GROWTH REGULATION OF REPLICATION-DEPENDENT GENES

这是一个研究G1-S调控机制的建议， 两个复制依赖基因的转录，使用孔 以组蛋白和胸苷激酶（tk）基因为模型系统。 该提案的主要目标是确定监管因素， 检测G1-S控制元件处的蛋白质相互作用 调解这一规定。 据记载，这些基因是 在G1-S期边界转录激活的机制， 取决于位于其启动子中的特定序列元件。 我们 最近成功定位了顺式调控元件 在体内赋予G1-S转录调控。 对于H3.2 基因，仓鼠核因子，命名为H3 abp 1，结合G1-S 调控位点GGCGAGTCAG，其类似于Jun蛋白结合位点。 最近，Jun蛋白被证明是特定需要的， 在血清刺激和异步生长中均进入S期 成纤维细胞 我们现在确定H3 abp 1复合物与 但在免疫学和功能上与先前的 描述了Jun/CREB/ATF 1蛋白。 此外，我们发现H3 abp 1 特异性结合H3启动子，其结合活性 双相，在G1-S边界急剧上升。 这其中的一个主要推动力 建议是纯化H3 abp 1。 H3 apb 1合成 谱，结合特性，细胞周期依赖性翻译后 修饰以及与其他共激活剂或任何已知细胞的相互作用 将检测周期调节蛋白。 提供直接证据 对于H3 abp 1和H3.2调节之间的功能性联系， 的纯化的H3 abp 1刺激H3转录在体外和在 体内和减少或过度表达H3 abp 1的影响将是 研究了 对于tk系统，已经将14 bp的蛋白结合位点 被鉴定为G1-S调节单位，其活性通过 邻近的CCAAT网站。 其中一种蛋白质的结合活性 与G1-S调控位点相互作用的复合物在 G1-S边界 我们提出的研究旨在确定 这些蛋白质复合物的组成部分。 此外，我们测试 假设人类tk和组蛋白H1 CCAAT位点可能有共同的 调节因子介导它们在S期的同时增加 转录。 这些研究将提供基本信息， 在复杂的，相互依存的分子事件，介导严格的 调节细胞周期进程。 我们的新方向包括 将我们的研究扩展到连续循环的细胞中， 离心淘析并与血清刺激的细胞进行比较。 这将提供关于细胞如何调节G1-S的重要信息 不同生理条件下的转录调控。

This is a proposal to study the mechanisms that govern the G1-S regulated transcription of two replication-dependent genes, using the well characterized histone and thymidine kinase (tk) genes as model systems. The major goal of this proposal is to identify the regulatory factors and to examine the protein interactions at the G1-S control elements mediating this regulation. It has been documented that these genes are transcriptionally activated at the G1-S phase border by a mechanism that depends on specific sequence elements located in their promoters. We have recently succeeded in localizing the cis-regulatory control elements that confer the G1-S transcription regulation in vivo. For the H3.2 gene, a hamster nuclear factor, designated H3 abp1, binds a G1-S regulatory site GGCGAGTCAG which resembles a Jun protein binding site. Recently, the Jun proteins are shown to be specifically required for entrance into S-phase in both serum stimulated and asynchronously growing fibroblasts. We now determined that the H3 abp1 complex is related to but is immunologically and functionally distinct from the previously described Jun/CREB/ATF1 proteins. Further, we discovered that H3 abp1 specifically binds to the H3 promoter and its binding activity is biphasic and rises sharply at the G1-S border. A major thrust of this proposal is the purification of the H3 abp1. The H3 apb1 synthesis profile, binding properties, cell-cycle dependent posttranslational modifications and interactions with other co-activators or any known cell cycle regulated proteins will be examined. To provide direct evidence for a functional link between H3 abp1 and H3.2 regulation, the ability of the purified H3 abp1 to stimulate H3 transcription in vitro and in vivo and the effect of reduced- or over-expression of H3 abp1 will be investigated. For the tk system, a 14 bp protein binding site has been identified as a G1-S regulatory unit and its activity is enhanced by an adjacent CCAAT site. The binding activities of one of the protein complexes interacting with the G1-S regulatory site changes sharply at the G1-S border. Our proposed studies are aimed at defining the components of these protein complexes. In addition, we test the hypothesis that the human tk and histone H1 CCAAT site may share common regulatory factor mediating their simultaneous increase in S-phase transcription. These studies will provide the fundamental information on the complex, interdependent molecular events which mediate stringent regulation of cell cycle progression. Our new direction includes expansion of our studies into continuously cycling cells separated by centrifugal elutriation and compared that to serum stimulated cells. This will provide important information on how cells adjust G1-S transcriptional control under different physiological conditions.