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

转录因子如何识别特定的DNA序列来调控基因的表达仍然是生物学中的一个重要问题。当考虑到转录因子大家族的成员时，这个问题尤其令人困惑，这些转录因子拥有高度相似的DNA结合结构域，但选择性地结合不同的DNA序列。另一个尚未被研究的重要问题是细胞内pH(Phi)如何调节无数的细胞行为，包括增殖、迁移、分化和细胞命运的转变。这个项目建立在先前的研究基础上，即phi动力学如何通过其活动对phi变化敏感的蛋白质来调节细胞行为。这项研究将探索Phi影响转录因子的DNA结合选择性，从而影响它们在调控基因表达中的功能的想法。该项目还为学生和博士后学者提供了从生物物理学到细胞生物学的跨学科培训机会。该项目测试了PHI动力学调节转录因子-DNA结合亲和力和选择性的新想法。在不同的家族中，至少有65个转录因子在DNA结合区含有一个保守的组氨酸，它与核苷酸碱基形成氢键。需要检验的假设是，随着pH的变化，组氨酸的质子化和去质子化调节转录因子的启动子结合选择性，这一假设得到了分子动力学模拟和生化分析的支持，表明pH通过重组FOXM1和FOXC2调节DNA结合。目标包括pH依赖的转录因子与DNA结合的计算预测(目标1)，pH依赖的DNA结合亲和力和基序偏好的生化分析(目标2)，以及研究PHI动态对细胞中转录因子靶基因选择性的影响(目标3)。这一结果可能揭示一种新的pH调节转录因子功能的机制，并为如何重复使用具有相似DNA结合结构域的转录因子来调节各种基因提供新的见解。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.