Characterization of novel modifiers of dE2F/RBF function

描述(申请人提供)：视网膜母细胞瘤蛋白(PRB)是一种重要的肿瘤抑制因子，通过抑制E2F转录因子的活性而发挥细胞增殖的负面调节作用。E2F转录因子是G1向S转变的关键调控因子，其活性对S进入细胞周期具有限速作用。在大多数肿瘤细胞中，pRb通路被认为是功能失活的；这突显了pRb和E2F在哺乳动物细胞增殖调控中的重要性。虽然pRb和E2F的关键作用已经确定，但这些蛋白质如何精确地调节细胞周期进程，这些蛋白质的作用，以及为什么它们的活动“如此重要，还不完全清楚”。雪上加霜的是，越来越多的蛋白质被证明与pRb相互作用。目前，该领域面临着一个重大挑战，即如何确定对PRB功能具有重要意义的相互作用。这项拟议的研究旨在利用果蝇作为模式生物来识别内源性E2F/RBF抑制因子活性的重要调节因子。在果蝇中，有两个E2F基因。DE2F1是一种强大的转录激活因子和细胞增殖的正向调节因子。在幼虫发育过程中，其活性被抑制因子dE2F2所抵消。我们的方法是基于我们之前的发现，即de2f1突变体中细胞增殖的强烈阻碍是由于dE2F2/RBF抑制物复合体的未受抑制的活性。我们建议对抑制de2f1突变表型的突变进行基因筛查。这些突变可能发生在其产物对dE2F2/RBF在体内阻断细胞周期具有重要功能的基因中。提出了两个特定的目标：(1)对3号染色体右臂de2f1突变表型的抑制子进行全面筛选，并制定策略并启动对其他常染色体臂的筛选。(2)对de2f1突变表型的抑制子进行了详细的表型分析和定位。我们的目标是确定dE2F2/RBF抑制物复合体的功能伙伴，它们在体内细胞增殖中对dE2F2/RBF依赖的阻断至关重要。鉴于pRb在肿瘤抑制中的关键作用，了解果蝇同系物dE2F2/RBF的功能是如何调控的，将为理解哺乳动物细胞生长的调控以及这种调控在人类癌症中可能出错的方式提供关键线索。

DESCRIPTION (provided by applicant): The retinoblastoma protein (pRB) is an important tumor suppressor that functions as a negative regulator of cell proliferation by restraining the activity of the E2F transcription factor. The E2F transcription factor is a critical regulator of the G1 to S transition and its activity is rate-limiting for S phase entry. In most tumor cells, the pRB pathway is believed to be functionally inactivated; this highlights the significance of pRB and E2F in the regulation of mammalian cell proliferation. While the critical role of pRB and E2F is well established, how precisely these proteins regulate cell cycle progression, what these proteins do, and why their activities are " so important, is not completely understood. Adding to the problem, there is an increasing number of proteins which have been shown to interact with pRB. Currently, the field faces a major challenge to identify the interactions which are functionally significant for pRB function. The proposed research aims to take advantage of Drosophila as a model organism to identify important regulators of endogenous E2F/RBF represser activity. In Drosophila, there are two E2F genes. dE2F1 is a strong activator of transcription and positive regulator of cell proliferation. During larval development, its activity is counterbalanced by the represser dE2F2. Our approach is based on our previous finding, that the strong block in cell proliferation in de2f1 mutants is due to the unchecked activity of the dE2F2/RBF represser complex. We propose to perform a genetic screen for mutations which suppress the de2f1 mutant phenotype. These mutations may be in genes whose products are functionally important for dE2F2/RBF to block cell cycle in vivo. Two specific aims are proposed: (1) To perform a full scale screen for suppressors of the de2f1 mutant phenotype on the right arm of chromosome 3, and to develop strategies for and to initiate screening of, other autosomal arms. (2) To carry out a detailed phenotypic analysis and mapping of suppressors of the de2f1 mutant phenotype. Our goal is to identify functional partners of the dE2F2/RBF represser complex which are critical for dE2F2/RBF- dependent block in cell proliferation in vivo. Given the pivotal role of pRB in tumor suppression, an understanding of how the function of the Drosophila homologues dE2F2/RBF is regulated will provide crucial clues in understanding the regulation of mammalian cell growth and the ways in which such controls may go wrong in human cancer.