Transcription Factor Collectives in Vertebrate Wnt Signaling

 描述（由申请人提供）：Wnt是通过几种途径调节细胞行为的分泌蛋白，其中最具特征的是Wnt/β-连环蛋白信号传导，其在动物发育和成体组织稳态中起许多重要作用。例如，在肠隐窝中，该途径是维持干细胞群所必需的，也是指定潘氏细胞所必需的，潘氏细胞分泌抗微生物蛋白以控制肠道细菌。该通路的失调与结直肠癌（CRC）和炎症性肠病有关。Wnt信号传导促进β-连环蛋白的核积累，然后通过转录因子（TF）的TCF/LEF 1（TCF）家族的成员将β-连环蛋白募集到Wnt响应性顺式调节模块（W-CRM）。一旦到达那里，β-连环蛋白就充当Wnt靶基因转录的有效激活剂。一些全基因组研究已经将TCF占用与基因组中的其他TF联系起来。TF的这种聚集被称为“TF集合”。然而，TCF和其他TF之间的功能相互作用的机制知之甚少。我们的特点是两个W-CRMs从人axin 2和c-myc基因的详细使用细胞培养。c-myc W-CRM之所以令人感兴趣，是因为它在与人类结直肠癌（CRC）增加相关的TCF位点中含有多态性。我们的初步数据支持一个模型，其中TCF与其他几个TF的工作，以实现Wnt激活这些W-CRM。我们将描述这些因素之间的物理和功能相互作用，以了解包含TCF的TF集体如何运作。我们已经发现c-myc W-CRM在小鼠胚胎的几种组织中具有活性，并且鉴于其与人类CRC的联系，我们将在成年小鼠肠道中检查其活性。我们还将测试在细胞培养中激活这种W-CRM的重要因素是否也需要在小鼠组织中。这些因子之一是Sox 9，其是一种TF，其通常被认为是Wnt/β-连环蛋白信号传导的拮抗剂，但也与TCF合作以激活c-myc W-CRM。这种合作可以解释为什么TCFs和Sox 9都是肠道潘氏细胞形成所必需的。此外，Sox 9是睾丸形成所必需的，具有Sox 9突变的XY个体通常发育为女性。我们正在表征Sox 9突变体，这些突变体专门影响其与Wnt信号传导作用或对抗Wnt信号传导的能力。我们建议用这些突变改造小鼠，以确定这些活动是否是其在潘氏细胞特化和性别决定中作用的基础。这项工作将增加我们对Wnt信号在CRC以及影响潘氏细胞功能的炎症性肠病中的作用的理解。

 DESCRIPTION (provided by applicant): Wnts are secreted proteins that regulate cell behavior through several pathways, the best characterized of which is Wnt/ß-catenin signaling, which plays many important roles in animal development and adult tissue homeostasis. For example, in intestinal crypts, the pathway is required to maintain stem cell populations, and is also needed to specify Paneth cells, which secrete anti-microbial proteins to keep gut bacteria in check. Misregulation of the pathway is associated with colorectal cancer (CRC) and inflammatory bowel disorders. Wnt signaling promotes nuclear accumulation of ß-catenin, which is then recruited to Wnt responsive cis- regulatory modules (W-CRMs) by members of the TCF/LEF1 (TCF) family of transcription factors (TFs). Once there, ß-catenin acts as a potent activator of Wnt target gene transcription. Several genome-wide studies have linked TCF occupancy with other TFs in the genome. This clustering of TFs has been termed a "TF collective". However, the mechanisms underlying functional interactions between TCFs and other TFs are poorly understood. We have characterized two W-CRMs from the human axin2 and c-myc genes in detail using cell culture. The c-myc W-CRM is of interest because it contains a polymorphism in a TCF site linked to increased colorectal cancer (CRC) in humans. Our preliminary data support a model where TCFs work with several other TFs to achieve Wnt activation of these W-CRMs. We will characterize the physical and functional interactions between these factors, to understand how a TF collective containing TCFs operates. We have found that the c-myc W-CRM is active in several tissues in mouse embryos and given its link to CRC in humans, we will examine its activity in the adult mouse intestine. We will also test whether the factors important for activating this W-CRM in cell culture are also required in mouse tissues. One of these factors is Sox9, a TF which is which is typically thought of as an antagonist of Wnt/ß-catenin signaling, but which also cooperates with TCFs to activate the c-myc W-CRM. This cooperation may explain why TCFs and Sox9 are both required for Paneth cell formation in the intestine. In addition, Sox9 is required for testis formation and XY individuals with Sox9 mutations often develop as females. We are characterizating Sox9 mutants that specifically affect its ability to act with or against Wnt signaling. We propose to engineer mice with these mutations, to determine whether these activities underlie its role in Paneth cell specification and sex determination. This work will increase our understanding of the role of Wnt signaling in CRC as well as inflammatory bowel disorders that affect Paneth cell function.