Regulation of transcription factor activity in neural crest development by pH dynamics

pH 动态对神经嵴发育中转录因子活性的调节

• 批准号: 10508784
• 负责人: DIANE L BARBER
• 金额: $ 24.23万
• 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: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10508784
• 关键词: Address; Adenine; Affinity; Binding; Biochemistry; Biology; Biophysics; Biosensor; Cell Lineage; Cells; Cellular biology; ChIP-seq; Charge; Congenital Abnormality; Consensus; Craniofacial Abnormalities; DNA; DNA Binding; DNA Binding Domain; Data; Defect; Development; Electrostatics; Embryo; Environmental Monitoring; Evolution; FOXC2 gene; FOXM1 gene; Family; Fluorescence Anisotropy; Gene Expression; Genetic Transcription; Histidine; Human; Hydrogen Bonding; Impairment; Ligands; Luciferases; Measures; Modeling; Molecular; Neural Crest; Neural Crest Cell; Nuclear; Nucleic Acid Binding; Nucleic Acids; Nucleotides; Outcome; Proteins; Publishing; Recombinant DNA; Recombinant Proteins; Reporter; Research Personnel; Role; Structure; System; Testing; Thymine; Time; Tissues; Titrations; Transcriptional Regulation; Work; Zebrafish; base; cell behavior; clinically relevant; craniofacial; genome-wide; in vivo; induced pluripotent stem cell; insight; malformation; member; mutant; preference; programs; promoter; sensor; stem cell differentiation; stem cells; success; tool; transcription factor

Abstract Our proposal tests a new idea on how transcription factor-DNA binding selectivity is regulated within the context of neural crest (NC) development and intracellular pH (pHi) dynamics. Transcription factors in FOX, SOX, MITF, MYC, and other families with established roles in NC development and craniofacial lineages have a conserved histidine that forms hydrogen bonds with DNA nucleotides. With the ability of histidine to titrate within the cellular pH range and nuclear and cytosolic pH values being similar, our idea proposes that histidine-nucleotide binding affinities (Kd’s) and hence promotor selectivity can be regulated by pHi dynamics. Despite this idea being applicable to at least 65 transcription factors across multiple families it has largely escaped the notice of investigators across different fields. The biophysical principles of our prediction are that when histidine is protonated at a lower pH it will be a hydrogen bond donor with a hydrogen bond acceptor nucleotide, such as thymine, and when histidine is deprotonated at a higher pH it will be a hydrogen bond acceptor with a hydrogen bond donor nucleotide, such as adenine. Hence, we will test the hypothesis that pHi dynamics regulates transcription factor-DNA binding selectivity for neural crest development by focusing on three transcription factors from different families, FOXD3, SOX10, and MITF, that have established roles in NC development and specification of craniofacial lineages. Our hypothesis is supported by pHi dynamics regulating gene expression for stem cell differentiation and lineage specification, abundant structure data, and our preliminary findings. Moreover, our hypothesis addresses a critical gap in our understanding of how transcription factors are used reiteratively in developmental programs. Contributing to the success of our proposal is our work pioneering a molecular understanding of how pHi dynamics regulates myriad cell behaviors by bridging protein electrostatics and cell biology. In Aim 1 we will determine pH-dependent DNA binding affinities and motif preferences for FOXD3, SOX10 and MITF. We will determine pH regulated Kd’s of recombinant DNA binding domains to previously identified motifs by using fluorescence anisotropy, supported by preliminary data on pH regulated binding affinities of FOXM1 and FOXC2, and identify pH-dependent genome-wide binding preferences with the unbiased approach of systemic evolution of ligand by exponential enrichment (SELEX). In Aim 2 we will determine the role of pHi dynamics in transcription factor-DNA binding in iPSC-derived neural crest cells and in zebrafish models. Cell studies will identify pHi regulated motif preferences by using a dual fluorescent reporter we developed and by ChIP-seq. Zebrafish studies, supported by our data showing spatial differences in pHi in zebrafish embryos during the period of NC development, will test rescue of defects with homozygous- null sox10 and mitf. If our predictions are correct, the outcome of our proposal would be identifying for the first time that transcription factors can be pH sensors with pHi regulating promoter selectivity for NC development, with our findings demonstrating in molecular detail how this occurs and with relevance to craniofacial anomalies.

抽象的 我们的提案测试了转录因子-DNA 结合选择性如何在上下文中调节的新想法 神经嵴 (NC) 发育和细胞内 pH (pHi) 动态。 FOX、SOX、MITF 中的转录因子， MYC 和其他在 NC 发育和颅面谱系中具有既定作用的家族具有保守的 组氨酸与 DNA 核苷酸形成氢键。具有组氨酸在细胞内滴定的能力 pH 范围以及核和细胞质 p​​H 值相似，我们的想法提出组氨酸-核苷酸结合 pHi 动力学可以调节亲和力 (Kd) 以及启动子选择性。尽管这个想法是 它适用于多个家族的至少 65 个转录因子，但它在很大程度上没有引起人们的注意 不同领域的研究者。我们预测的生物物理学原理是，当组氨酸 在较低 pH 下质子化，它将成为具有氢键受体核苷酸的氢键供体，例如 胸腺嘧啶，当组氨酸在较高 pH 下去质子化时，它将成为带有氢的氢键受体 键供体核苷酸，例如腺嘌呤。因此，我们将检验 pHi 动力学调节的假设 转录因子-DNA 结合选择性通过关注三种转录来促进神经嵴发育 来自不同家族（FOXD3、SOX10 和 MITF）的因子，这些因子在 NC 发展和 颅面谱系的规范。我们的假设得到了调节基因表达的 pHi 动力学的支持 用于干细胞分化和谱系规范、丰富的结构数据以及我们的初步发现。 此外，我们的假设解决了我们对转录因子如何使用的理解中的一个关键差距 在发展计划中反复强调。我们的工作开创性地为我们的提案的成功做出了贡献 从分子角度理解 pHi 动力学如何通过桥接蛋白质静电来调节无数细胞行为 和细胞生物学。在目标 1 中，我们将确定 pH 依赖性 DNA 结合亲和力和基序偏好 FOXD3、SOX10 和 MITF。我们将确定重组 DNA 结合域的 pH 调节 Kd 先前通过使用荧光各向异性识别的基序，并得到 pH 调节初步数据的支持 FOXM1 和 FOXC2 的结合亲和力，并利用 通过指数富集进行配体系统进化的无偏方法（SELEX）。在目标 2 中，我们将 确定 pHi 动力学在 iPSC 衍生神经嵴细胞转录因子-DNA 结合中的作用 以及斑马鱼模型。细胞研究将通过使用双荧光来识别 pHi 调节的基序偏好 我们开发的报告器并通过 ChIP-seq 进行。斑马鱼研究，由我们显示空间差异的数据支持 在 NC 发育期间斑马鱼胚胎的 pHi 中，将测试纯合子缺陷的挽救 空 sox10 和 mitf。如果我们的预测是正确的，我们提案的结果将是确定第一个 转录因子可以成为 pH 传感器，具有 pHi 调节启动子选择性以用于 NC 开发的时间， 我们的研究结果以分子细节证明了这是如何发生的以及与颅面异常的相关性。