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 我们的提案测试了一个关于转录因子-DNA结合选择性如何在上下文中调节的新想法 神经rest（NC）发育和细胞内pH（PHI）动力学。 Fox，Sox，Mitf的转录因子， MYC和其他在NC开发和颅面谱系中既有角色的家庭都有保守的 与DNA核唯一形成氢键的组氨酸。组氨酸在细胞内滴定的能力 pH范围，核和胞质pH值相似，我们的想法提议是组氨酸核苷酸结合 亲和力（KD）及其启动子选择性可以通过PHI动态来调节。尽管这个想法是 适用于多个家庭的至少65个转录因子 跨不同领域的调查人员。我们预测的生物物理原理是，当组氨酸为 在较低的pH下质子化，它将是具有氢键受体核苷酸的氢键供体，例如 胸骨，当组氨酸在较高的pH下去质子化时，它将是氢键受体 键供体核苷酸，例如腺嘌呤。因此，我们将测试PHI动力学调节的假设 转录因子-DNA结合选择性通过专注于三个转录来为神经rest发育 来自不同家庭FOXD3，SOX10和MITF的因素，在NC开发中确立了角色 颅面谱系的规范。我们的假设得到调节基因表达的PHI动力学支持 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如何使用转录因素的关键差距 重申发展计划。为我们的提案的成功做出贡献是我们的工作开创 分子理解PHI动力学如何通过桥接蛋白质静电来调节众多细胞行为 和细胞生物学。在AIM 1中，我们将确定pH依赖性的DNA结合亲和力和基序的偏好 FOXD3，SOX10和MITF。我们将确定重组DNA结合域与 先前通过使用荧光各向异性鉴定的基序，并由PH受调节的初步数据支持 FOXM1和FOXC2的结合亲和力，并与pH依赖性基因组结合偏好与 通过指数富集（SELEX）对系统进化的无偏见方法。在目标2中，我们将 确定PHI动力学在转录因子-DNA结合IPSC衍生的神经Crest细胞中的作用 和斑马鱼模型。细胞研究将通过使用双荧光来识别PHI调节的基序偏好 我们开发的记者和Chip-Seq。斑马鱼研究，并得到我们显示空间差异的数据的支持 在NC开发期间，在斑马鱼胚胎中的Phi中，将测试纯合子的缺陷。 NULL SOX10和MITF。如果我们的预测是正确的，我们的提议的结果将是第一个 转录因子可以是具有pHI调节剂选择性的pH传感器的时间， 我们的发现详细说明了这种情况如何发生并与颅面异常相关。