TAF7: A Check-point Regulator in Transcription Initiation

TAF7：转录起始中的检查点调节器

• 批准号: 9556789
• 负责人: Dinah S. Singer
• 金额: $ 30.82万
• 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/9556789
• 关键词: Ablation; Acetyltransferase; Affect; Attention; Binding; Blood Coagulation Factor VII; Cell Differentiation process; Cell Nucleus; Cell Proliferation; Cell physiology; Cells; Complex; Cytoplasm; DNA; DNA Polymerase II; DNA-Directed RNA Polymerase; Development; Dissociation; Elements; Embryo; Enhancers; Event; Excision; Gel Chromatography; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; HIV; Immunoprecipitation; In Vitro; Kinetics; Knock-out; Knockout Mice; Lateral; MHC Class I Genes; Malignant Neoplasms; Maps; Mediating; Mediator of activation protein; Modeling; Molecular; Molecular Chaperones; Molecular Profiling; Mutation; Nuclear; Nucleotides; Peripheral; Phosphorylation; Phosphotransferases; Play; Polyribosomes; Positive Transcriptional Elongation Factor B; Process; Proliferating; Proteins; RNA; RNA Splicing; RNA Transport; RNA chemical synthesis; RNA-Binding Proteins; Regulation; Repression; Research; Ribosomal Proteins; Role; Series; Signal Transduction; Site; Small Interfering RNA; Stimulus; T-Cell Development; T-Lymphocyte; TAF1 gene; TAF7 gene; TATA-Binding Protein Associated Factors; TATA-Box Binding Protein; Technology; Thymocyte Development; Thymus Gland; Trans-Activators; Transcript; Transcription Elongation; Transcription Initiation; Transcription Process; Translations; Validation; cell growth; cell type; factor EF-P; in vivo; interest; malignant phenotype; melting; novel; prevent; programs; promoter; response; small hairpin RNA; thymocyte; transcription factor; transcription factor S-II; transcription factor TFIIE; transcription factor TFIIF; transcription factor TFIIH

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. The largest TFIID component, TAF1 has both acetyltransferase (AT) and kinase activities. We demonstrated previously that TAF1 is necessary for the transcription of an MHC class I gene, and that its intrinsic acetyltransferase activity is essential for both in vitro transcription of naked DNA and in vivo transcription. Of particular interest, MHC class I transcription is inhibited, both in vitro and in vivo, by the transactivator, HIV Tat, which we demonstrated binds to the TAF1 AT domain and inhibits its enzymatic activity. Similarly, TAF7, a 55 kD TFIID component, binds to TAF1 and inhibits its AT activity, resulting in repression of MHC class I transcription. Thus, TAF7 is an intrinsic regulator of transcription. Upon PIC assembly, TAF7 is released from TFIID, relieving the repression of the TAF1 AT activity and allowing transcription initiation. We have shown that after its dissociation from the PIC, TAF7 functionally interacts with the general transcription factor TFIIH, the CTD kinase BRD4 and the elongation factor P-TEFb. Association of TAF7 with TFIIH inhibits its CDK7 kinase activity with the resultant inhibition of TFIIH-mediated phosphorylation of the Pol II CTD Ser-5; binding of TAF7 to BRD4 inhibits its phosphorylation of Ser-2 of the Pol II CTD preventing pause release, binding of TAF7 to the P-TEFb elongation complex inhibits its phosphorylation of Ser-2, 5 of the Pol II CTD associated with elongation. Importantly, we have shown that TAF7 functions in vitro to inhibit transcription at steps after PIC assembly and in vivo co-localizes with P-TEFb and Pol II downstream of the promoter. Thus, in addition to its role in transcription initiation as a TFIID component, TAF7 also functions in the transition from PIC assembly to initiation and elongation. We propose a novel model in which TAF7 regulates the orderly progression of events in transcription, preventing transcription elongation until the steps of transcription initiation are completed and the transcription elongation complex (TEC) is fully assembled. In addition to the regulatory role it plays within TFIID, TAF7 also functions to regulate TAF1-independent transcription. The first evidence for this came from our observation that TAF7 exists in a TFIID-independent form that coelutes with complexes in the 230 Kd range on gel filtration. Interestingly, the kinetics and spectrum of genes affected by siRNA mediated depletion of TAF7 or TAF1 differs between the two. This finding supports the conclusion that TAF7 functions at multiple points in transcription, not just in PIC assembly. 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. Consistent with the critical role of TAF7 in cell growth, expression profiling reveals that depletion of TAF7 by siRNA results in reduced expression of a large number of genes. We have discovered that in addition to the regulatory role it plays in transcription, TAF7 also functions as an chaperone that delivers newly synthesized RNA from the site of transcription to polysomes in the cytoplasm. Thus, TAF7 is an RNA binding protein which shuttle from the nucleus to the cytoplasm. Consistent with TAF7 playing a functional role in the cytoplasm, we have mapped both NLS and NES elements within the protein. Mutation of the NES sequence results in reduced translation, consistent with a requirement for TAF7 delivery of RNA to polysomes. The cytoplasmic TA7 is found in a large 450kD complex that also contains many ribosomal proteins. Immunoprecipitation studies have validated the binding of TAF7 to ribosomal proteins and demonstrated that this binding is RNA dependent. Further validation is that TAF7 is recovered from purified polysomes. Taken together, these findings demonstrate that TAF7 is not only a checkpoint regulator of transcription but also functions to promote translation. Intriguingly, the TAF7 cytoplasmic/nuclear ratio differs among different cell types, increasing with increasingly malignant phenotypes. Current studies are directed at elucidating the role of TAF7 in malignancy. To further examine the role of TAF7 in global gene regulation and development we have generated conditional TAF7 knock-out mice. Complete ablation of TAF7 results in embryonic lethality; embryos do not develop beyond d.3-4. Excision of TAF7 in MEFs derived from conditional knock-outs results in aborted cell proliferation and a global reduction in transcription initiation. TAF7 is also essential for the differentiation of immature thymic precursors that undergo massive proliferation during their differentiation. Thus, TAF7 deletion mediated by cre, expressed under the control of the Lck promoter that functions in very early DN thymocytes, ablates all T cell development. In sharp contrast, TAF7 is not required for the subsequent proliferation-independent differentiation of lineage committed thymocytes. Deletion of TAF7 by cre under the control of a EIII8 enhancer, which only functions at the DP stage of thymocyte development spares the maturation of single positive cells which are able to egress into the periphery. However, although only a small number of transcripts is affected by TAF7 deletion, TAF7-deficient peripheral T cells are not able to undergo activation and expansion in response to antigenic stimuli. We conclude that TAF7 plays a critical role in transcription in cells that undergo proliferation, but that its role is more restricted in non-proliferating differentiated cells.

一般的转录因子TFIID由与一系列与TBP相关因子（TAF）相关的TATA结合蛋白（TBP）组成，这些因子（TAF）共同参与了转录预发起络合物的组装。 TAF1最大的TFIID成分具有乙酰转移酶（AT）和激酶活性。我们先前证明了TAF1对于MHC I类基因的转录是必需的，并且其内在的乙酰基转移酶活性对于裸DNA的体外转录和体内转录都是必不可少的。尤其感兴趣的是，Intractivator HIV TAT抑制了MHC I类转录的体外和体内，我们证明了它与域的TAF1结合并抑制其酶促活性。同样，TAF7是55 kD TFIID成分，与TAF1结合并抑制其AT活性，从而抑制MHC I类转录。因此，TAF7是转录的内在调节剂。 PIC组装后，TAF7从TFIID中释放出来，从而缓解了在活动时TAF1的抑制并允许转录启动。我们已经表明，在与PIC解离后，TAF7在功能上与一般转录因子TFIIH，CTD激酶BRD4和伸长因子P-TEFB相互作用。 TAF7与TFIIH的缔合抑制其CDK7激酶活性，从而抑制了TFIIH介导的POL II CTD Ser-5的TFIIH介导的磷酸化； TAF7与BRD4的结合抑制了其在Pol II CTD的Ser-2磷酸化，以防止停顿释放，TAF7与P-TEFB伸长率的结合抑制了与伸长率相关的Pol II CTD的Ser-2，5。重要的是，我们已经表明，TAF7在体外功能可在PIC组装后的步骤和体内与启动子下游的P-TEFB和POL II共定位。因此，除了其作为TFIID成分的转录启动中的作用外，TAF7还起作用从PIC组装到启动和伸长的过渡。我们提出了一个新型模型，其中TAF7调节转录中事件的有序进展，防止转录伸长，直到转录启动的步骤完成并完全组装了转录伸长率（TEC）。除了它在TFIID中扮演的调节作用外，TAF7还起作用可调节非依赖TAF1的转录。第一个证据来自我们的观察结果，即TAF7以TFIID独立的形式存在，该形式与凝胶过滤的230 kD范围内的复合物相结合。有趣的是，两者之间受siRNA介导的TAF7或TAF1耗竭影响的基因的动力学和光谱在两者之间有所不同。该发现支持以下结论：TAF7在转录中的多个点起作用，而不仅仅是在PIC组装中。 TAF7在调节正常转录中的重要性是通过我们的发现，即通过siRNA或SHRNA技术耗尽TAF7的细胞繁殖较差。与TAF7在细胞生长中的临界作用一致，表达分析表明，siRNA对TAF7的耗竭导致大量基因的表达降低。我们已经发现，除了它在转录中所扮演的调节作用外，TAF7还充当伴侣，可提供从转录部位提供新合成的RNA，从细胞质中的多聚群。因此，TAF7是一种RNA结合蛋白，它从核从细胞核穿过细胞质。与TAF7在细胞质中起功能作用一致，我们在蛋白质中绘制了NLS和NES元素。 NES序列的突变导致翻译减少，这与TAF7递送RNA向多个多粒子的需求一致。细胞质TA7在一个大型的450kD络合物中发现，还含有许多核糖体蛋白。免疫沉淀研究验证了TAF7与核糖体蛋白的结合，并证明该结合取决于RNA。进一步的验证是，从纯化的多聚体中回收了TAF7。综上所述，这些发现表明TAF7不仅是转录的检查点调节因子，而且是促进翻译的功能。有趣的是，TAF7细胞质/核比率在不同的细胞类型之间有所不同，随着恶​​性表型的增加而增加。当前的研究旨在阐明TAF7在恶性肿瘤中的作用。为了进一步研究TAF7在全球基因调节和发育中的作用，我们产生了有条件的TAF7敲除小鼠。 TAF7的完全消融会导致胚胎致死性；胚胎不会超过D.3-4。从条件敲除的MEF中切除TAF7导致流产的细胞增殖和转录开始的全球减少。 TAF7对于分化在分化过程中发生巨大增殖的未成熟胸腺前体的分化也是必不可少的。因此，由CRE介导的TAF7缺失在LCK启动子的控制下表达，该启动子在非常早期的DN胸腺细胞中起作用，消融所有T细胞的发育。相比之下，随后的谱系胸腺细胞非依赖性依赖性分化并不需要TAF7。在EIII8增强子的控制下，CRE删除TAF7，仅在胸腺细胞发育的DP阶段起作用，可以使能够出口到周围的单个阳性细胞的成熟。然而，尽管只有少数转录本受到TAF7缺失的影响，但TAF7缺乏的外围T细胞无法响应抗原性刺激而受到激活和扩张。我们得出的结论是，TAF7在经历增殖的细胞中在转录中起关键作用，但其在非增殖分化细胞中的作用更受限制。