Transcriptional Regulation by the pRB and E2F Families

描述(申请人提供)：要全面了解正常哺乳动物细胞的生长控制，我们需要知道基因调控电路是如何作为细胞周期的函数运作的。转录因子E2F和pRb，一种已知可以抑制E2F活性的肿瘤抑制因子，是研究得最好的细胞周期依赖基因表达的调节因子。已知的是，在大多数人类癌症中，涉及pRb的通路都是去调控的。尽管E2F和pRB在基因上与生长控制有关，但这些因子调节内源基因表达和全球调控细胞周期进程的机制尚不清楚。对它们功能的完整描述不仅要求我们了解在给定的细胞周期调控启动子上发生的生理变化，而且还需要了解在细胞周期中以可逆的方式将基因从关闭状态切换到开启状态的复杂的生化机制。只有在细胞被刺激生长或持续循环的条件下，才能通过研究自然(染色质)环境中E2F靶标的调节来实现详细的描述。我们的假设表明，在细胞周期的每个阶段，必须存在区分基因表达谱的离散机制。鉴于pRb在肿瘤抑制中的关键作用，对这些机制的理解将为理解正常细胞生长的调控和人类癌症控制可能出错的方式提供必要的关键线索。这项研究提案利用多种互补的方法来测试这一想法。在目标1中，将使用基因组学和生物化学相结合的方法来确定E2F和PRB家族的靶标以及细胞周期每个阶段增殖细胞中的染色质修饰酶，以确定那些调节处于活跃和非活跃状态的基因的因素。利用迅速出现的生物信息学和计算工具，目标2中的方法将试图识别那些连接由E2F、PRB和染色质修饰酶共同调节的基因的模式。AIM 3中的生化实验将测试对这些模式的预测，并将揭示那些在细胞周期的不同阶段区分天然E2F目标基因表达谱的机制。

DESCRIPTION (provided by applicant): A comprehensive understanding of growth control in normal mammalian cells requires that we know how gene regulatory circuits operate as a function of the cell cycle. The transcription factor, E2F, and pRB, a tumor suppressor known to restrain E2F activity, are among the best-studied regulators of cell cycle dependent gene expression. Pathways that involve pRB are known to be de-regulated in a majority of human cancers. Despite the fact that E2F and pRB have been linked genetically to growth control, the mechanisms by which these factors modulate endogenous gene expression and globally regulate cell cycle progression are not well understood. A complete description of their function requires that we understand not only physiological changes that occur at a given cell cycle regulated promoter but also the intricate biochemical mechanisms that switch a gene from the 'OFF' to 'ON' state in a reversible manner during the cell cycle. A detailed description can be achieved only by studying the regulation of E2F targets in a natural (chromatin) setting under conditions where cells are either stimulated to grow or are continuously cycling. Our hypothesis suggests that discrete mechanisms must exist that distinguish gene expression profiles during each stage of the cell cycle. Given the pivotal role of pRB in tumor suppression, an understanding of these mechanisms will provide crucial clues necessary for understanding the regulation of normal cell growth and the ways in which controls may go awry in human cancer. This Research Proposal takes advantage of multiple, complementary approaches to test this idea. In Aim 1, a combination of genomics and biochemistry will be used to identify targets of E2F and pRB families and chromatin modifying enzymes in proliferating cells at each stage of the cell cycle to determine those factors that regulate genes in both active and inactive states. Using rapidly emerging bioinformatics and computational tools, approaches in Aim 2 will attempt to identify those patterns that link genes co-regulated by E2F, pRB, and chromatin modifying enzymes. Biochemical experiments in Aim 3 will test predictions about these patterns and will reveal those mechanisms that distinguish expression profiles of native E2F target genes at different stages of the cell cycle.