CELLULAR TRANSFORMATION BY AP-1 TRANSCRIPTION FACTORS

编码DNA结合的癌基因，转录因子很可能 通过直接改变基因调控而导致癌症状态。 Jun和Fos癌蛋白，属于转录家族 结合AP-1位点的因子(TGACTCA)已被广泛研究 特色化的。突变分析确定了Jun和Fos的区域 对肿瘤的发生很重要，但AP的功能复杂性- 1因素使得很难区分潜在的分子 机械装置。此外，尽管在许多启动子中发现了AP-1位点 并且可以影响转录，致癌相关的靶基因没有 但已被确认身份。我们开发了一种新的研究方法 酵母Gcn4、And参与的AP-1因子介导的肿瘤发生 自主和异源的AP-1转录因子，其功能是 不受Jun-Fos家族监管复杂性的影响。在大鼠中 胚胎成纤维细胞，GCN4通过AP-1诱导转录激活 站点，但与Jun和Fos1不同的是，它无法调解细胞 与哈拉斯合作进行转型。嵌合蛋白的分析 含有Gcn4 DNA结合域的Jun上定义的致癌功能 和Fos，不同于一般的转录激活结构域。 这项提议总目标是使用这些和其他AP-1 以衍生物为探针识别致癌相关的靶基因 以及确定Fos和Jun如何转换细胞。要做到这一点， 我们建议进行以下实验。首先，与肿瘤相关 靶基因将通过差异影响进行识别。 通过GCN4的致癌和非致癌衍生物 通过AP-1位点有效地转录激活。大鼠胚胎 成纤维细胞将与表达Ha-ras、a 细胞表面标记和目的GGN4衍生物，并转染 将通过摇摄(使用细胞表面的抗体)来分离细胞 标记)。差异表达的基因将通过直接识别 用聚合酶链式反应分析可能对细胞生长有重要作用的候选基因 基于RNA展示技术，并利用消减文库。第二, 对于选定的一些目标基因，我们将分离启动子 区域并确定它们是否可以概括该表达式 图案。进一步的启动子分析将集中在识别AP-1上 和其他参与区分Gcn4的DNA序列 衍生品。长期目标是确定转录 与细胞转化特别相关的机制。 第三，使用酵母双杂交系统，我们将寻找能够 与Fos的N末端环残基相互作用，该区域 融合到Gcn4 bZIP结构域时转换细胞。致癌相关性 将使用我们现有的一套 与L或2不同的变换与非变换的Fos导数 氨基酸。通过致癌相关性测试的基因将是 测序，编码的蛋白质将被测试其 基因启动子转录特性的鉴定和表征 目标是L和2。总体而言，这些研究代表了一种新的方法 AP-1肿瘤相关靶点的鉴定与鉴定 转录因子。这一结果应该具有广泛的意义，因为 其他癌基因和抑癌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.