Gene regulation in ARMS pathology; the role of FOXO1 in Pax3-FOXO1 DNA binding

ARMS 病理学中的基因调控；

• 批准号: 8514641
• 负责人: Kelly Elaine Johanson
• 金额: $ 13.76万
• 依托单位: XAVIER UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8514641
• 关键词: Adolescent; Affect; Alveolar Rhabdomyosarcoma; Amino Acid Sequence; Amino Acids; Base Pairing; Behavior; Beryllium; Binding; Cell Line; Child; Chimeric Proteins; Chromosomal translocation; Code; Computer Simulation; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; DNA-Protein Interaction; Data; Databases; Development; Elements; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Targeting; Generations; Genes; Goals; Grant; In Vitro; Indium; Lead; Malignant Skeletal Muscle Neoplasm; Mediating; Mentors; Modeling; Molecular; Mutate; Mutation; Nature; Oncogenic; Pathology; Patients; Pilot Projects; Positioning Attribute; Production; Promoter Regions; Proteins; Regulatory Element; Research; Research Personnel; Response Elements; Rhabdomyosarcoma; Role; Sequence Analysis; Specificity; Survival Rate; TFAP2B gene; Testing; Transcript; Transcription Coactivator; Transcriptional Activation; Work; in vivo; innovation; mortality; mouse model; novel; promoter; t(2; 13)(q35; q14); therapeutic target; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): Alveolar Rhabdomyosarcoma (ARMS) patients carrying the t (2; 13) (q35; q14) chromosomal translocation have the lowest survival rate of all rhabdomyosarcomas; however, the reason for this fact is not clear. The translocation encodes an oncogenic fusion protein Pax3-FOXO1, and the strong correlation between the presence of the translocation and increased mortality indicates that this fusion protein directly participates n the mechanism of ARMS development. The contributions of the Pax3 portion of the fusion protein to oncogenesis have been extensively studied. By contrast little is known about the role the FOXO1 portion makes to ARMS development. Despite a lack of experimental evidence, the truncated FOXO1 DNA binding domain has been assumed to be nonfunctional. However, sequence analysis reveals that key amino acids that mediate FOXO1-dependent DNA interactions are present within the fusion protein. Therefore, this evidence emphasizes the need to examine the role of the truncated FOXO1 DBD in targeting promoter regions to aid in identifying novel Pax3-FOXO1 transcriptional targets that contribute to the development of ARMS. The long-term goal is to determine the mechanism by which Pax3-FOXO1 binds to and activates regulatory elements, and how activation of these specific genes leads to the development of ARMS. The initial step in achieving this goal is to determine the contribution made by the truncated FOXO1 DBD to Pax3-FOXO1 DNA binding by examining both the DNA and amino acid contacts necessary for this interaction with the following two specific aims: Specific Aim 1: Determine structural elements necessary for Pax3-FOXO1 DNA binding and transcriptional activation in vitro. Using synthetic promoter constructs with both Pax3 and FOXO1 recognition sequences, we will examine the effect of mutating the FOXO1 portion of the sequence and amino acids necessary for FOXO1 binding on DNA binding and transcriptional activation. Specific Aim 2: Identify the contribution made by the FOXO1 element to the activation of known Pax3-FOXO1 direct transcriptional targets. We will test the importance of putative FOXO1 DNA sequence elements in the ability of Pax3-FOXO1 to bind and activate CNR1, TFAP2B, PRRX1, and MEOX2 in a rhabdomyosarcoma cell line. The significance of the proposed project is that it will provide both the data and the appropriate mentoring required to position the PI to undertake subsequent definitive studies on the role of the truncated FOXO1 DNA- binding domain in mediating DNA binding by Pax3-FOXO1 at the SC1 level. The research proposed in this application is innovative because it focuses on the contributions that the truncated FOXO1 DNA-binding domain makes to Pax3-FOXO1 DNA binding and gene expression. These data will provide basic information about the molecular mechanism by which Pax3-FOXO1 recognizes and regulates the expression of genes involved in ARMS development.

描述（由申请人提供）：携带 t (2; 13) (q35; q14) 染色体易位的腺泡型横纹肌肉瘤 (ARMS) 患者在所有横纹肌肉瘤中存活率最低；然而，造成这一事实的原因尚不清楚。该易位编码致癌融合蛋白Pax3-FOXO1，易位的存在与死亡率增加之间的强相关性表明该融合蛋白直接参与ARMS发育机制。融合蛋白的 Pax3 部分对肿瘤发生的贡献已被广泛研究。相比之下，人们对 FOXO1 部分对 ARMS 开发的作用知之甚少。尽管缺乏实验证据，截短的 FOXO1 DNA 结合域仍被认为是无功能的。然而，序列分析表明，介导 FOXO1 依赖性 DNA 相互作用的关键氨基酸存在于融合蛋白中。因此，这一证据强调需要检查截短的 FOXO1 DBD 在靶向启动子区域中的作用，以帮助识别有助于 ARMS 发展的新 Pax3-FOXO1 转录靶标。长期目标是确定 Pax3-FOXO1 结合并激活调控元件的机制，以及这些特定基因的激活如何导致 ARMS 的发展。实现这一目标的第一步是通过检查这种相互作用所需的 DNA 和氨基酸接触来确定截短的 FOXO1 DBD 对 Pax3-FOXO1 DNA 结合的贡献，具体目标如下： 具体目标 1：确定结构Pax3-FOXO1 DNA 结合和体外转录激活所需的元件。使用具有 Pax3 和 FOXO1 识别序列的合成启动子构建体，我们将检查 FOXO1 序列部分和 FOXO1 结合所需的氨基酸突变对 DNA 结合和转录激活的影响。具体目标 2：确定 FOXO1 元件对已知 Pax3-FOXO1 直接转录靶点激活的贡献。我们将测试假定的 FOXO1 DNA 序列元件对于 Pax3-FOXO1 在横纹肌肉瘤细胞系中结合和激活 CNR1、TFAP2B、PRRX1 和 MEOX2 的能力的重要性。拟议项目的重要性在于，它将提供所需的数据和适当的指导，使 PI 能够对截短的 FOXO1 DNA 结合域在 SC1 上介导 Pax3-FOXO1 DNA 结合中的作用进行后续确定性研究。等级。本申请中提出的研究具有创新性，因为它重点关注截短的 FOXO1 DNA 结合结构域对 Pax3-FOXO1 DNA 结合和基因表达的贡献。这些数据将提供有关 Pax3-FOXO1 识别和调节 ARMS 发育相关基因表达的分子机制的基本信息。