Genetic modulation of Wingless/Wnt signaling

描述(申请人提供)：WNT信号决定了所有动物胚胎发育期间的细胞命运，包括人类，在生命的后期需要维持干细胞数量。Wnt活性过高与人类癌症，尤其是结肠癌有关。为了了解Wnt信号是如何导致癌症的，通常控制Wnt途径活性的因素必须得到充分的表征。Bejsovec实验室的长期目标是利用果蝇遗传模型系统和培养的人类细胞来剖析这一重要信号通路的机制。该实验室通过突变分析确定了许多重要的途径成分，如激活Wnt靶基因的转录因子Tcf和负调控Wnt途径的人类肿瘤抑制同系物Apc2。这项建议关注的是在同一突变筛选中发现的另一个负调控基因：Tum是人类GTP酶激活蛋白(GAP)racGap1的同源基因。令人惊讶的是，该基因的GAP活性是细胞分裂所必需的，但它的GAP活性并不是其在Wnt调控中所必需的。在酵母双杂交筛选中发现的两个Tum相互作用的分子，强烈调节人类细胞中的Wnt途径的活性。Pavarotti(Pav)是一种激动素样蛋白，其作用方式与Tum相似，而另一种相互作用蛋白，一种新的苍蝇蛋白，暂定为Sills，增强Wnt诱导的靶基因表达。本申请提出三个目的来确定Tum及其结合伙伴的作用机制。在细胞分裂过程中，Tum和Pav在组织细胞骨架中都扮演着Wnt不依赖的角色，因此一个主要的问题是它们的Wnt依赖的角色是否也存在于细胞质中。目标1将确定与其Wnt调节活性相关的亚细胞位置。目标2将解决Tum和Pav是否与其他Wnt途径组件直接相互作用，或与Wnt靶基因启动子上形成的转录复合体相互作用。目标3将描述Sills及其人类同源物的细胞功能，使用苍蝇胚胎和培养细胞来确定它是如何增强Wnt活性的。这些实验将有助于更清楚地了解活组织如何调节对Wnt信号的反应。公共卫生相关性：WNT信号对所有动物的正常发育都是必不可少的，但异常的WNT信号与各种形式的癌症，特别是结直肠癌有关。这里提出的实验将使用果蝇模型系统和培养的人类细胞来研究新发现的调节Wnt信号的遗传因素。

DESCRIPTION (provided by applicant): Wnt signaling determines cell fates during embryonic development in all animals, including humans, and is required later in life to maintain stem cell populations. Excess Wnt activity is associated with human cancers, particularly colon cancer. To understand how Wnt signaling causes cancer, the factors that normally control Wnt pathway activity must be fully characterized. The long term objective of the Bejsovec laboratory is to dissect the mechanics of this important signaling pathway, using both the Drosophila genetic model system and cultured human cells. This lab has identified by mutational analysis many important pathway components, such as Tcf, the transcription factor that activates Wnt target genes, and Apc2, a human tumor suppressor homolog that negatively regulates the Wnt pathway. This proposal focuses on another negative regulatory gene identified in the same mutational screen: tumbleweed (tum) is a homolog of the human RacGap1, a GTPase activating protein (GAP). Surprisingly, the GAP activity of this gene is essential for cell division, but its GAP activity is not required for its role in Wnt regulation. Two Tum-interacting molecules, identified in yeast two- hybrid screens, strongly modulate Wnt pathway activity in human cells. Pavarotti (Pav), a kinesin-like protein, acts as a repressor in a similar fashion to Tum, while the other interactor, a novel fly protein tentatively named Sills, enhances Wnt-induced target gene expression. This application proposes three aims to determine the mechanism of action for Tum and its binding partners. Both Tum and Pav play a Wnt-independent role in organizing the cytoskeleton during cell division, so a primary issue is whether their Wnt- dependent role also occurs in the cytoplasm. Aim 1 will determine the subcellular locations relevant to their Wnt-modulating activity. Aim 2 will address whether Tum and Pav interact directly with other Wnt pathway components or with the transcription complex that forms on Wnt target gene promoters. Aim 3 will characterize the cellular function of Sills and its human homolog, using both fly embryos and cultured cells to determine how it enhances Wnt activity. These experiments will help formulate a clearer picture of how living tissue modulates the response to Wnt signal. PUBLIC HEALTH RELEVANCE: Wnt signaling is essential for normal development in all animals, but abnormal Wnt signaling is associated with various forms of cancer, particularly colorectal cancer. Experiments proposed here will use both the fruitfly model system and cultured human cells to study newly-discovered genetic factors that regulate Wnt signaling.