ROLE OF E2F IN REGULATION OF CELL CYCLE AND TUMOR DEVELOPMENT

视网膜母细胞瘤的发育和抑瘤特性 蛋白质(PRB)似乎都至少部分依赖于其 能够调节一个离散的子集的转录活动 E2F转录因子。动物模型将被用来研究 PRB特异性E2F的发育作用并确定其 调节有助于视网膜母细胞瘤蛋白的特性。 具有E2F-2和E2F-3基因胚系突变的新小鼠品系 将使用标准的基因打靶技术来创造。分析 这些小鼠将确定单个或多个pRb的丢失是如何- 特定的E2F影响胚胎和成年小鼠的发育。 然后将使用杂交来确定这些基因的突变是否 基因足以阻止肿瘤的形成 Rb+/-小鼠或在Rb-/-胚胎中发生的广泛的细胞凋亡。 这将有助于确定E2F-监管如何有助于 PRb的发育和抑瘤特性。的影响 这些突变也将在分子水平上使用 单个和复合突变小鼠品系，以产生原代细胞系。 在每种情况下，E2F复合体的光谱，程度和时间 E2F应答基因的激活及其突变株的生长特性 将对细胞进行分析。这些研究将有助于将公共关系科专门的 E2F来激活特定的靶基因，并确定这些 个别事件有助于儿童的生长刺激特性 内源性E2F。最后，我们将继续在体外开发 转录化验将使我们能够研究直接后果 PRb与其靶E2F之间的相互作用。特别是，这一点 系统将进行优化，以研究公共关系科废除 与之结合的启动子的基础转录。这些项目的目标是 体外研究是分离E2F特异性和全局抑制 PRB的特性，以建立这些特性的分子机制 影响是中介的，并确定每个影响如何对 这种蛋白质的生长抑制特性。

The developmental and tumor-suppressive properties of the retinoblastoma protein (pRB) both appear to be at least partially dependent upon its ability to regulate the transcriptional activity of a discrete subset of the E2F transcription factors. Animal models will be used to study the developmental role of the pRB-specific E2Fs and determine how their regulation contributes to the properties of the retinoblastoma protein. Novel mouse strains with germline mutations in the E2F-2 and E2F-3 genes will be created using standard gene targeting technology. Analysis of these mice will establish how loss of either single or multiple pRB- specific E2Fs affects the development of embryonic and adult mice. Crossbreeding will then be used to determine whether mutation of the these genes is sufficient to block either the tumor formation that occurs in the Rb+/- mice or the widespread apoptosis that occurs in the Rb-/- embryos. This will help to establish how E2F-regulation contributes to the developmental and tumor-suppressive properties of pRB. The effect of these mutations will also be examined at the molecular level using the single and compound mutant mouse strains to generate primary cells lines. In each case, the spectrum of E2F complexes, the degree and timing of activation of E2F responsive genes and growth properties of these mutant cells will be analyzed. These studies will help to link the pRB-specific E2Fs to the activation of specific target genes and determine how these individual events contribute to growth stimulatory properties of the endogenous E2F. Finally, we will continue to develop in vitro transcription assays that will allow us to study the direct consequences of the interaction between pRB and its target E2Fs. In particular, this system will be optimized to study the mechanism by which pRB abolishes the basal transcription of promoters to which it is bound. The goal of these in vitro studies is to separate the E2F-specific and global repression properties of pRB, to establish the molecular mechanisms by which these effects are mediated, and to determine how each one contributes to the growth suppressive properties of this protein.