pH dynamics determining DNA binding specificity of FOX transcription factors

项目总结/摘要 哺乳动物FOX家族转录因子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FOX家族成员结合一组不同的靶基因来调节无数的细胞行为。当前 对不同FOX家族转录因子的DNA结合选择性的理解是， 由辅因子缔合、翻译后修饰和细胞特异性表达赋予。这项建议 旨在解决一个新的想法，即细胞内pH（pHi）动力学是一个额外的机制调节目标 FOX家族转录因子的基因选择性。尽管所有FOX蛋白都含有不变的组氨酸 DNA结合域中直接与核苷酸和组氨酸残基形成氢键的残基 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还没有被证实。 报道基于初步数据证明FoxM 1与共同共识的pH依赖性结合 序列，这项建议测试的总体假设，不变的组氨酸在FOX DNA结合 结构域是pH调节的靶基因结合选择性的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- 将使用分子动力学在计算机中测试许多FOX家族成员的结合特异性 模拟，将告知体外生物化学方法，以确定结合亲和力使用荧光 各向异性此外，全基因组pH依赖性结合偏好将使用系统性方法确定。 配体指数富集进化（SELEX）。目标2的目的是确定pHi的作用 在细胞中调节FoxM 1靶基因的动力学，重点是幼稚小鼠胚胎干细胞和癌症 细胞，因为它们具有由pHi动力学调节的行为。全局FoxM 1启动子结合偏好 在胚胎干细胞和癌细胞中， 将通过使用RT-qPCR确定pHi调节的基因表达来告知机制分析。除了 揭示了FOX蛋白质转录调节的新范式，所测试的原理具有 对于在DNA结合结构域中含有组氨酸的其他转录因子的活性具有重要意义， 包括IRF、加塔和ETV家庭的成员。此外，由于pH调节的转录因子活性 和基因表达仍然未得到充分研究，结果将增加对pHi动力学如何 调节细胞行为。最后，由于FOX转录活性和pHi动力学失调， 对于许多疾病，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.