pH dynamics determining DNA binding specificity of FOX transcription factors

项目摘要/摘要 哺乳动物狐狸转录因子家族的成员调节广泛的细胞过程 包括细胞分裂、分化和凋亡。尽管共享高度保守的DNA结合序列， 每个狐狸家族成员都绑定了一组不同的靶基因，以调节无数的细胞行为。海流 对不同Fox家族转录因子的DNA结合选择性的理解是，调控是 由辅因子关联、翻译后修饰和细胞特异性表达决定。这项建议 旨在解决一种新的观点，即细胞内pH(Phi)动态是一个额外的调节靶点的机制 Fox家族转录因子的基因选择性。虽然所有的狐狸蛋白都含有不变的组氨酸 DNA结合区中的残基，直接与核苷酸和组氨酸残基形成氢键 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得到 据报道。基于初步数据表明FOXM1与共同共识的结合依赖于pH 序列，这一建议检验了狐狸DNA结合中不变组氨酸的总体假设 结构域是一个pH传感器，用于调节Phi对靶基因的结合选择性。目标1的目标是 测定pH调节的Fox家族蛋白的DNA结合选择性。关于pH调节的DNA的预测- 将使用分子动力学在电子计算机中测试一些Fox家族成员的结合特异性 模拟将为使用荧光来确定结合亲和力的体外生化方法提供信息 各向异性。此外，全基因组范围内依赖于pH的结合偏好将使用系统 指数富集法配基进化(SELEX)。目标2的目标是确定Phi的角色 FOXM1靶基因在细胞中的调控动态，主要集中在幼稚的小鼠胚胎干细胞和肿瘤 细胞，因为它们的行为受到phi动力学的调节。全球FOXM1启动子结合偏好 作为对Phi的响应，将通过使用CHIP-SEQ来鉴定胚胎干细胞和癌细胞中的动态 将通过使用RT-qPCR确定Phi调节的基因表达来进行机制分析。除了……之外 揭示了狐狸蛋白转录调控的新范式，正在测试的原理有 对DNA结合域中含有组氨酸的其他转录因子的活性的意义， 包括IRF、GATA和ETV家族的成员。此外，由于Phi调节转录因子的活性 和基因表达仍然研究不足，结果将增加对phi动力学如何机制的理解 调节细胞行为。最后，由于Fox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 限制癌症进展，也用于控制再生医学。

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.