GROWTH REGULATION OF REPLICATION-DEPENDENT GENES

这是一项研究G1-S调控机制的建议 利用Well转录两个依赖于复制的基因 以组蛋白和胸苷激酶(Tk)基因为模型系统。 这项提案的主要目标是确定监管因素和 研究G1-S调控元件上的蛋白质相互作用 调解这一规定。已有文献证明，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 最近成功地将顺式监管控制元素本地化 这就赋予了体内G1-S转录调控的能力。对于H3.2 仓鼠核因子基因，命名为H3abp1，结合G1-S 调控位点GGCGAGTCAG，类似于Jun蛋白结合位点。 最近，Jun蛋白被证明是特定需要的 血清刺激期和非同步生长期进入S期 成纤维细胞。我们现在确定H3abp1复合体与 但在免疫和功能上与以前的 描述了Jun/CREB/atf1蛋白。此外，我们发现H3abp1 与H3启动子特异性结合，其结合活性为 在G1-S交界处急剧上升。这件事的一个主要推动力 建议对H3abp1进行提纯。H3apb1的合成 简档、结合属性、细胞周期依赖的翻译后 与其他共激活剂或任何已知细胞的修饰和相互作用 将对周期调节蛋白进行检测。提供直接证据 对于H3abp1和H3.2调控之间的功能联系，能力 纯化的H3abp1在体外和体内刺激H3转录 H3abp1的表达下调或过表达的影响将是 调查过了。对于tk系统，已经有一个14bp的蛋白结合位点 被确定为G1-S调节单位，其活性通过一种 毗邻CCAAT地点。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体在 G1-S边界。我们建议的研究旨在界定 这些蛋白质复合体的成分。此外，我们还测试了 人类tk和组蛋白H1 CCAAT位点可能共有的假说 调控因素在S期调节它们的同时升高 抄写。这些研究将提供基本信息 关于复杂的、相互依赖的、调解严格的分子事件 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 离心洗脱，并与血清刺激细胞进行比较。 这将为细胞如何调整G1-S提供重要信息 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.