Protein biophysics for pH-regulated transcription factor-DNA binding selectivity

转录因子如何识别特定的DNA序列来调节基因表达仍然是生物学中的一个重要问题。当考虑到具有高度相似的DNA结合域但选择性结合不同DNA序列的转录因子大家族的成员时，这个问题尤其令人困惑。另一个尚未充分研究的重要问题是细胞内pH （pHi）动力学如何调节无数细胞行为，包括增殖、迁移、分化和细胞命运转变。该项目建立在先前关于pHi动态如何通过对pHi变化敏感的蛋白质调节细胞行为的研究基础之上。本研究将探讨pHi影响转录因子的DNA结合选择性，从而影响转录因子调控基因表达的功能。该项目还为学生和博士后提供从生物物理学到细胞生物学的跨学科培训机会。本项目验证了pHi动力学调节转录因子- dna结合亲和力和选择性的新思路。至少65个不同家族的转录因子在DNA结合域中含有与核苷酸碱基形成氢键的保守组氨酸。待验证的假设是组氨酸的质子化和去质子化随pH的变化调节转录因子的启动子结合选择性，分子动力学模拟和生化实验表明pH调节重组FOXM1和FOXC2的DNA结合。目标包括ph依赖性转录因子与DNA结合的计算预测（目标1），ph依赖性DNA结合亲和力和基序偏好的生化分析（目标2），以及研究pHi动力学对细胞中转录因子靶基因选择性的影响（目标3）。这些结果可能揭示ph介导的转录因子功能调节的新机制，并为具有相似dna结合结构域的转录因子如何重复地调节多种基因提供新的见解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

How transcription factors recognize specific DNA sequences to regulate gene expression remains a significant question in biology. This question is particularly perplexing when considering members of large families of transcription factors that share highly similar DNA- binding domains but bind selectively to different DNA sequences. Another significant question that remains understudied is how intracellular pH (pHi) dynamics regulate myriad cell behaviors, including proliferation, migration, differentiation and cell fate transitions. This project builds on prior studies of how pHi dynamics regulate cell behaviors via proteins whose activities are sensitive to changes in pHi. The research will explore the idea that pHi affects the DNA binding selectivity of transcription factors and thereby their function in regulating gene expression. The project also offers cross-disciplinary training opportunities from biophysics to cell biology for students and postdoctoral scholars.This project tests a new idea that pHi dynamics regulate transcription factor-DNA binding affinity and selectivity. At least 65 transcription factors in different families contain a conserved histidine in the DNA binding domain that forms hydrogen bonds with nucleotide bases. The hypothesis to be tested is that protonation and deprotonation of the histidine with changing pH regulates promoter binding selectivity of transcription factors, which is supported by molecular dynamics simulations and biochemical assays indicating pH modulates DNA binding by recombinant FOXM1 and FOXC2. The objectives include computational predictions of pH-dependent transcription factor binding to DNA (Aim 1), biochemical analysis of pH-dependent DNA binding affinities and motif preferences (Aim 2), and investigating the effects of pHi dynamics in transcription factor target gene selectivity in cells (Aim 3). The outcomes could reveal a new mechanism for pH-mediated regulation of transcription factor function, and offer new insights on how transcription factors with similar DNA-binding domains are used reiteratively to regulate a variety of genes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.