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

基本信息

批准号：
10656499
负责人：
DIANE L BARBER
金额：
$ 20.19万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10656499
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 Specific qualifier value Structure System Testing Thymine Time Tissues Titrations Transcriptional Regulation Work Zebrafish 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

项目摘要

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 FOX、SOX、MITF 中神经嵴 (NC) 发育和细胞内 pH (pHi) 动力学的研究。 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范围与核和细胞质pH值相似，我们的想法提出组氨酸-核苷酸结合尽管有这样的想法，但亲和力（Kd）和启动子选择性可以通过 pHi 动力学来调节。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我们预测的生物物理学原理是，当组氨酸为在较低 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我们在发展计划中反复强调我们的工作是开创性的，为我们的建议的成功做出了贡献。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。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，并利用在目标 2 中，我们将通过指数富集进行配体系统进化的无偏方法（SELEX）。确定 pHi 动力学在 iPSC 衍生神经嵴细胞转录因子-DNA 结合中的作用在斑马鱼模型中，细胞研究将通过使用双荧光来识别 pHi 调节的基序偏好。我们开发的报告器和斑马鱼研究，并得到我们显示空间差异的数据的支持。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 null 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。