Developmental Functions of Rb family proteins

描述(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)调控网络的基因在人类肿瘤发生过程中经常成为突变或放松调控的目标。尽管对小鼠的生化、组织培养和转基因研究表明pRb家族成员参与了广泛的活动，但pRb在正常发育过程中的真实功能尚未得到解决。此外，新的研究表明，pRb可能通过多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，并以机械方式将PRB途径与其他细胞网络联系起来。为此，我们使用了一系列的遗传学和分子方法来研究线虫中唯一的RB同源基因Lin-35的新功能，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，利用转录组图谱，我们已经确定了许多代表新的候选细胞周期调控因子的LIN-35靶基因。我们的主要目标分为三类。在目标1中，我们将致力于进一步阐明控制咽部形态发生的LIN-35调控网络。具体地说，我们将探索几个新基因在这一途径中发挥作用的作用，并将通过既定的筛选方法确定其他成分。在目标2中，我们将研究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，并将包括直接研究，以检查在全基因组RNAi抑制筛查中确定的LIN-35和基因之间的功能联系。在目标3中，我们将跟进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的遗传背景和体内的说法来确定，这将使我们能够确定这些基因在细胞周期进程和调节中的作用。这些目标的成功完成将加深我们对pRb家族成员细胞周期和非细胞周期功能的基本了解，并将为我们提供对Lin-35及其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方面的作用的机械性见解。这项拟议的研究还将支持为pRb家族成员确定真实的体内活动的努力，并确定可能增强或减少rb突变的表型影响的遗传修饰物。因此，这些研究对于理解pRb的肿瘤抑制功能和构成人类更大的pRb调控网络的许多细胞因素具有很强的相关性。我们发表的发现和广泛的初步结果为开展拟议的实验提供了坚实的基础和逻辑框架。

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.