SV40 Tumor Antigen

描述（由申请人提供）： 对动物肿瘤病毒如SV 40的研究已经阐明了病毒和细胞基因调控的许多机制。第一个被分离和鉴定的真核序列特异性DNA结合蛋白是SV 40大T-ag。对SV 40 T-ag的早期研究揭示了其通过介导DNA复制、转录和肿瘤转化的能力而具有的显著的多功能性质。T-ag的研究今天继续与最近的高分辨率X射线结构的T-ag十二聚体/ATP酶，这是负责DNA结合和启动病毒DNA合成。SV 40 T-ag在控制早期病毒和细胞基因调控中的关键功能促使我们的实验室启动了对细胞机制的系统分析，该细胞机制与这种病毒反式调节因子一起指导动物细胞中的基因表达。这些研究已经导致超过75种转录因子的发现和生化表征，包括序列特异性增强子/启动子识别因子;前起始RNA聚合酶II复合物的核心启动子/基础组分;多亚基共激活子和染色质重塑辅因子。这些生物化学研究已经与基于细胞的测定和小鼠中的基因靶向相结合，以揭示一种精细的机制，其包括：核心启动子识别复合物的细胞类型特异性和基因选择性亚基。在这次资助更新中，我们建议继续努力剖析后生动物中转录装置调控基因表达的机制。我们将继续努力利用染色质化的DNA模板建立一个高度纯化的体外转录系统，重点是揭示核心转录机制的细胞类型特异性组分（即TAFII 105，TRF 2等）的机制。除了研究激活，我们将重新努力研究病毒和细胞蛋白质如何在抑制中发挥作用。与以前的赠款不同，我们的工作中有很大一部分将在体内进行（即KO小鼠，免疫荧光，FISH等）。最后，我们将增加我们在结构分析领域的努力，结合电子显微镜和单颗粒三维重建与X射线晶体学，以解决大型多亚基蛋白质复合物的三维结构。这些研究应该为我们提供一个全面的图片，病毒和细胞基因组是如何表达在一个高度多样化的转录装置，在动物细胞中进化的调节方式。了解基因调控机制已经为开发各种新型抗癌治疗策略提供了基础，我们预计，随着新的靶分子在导致肿瘤发生和疾病的关键路径上被鉴定，将出现旨在干预基因调控机制的其他治疗干预措施。

DESCRIPTION (provided by applicant): Studies of animal tumor viruses such as SV40 have elucidated numerous mechanisms of both viral and cellular gene regulation. The first eukaryotic sequence-specific DNA binding protein to be isolated and characterized was the SV40 large T-ag. Early work on the SV40 T-ag revealed its remarkable multifunctional nature by its ability to mediate DNA replication, transcription and neoplastic tranformation. The studies of T-ag continue today with the recent high resolution X-ray structure of the T-ag dodecamer/ATPase which is responsible for DNA binding and initiation of viral DNA synthesis. The critical functions of the SV40 T-ag in controlling early viral and cellular gene regulation prompted our lab to launch a systematic analysis of the cellular machinery that works in conjunction with this viral trans-regulator to direct gene expression in animal cells. These studies have led to the discovery and biochemical characterization of over 75 transcription factors including sequence-specific enhancer/promoter recognition factors; core promoter/basal components of the pre-initiation RNA polymerase II complex; multi-subunit co-activators and chromatin remodeling co-factors. These biochemical studies have been combined with cell-based assays and gene targeting in the mouse to reveal an elaborate machinery which includes: cell-type specific and gene selective subunits of the core promoter recognition complex. In this grant renewal, we propose to continue our efforts to dissect the mechanisms by which the transcriptional apparatus operates to regulate gene expression in metazoan organisms. We will extend our effort to establish a highly purified in vitro transcription system using chromatinized DNA templates, focusing on uncovering the mechanisms of cell-type specific components of the core transcriptional machinery (i.e.TAFII105, TRF2, etc.). In addition to studying activation, we will renew our effort to study how viral and cellular proteins function in repression. Unlike previous grants, a significantly greater proportion of our work will be conducted in vivo (i.e. KO mice, immunofluorescence, FISH etc.). Finally, we will increase our effort in the area of structural analysis incorporating electron microscopy and single particle 3D reconstruction with X-ray crystallography to solve the 3D structure of large multi-subunit protein complexes. These studies should provide us with a comprehensive picture of how both viral and cellular genomes are expressed in a regulated fashion by the highly diversified transcriptional apparatus that has evolved in animal cells. Understanding the gene regulatory machinery has already provided the basis for developing various novel anti-cancer therapeutic strategies and we anticipate that as new target molecules are identified on the critical path leading to oncogenesis and disease, additional therapeutic interventions designed to intercede with gene regulatory mechanisms will emerge.