Regulation of TAFI Activity by TAF7

TAF7 对 TAFI 活动的调节

• 批准号: 7594827
• 负责人: DINAH SINGER
• 金额: $ 62.4万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594827
• 关键词: Attention; Binding; Biological Assay; CDK9 Protein Kinase; Carboxypeptidase U; Cell physiology; Cells; Class; Complex; Cytochrome P-450 CYP2E1; DNA; DNA Polymerase II; DNA-Directed RNA Polymerase; Dissociation; Event; Gene Expression; General Transcription Factors; Genes; Genetic Transcription; Goals; In Vitro; Individual; Lateral; Libraries; MHC Class I Genes; Mediating; Mediator of activation protein; Modeling; Molecular; Molecular Profiling; Nucleotides; Phosphorylation; Positive Transcriptional Elongation Factor B; Process; Proliferating; Proteins; RNA; RNA Splicing; RNA chemical synthesis; Recombinants; Regulation; Regulatory Pathway; Repression; Research; Role; Series; Signal Transduction; Site; Small Interfering RNA; TAF1 gene; TAF7 gene; TATA-Binding Protein Associated Factors; TATA-Box Binding Protein; Technology; Transcription Elongation; Transcription Factor TFIID; Transcription Initiation; Transcription Process; Transferase; Yeasts; base; cell growth; factor EF-P; human TAF1 protein; in vivo; inhibitor/antagonist; melting; programs; promoter; small hairpin RNA; transcription factor S-II; transcription factor TFIIE; transcription factor TFIIF; transcription factor TFIIH; yeast two hybrid system

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 dNTPs 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. The importance of TAF7 in regulating normal transcription is documented by our finding that cells depleted of TAF7, by siRNA or shRNA technology, proliferate poorly. Given the critical role of TAF7 in cell growth, it is surprising that, using expression profiling, we find that only a subset of genes are regulated by TAF7, consistent with the interpretation that multiple molecular complexes mediate different regulatory pathways.

一般转录因子TFIID由TATA结合蛋白（TBP）和一系列TBP相关因子（TAF）组成，这些因子共同参与转录前起始复合物的组装。TAF之一具有乙酰转移酶（AT）活性，其是MHC I类基因转录所必需的：AT活性的抑制抑制转录。为了鉴定可能调节TAFI的AT活性的潜在细胞因子，用跨越其AT结构域和RAP 74结合结构域的一部分的TAFI区段（848-1279 aa）筛选酵母双杂交文库。分离TFIID组分TAF 7并发现其抑制TAF 1AT活性。重要的是，将重组TAF 7添加到体外转录测定中抑制TAFI依赖性MHC I类转录。因此，TAF 7能够通过调节其AT活性来调节TAFI功能。为了研究TAF 7抑制TAF 1 AT活性在转录中的作用，我们开发了一种体外前起始复合物（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。在该试验中，I类PIC的转录起始于体内观察到的位点，依赖于所有4种dNTP，并被转录抑制剂抑制。我们的研究主要集中在TFIID组分TAF 7在调节从PIC组装到起始和延伸的过渡中的作用。我们发现TAF 7在PIC形成过程中仍然与TFIID相关，但在转录起始和延伸时释放。TAF 7的释放是TAF 1自身磷酸化的结果。令人惊讶的是，伴随着从TAF 1释放，TAF 7结合到转录延伸因子P-TEFb。TAF 7与P-TEFb的结合伴随着TAF 7和CDK 9（P-TEFb的激酶亚基）的磷酸化。重要的是，TAF 7在转录延伸期间保持与P-TEFb和转录延伸复合物相关联。基于这些发现，我们提出了一个模型，其中TAF 7作为一个检查点调节器的功能：抑制转录起始，直到PIC组装完成，然后激活延伸。TAF 7在调节正常转录中的重要性被我们的发现所证明，即通过siRNA或shRNA技术去除TAF 7的细胞增殖不良。鉴于TAF 7在细胞生长中的关键作用，令人惊讶的是，使用表达谱分析，我们发现只有一部分基因受TAF 7调控，这与多个分子复合物介导不同调控途径的解释一致。