Developmental Functions of Rb family proteins

描述（由申请人提供）：在控制细胞增殖和分化的许多途径中，视网膜母细胞瘤蛋白（pRb）调控网络的基因作为人类肿瘤发生过程中突变或失调的常见（如果不是强制性的）靶点而突出。虽然生化，组织培养和转基因研究在小鼠中有牵连的pRb家族成员在广泛的活动，在正常发育过程中的真实pRb功能的全谱尚未得到解决。此外，新的研究表明，pRb可能通过各种不同的机制发挥其抗肿瘤作用，尽管pRb在肿瘤抑制中的关键功能仍有待确定。我们的长期目标是了解pRb家族蛋白的分子、细胞和发育功能，并将pRb通路与其他细胞网络机械地联系起来。为此，我们使用了一系列的遗传和分子方法来表征lin-35的新功能，lin-35是C. elegans，包括在器官形态发生，肠道内稳态和细胞命运决定中的作用。此外，使用转录组分析，我们已经确定了许多LIN-35的靶基因，代表新的候选细胞周期调节因子。我们的主要目标分为三类。在目标1中，我们将集中于进一步阐明LIN-35调控网络控制咽部形态发生。具体来说，我们将探索几个新的基因，我们已经牵连作为这个通路中发挥作用的作用，并将通过建立筛选方法确定其他组件。目的二：研究LIN-35和锌指蛋白SLR-2在调节肠道功能和基因表达中的作用。这将使用正向遗传学和分子方法来完成，并将包括定向研究，以检查LIN-35与全基因组RNAi抑制剂筛选中鉴定的基因之间的功能联系。在目标3中，我们将跟踪微阵列和生物信息学分析，以确定新的细胞周期成分。候选基因的功能将使用各种致敏遗传背景和体内分析来确定，这将使我们能够确定这些基因在细胞周期进展和调控中的作用。这些目标的成功完成将增强我们对pRb家族成员的细胞周期和非细胞周期功能的基本理解，并将为LIN-35和相关途径在控制参与营养吸收和利用的两个主要器官的形成和功能中的作用提供机制见解。这项拟议的研究也将支持努力，以确定真实的pRb家族成员的体内活动，并确定遗传修饰，可能会增加或减少Rb突变的表型效应。因此，这些研究与理解pRb的肿瘤抑制功能以及构成人类更大pRb调控网络的许多细胞因子具有很强的相关性。我们发表的研究结果和广泛的初步结果提供了一个坚实的基础和逻辑框架进行拟议的实验。

DESCRIPTION (provided by applicant): Among the many pathways controlling cell proliferation and differentiation, genes of the retinoblastoma protein (pRb) regulatory network stand out as frequent, if not obligatory, targets for mutation or deregulation during human tumorigenesis. Although biochemical, tissue culture, and transgenic studies in mice have implicated pRb family members in a wide range of activities, the full spectrum of authentic pRb functions during normal development has yet to be resolved. In addition, new studies suggest that pRb may exert its anti-oncogenic effects through a variety of distinct mechanisms, although the critical functions of pRb in tumor suppression remain to be determined. Our long-term objectives are to understand the molecular, cellular, and developmental functions of pRb family proteins and to mechanistically link the pRb pathway to other cellular networks. To this end, we have used a range of genetic and molecular methods to characterize novel functions of lin-35, the sole Rb ortholog in C. elegans, including roles in organ morphogenesis, intestinal homeostasis, and cell fate determination. In addition, using transcriptome profiling, we have identified many LIN-35 target genes that rep- resent novel candidate cell cycle regulators. Our main objectives fall into three categories. In Aim 1, we will fo- cus on further elucidating the LIN-35 regulatory network controlling pharyngeal morphogenesis. Specifically, we will explore the roles of several new genes that we have implicated as functioning within this pathway and will identify additional components through established screening methods. In Aim 2, we will study the roles of LIN-35 and SLR-2, a Zn-finger protein, in regulating intestinal function and gene expression. This will be accomplished using forward genetics and molecular approaches and will include directed studies to examine the functional connection between LIN-35 and genes identified in a genome-wide RNAi suppressor screen. In Aim 3, we will follow up on microarray and bioinformatical analyses to identify novel cell cycle components. Functions for candidate genes will be determined using a variety of sensitized genetic backgrounds and in vivo as- says, which will allow us to determine roles for these genes in cell cycle progression and regulation. The successful completion of these aims will enhance our basic understanding of both cell cycle and non-cell cycle functions for pRb family members and will provide mechanistic insights into the roles of LIN-35 and associated pathways in controlling the formation and function of two principal organs involved in nutrient uptake and utilization. This proposed research will also support efforts to define authentic in vivo activities for pRb family members and to identify genetic modifiers that may augment or diminish the phenotypic effects of Rb mutations. As such, these studies have strong relevance to understanding the tumor-suppressing functions of pRb and the many cellular factors that comprise the greater pRb regulatory network in humans. Our published findings and extensive preliminary results provide a solid foundation and logical framework for carrying out the proposed experiments.