CELLULAR TRANSFORMATION BY AP-1 TRANSCRIPTION FACTORS

编码DNA结合转录因子的癌基因可能 通过直接改变基因调节而导致癌症状态。 Jun和Fos癌蛋白属于转录家族， 结合AP-1位点的因子（TGACTCA），已被广泛 表征了 突变分析确定了Jun和Fos的区域 对肿瘤发生很重要，但AP的功能复杂性- 1个因素使得难以区分潜在的分子 机制等 此外，尽管AP-1位点存在于许多启动子中， 并能影响转录，致癌相关的靶基因没有 尚未被确认。 我们开发了一种新的研究方法 AP-1因子介导的肿瘤发生，涉及酵母GCN 4， 自主和异源AP-1转录因子，其功能是 不受Jun-Fos家族复杂监管的限制 大鼠 胚胎成纤维细胞，GCN 4通过AP-1诱导转录激活 位点，但与Jun和Fos 1不同的是，它不能介导细胞凋亡。 与Ha-ras合作进行改造。 嵌合蛋白的分析 含有GCN 4 DNA结合结构域定义的致癌功能， 和Fos，它们不同于一般的转录激活结构域。 该提案的总体目标是使用这些和其他AP-1 作为鉴定致癌相关靶基因的探针的衍生物 以及确定Fos和Jun如何转化细胞。 为了实现这一点， 我们提出以下实验。 第一，致癌相关 靶基因将通过差异影响来鉴定 GCN 4的致癌和非致癌衍生物， 通过AP-1位点有效地激活转录。 大鼠胚胎 成纤维细胞将与表达Ha-ras的DNA平行转染， 细胞表面标志物和感兴趣的GGN 4衍生物，并转染 通过淘选（使用针对细胞表面的抗体 标记）。 差异表达的基因将通过直接测序来鉴定。 通过PCR分析可能在细胞生长中重要的候选基因- 基于RNA展示技术和消减cDNA文库。 第二、 对于选定数量的靶基因，我们将分离启动子 区域，并确定它们是否可以概括表达 格局 进一步的启动子分析将集中在鉴定AP-1 和其他DNA序列参与GCN 4之间的区别， 衍生物. 长期目标是确定转录 这些机制与细胞转化密切相关。 第三，使用酵母双杂交系统，我们将寻找蛋白质， 与Fos的N-末端环残基相互作用， 当与GCN 4 bZIP结构域融合时转化细胞。 致癌相关性 将使用我们现有的一套 相差1或2的变换和非变换Fos导数 个氨基酸 通过致癌相关性测试的基因将被 测序，编码的蛋白质将被测试其 启动子上的转录特性， 目标1和2。总的来说，这些研究代表了一种新的方法， 鉴定和表征AP-1的致癌相关靶标 转录因子 结果应该具有广泛的意义，因为 其他癌基因和肿瘤抑制基因的靶点基本上是 未知

Oncogenes that encode DNA-binding, transcription factors are likely to contribute to the cancerous state by directly altering gene regulation. The Jun and Fos oncoproteins, which belong to the family of transcription factors that bind AP-1 sites (TGACTCA), have been extensively characterized. Mutational analyses have defined regions of Jun and Fos that are important for oncogenesis, but the functional complexities of AP- 1 factors make it difficult to discriminate among potential molecular mechanisms. Moreover, although AP-1 sites are found in numerous promoters and can affect transcription, oncogenically relevant target genes have not yet been identified. We have developed a novel approach to studying oncogenesis mediated by AP-1 factors that involves yeast GCN4, an autonomous and heterologous AP-1 transcription factor whose function is not subject to the regulatory complexities of the Jun-Fos family. In rat embryo fibroblasts, GCN4 induces transcriptional activation through AP-1 sites, but unlike Jun and Fos1 it is unable to mediate cellular transformation in cooperation with Ha-ras. Analysis of chimeric proteins containing the GCN4 DNA-binding domain defined oncogenic functions on Jun and Fos that are distinct from generic transcriptional activation domains. The overall goal of this proposal is to use these and other AP-1 derivatives as probes for identifying oncogenically relevant target genes and for determining how Fos and Jun transform cells. To accomplish this, we propose the following experiments. First, oncogenically-relevant target genes will be identified by virtue of being differentially affected by oncogenic and non-oncogenic derivatives of GCN4 that are equally efficient at transcriptional activation through AP-1 sites. Rat embryo fibroblasts will be transfected in parallel with DNAs expressing Ha-ras, a cell-surface marker, and the GGN4 derivative of interest, and transfected cells will be isolated by panning (using antibodies to the cell surface marker). Differentially expressed genes will be identified by direct analysis of candidate genes likely to be important in cell growth, by PCR- based RNA display technology, and by subtractive cDNA libraries. Second, for a selected number of target genes, we will isolate the promoter regions and determine whether they can recapitulate the expression pattern. Further promoter analysis will be focused on identifying AP-1 and other DNA sequences involved in the discrimination between the GCN4 derivatives. The long-term goal is to determine the transcriptional mechanisms that are specifically correlated to cellular transformation. Third, using the yeast 2-hybrid system, we will search for proteins that interact with the N-terminal loop residues of Fos, a region that transforms cells when fused to the GCN4 bZIP domain. Oncogenic relevance of the identified proteins will be tested using our existing set of transforming and non-transforming Fos derivatives that differ by l or 2 amino acids. Genes passing the oncogenic relevance test will be sequenced, and the encoded proteins will be tested for their transcriptional properties on promoters identified and characterized in aims l and 2. Overall, these studies represent a novel approach for identifying and characterizing oncogenically-relevant targets of AP-1 transcription factors. The results should have broad significance because targets of other oncogenes and tumor suppressor genes are essentially unknown.