制。我们的研究集中在TFIID成分TAF7在调节PIC组装到起始和延伸的转变中的作用。我们发现TAF7在PIC形成过程中仍然与TFIID相关，但在转录起始和延伸时被释放。TAF7的释放源于TAF1的自磷酸化。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TAF7和CDK9 （P-TEFb的激酶亚基）的磷酸化。重要的是，在转录延伸过程中，TAF7仍然与P-TEFb和转录延伸复合物相关。基于这些发现，我们提出了一个TAF7作为检查点调节器的模型：抑制转录起始，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.