pH dynamics determining DNA binding specificity of FOX transcription factors

pH 动态决定 FOX 转录因子的 DNA 结合特异性

• 批准号: 10389680
• 负责人: Kyle Kisor
• 金额: $ 4.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10389680
• 关键词: Adhesions; Affinity; Apoptosis; Barbering; Behavior; Binding; Biochemical; Cell division; Cell physiology; Cells; ChIP-seq; Collaborations; Complex; Consensus Sequence; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; Data; Disease; Dysplasia; Evolution; FOXM1 gene; Family; Family member; Fluorescence Anisotropy; Gene Expression; Genes; Genetic Transcription; Histidine; Human Pathology; Hydrogen Bonding; In Vitro; Interferons; Kinetics; Ligands; Maintenance; Major Groove; Malignant Neoplasms; Molecular; Mus; Neoplasm Metastasis; Nucleic Acid Binding; Nucleic Acids; Nucleotides; Oncogenes; Outcome; Outcome Study; Pathology; Play; Post-Translational Protein Processing; Promoter Regions; Protein Dynamics; Protein Family; Proteins; Recombinants; Regenerative Medicine; Regulation; Reporting; Role; Specificity; Structure; Testing; Therapeutic; Titrations; Transcriptional Regulation; Work; base; cancer cell; cell behavior; cell motility; clinically relevant; daughter cell; embryonic stem cell; genome-wide; human disease; in silico; member; molecular dynamics; novel; novel therapeutic intervention; preference; promoter; protein function; protein structure; protonation; response; sensor; simulation; stem cell differentiation; stem cell self renewal; stem cells; transcription factor; tumor progression; tumorigenesis

Project Summary/Abstract Members of the mammalian FOX family of transcription factors regulate a broad range of cellular processes including cell division, differentiation, and apoptosis. Despite sharing a highly conserved DNA-binding sequence, each FOX family member binds a distinct set of target genes to regulate myriad cell behaviors. The current understanding of DNA-binding selectivity of different FOX family transcription factors is that regulation is conferred by co-factor association, post-translational modifications, and cell specific expression. This proposal aims to resolve a new idea that intracellular pH (pHi) dynamics is an additional mechanism regulating target gene selectivity of FOX family transcription factors. Although all FOX proteins contain an invariant histidine residue in the DNA-binding domain that directly forms hydrogen bonds with nucleotides and histidine residues can titrate with pHi dynamics, whether pHi regulates nucleotide binding specificity of FOX proteins has not been reported. Building on preliminary data demonstrating pH-dependent binding of FoxM1 to a common consensus sequence, this proposal tests the overall hypothesize that the invariant histidine in FOX DNA-binding domains is a pH sensor for pHi-regulated binding selectivity to target genes. The objective of Aim 1 is to determine pH-regulated DNA binding selectivity of FOX family proteins. Predictions on pH-regulated DNA- binding specificity of a number of FOX family members will be tested in silico using molecular dynamic simulations that will inform in vitro biochemical approaches to determine binding affinities using fluorescence anisotropy. Additionally, genome-wide pH-dependent binding preferences will be determined using systematic evolution of ligand by exponential enrichment (SELEX). The objective of Aim 2 is to determine the role of pHi dynamics in regulating FoxM1 target genes in cells, focusing on naïve mouse embryonic stem cells and cancer cells because they to have behaviors regulated by pHi dynamics. Global FoxM1 promoter binding preferences in response to pHi dynamics will be identified in embryonic stem cells and cancer cells by using ChiP-seq that will inform mechanistic analysis by determining pHi regulated gene expression using RT-qPCR. In addition to revealing a new paradigm for transcriptional regulation of FOX proteins the principles being tested have significance for the activity of other transcription factors that contain histidines in the DNA binding domain, including members of IRF, GATA and ETV families. Moreover, because pHi-regulated transcription factor activity and gene expression remain understudied, outcomes will add mechanistic understanding of how pHi dynamics regulates cell behaviors. Finally, because FOX transcriptional activity and pHi dynamics are dysregulated in many diseases, these findings will have clinical relevance toward new therapeutic approaches, particularly to limit cancer progression and also for controlling regenerative medicine.

项目摘要/摘要 转录因子的哺乳动物Fox家族的成员调节了广泛的细胞过程 包括细胞分裂，分化和凋亡。尽管共享一个高度保守的DNA结合序列，但 每个FOX家族成员都结合了一组独特的靶基因，以调节多种细胞行为。电流 了解不同FOX家族转录因子的DNA结合选择性的原因是调节是 由共同因素关联，翻译后修饰和细胞特异性表达方式。这个建议 旨在解决一个新想法，即细胞内pH（PHI）动力学是调节目标的附加机制 FOX家族转录因子的基因选择性。尽管所有狐狸蛋白都包含一个不变的组氨酸 居住在DNA结合结构域中，直接与核苷酸和组氨酸残留物形成氢键 可以用PHI动力学滴定，PHI是否调节FOX蛋白的核苷酸结合特异性尚未是 报告。以初步数据为基础，证明了FOXM1与共同共识的pH依赖性结合 序列，该提案测试了总体假设，即FOX DNA结合中不变的组氨酸 结构域是一种与靶基因的PHI调节结合选择性的pH传感器。目标1的目的是 确定FOX家族蛋白的pH调节的DNA结合选择性。对pH调节的DNA-的预测 许多FOX家族成员的结合特异性将在硅中使用分子动态进行测试 模拟将告知体外生化方法，以确定使用荧光的结合亲和力 各向异性。此外，将使用系统的 配体通过指数富集（SELEX）的进化。目标2的目的是确定PHI的作用 确定细胞中FOXM1靶基因的动力学，重点是幼稚的小鼠胚胎干细胞和癌症 细胞是因为它们具有由PHI动力学调节的行为。全局FOXM1启动子绑定偏好 通过使用Chip-Seq，将在胚胎干细胞和癌细胞中鉴定出PHI动力学 将通过使用RT-QPCR确定PHI调节基因表达来为机械分析提供信息。此外 揭示了针对FOX蛋白的转录调节的新范式 在DNA结合结构域中包含组氨酸的其他转录因子活性的重要性， 包括IRF，GATA和ETV家庭的成员。此外，因为Phi调节的转录因子活性 基因表达仍然可以理解，结果将增加对PHI动力学的理解 调节细胞行为。最后，因为FOX的转录活性和PHI动力学在 许多疾病，这些发现将对新的治疗方法具有临床意义，特别是 限制癌症的进展，还可以控制再生医学。