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

摘要 我们的建议测试了一个新的想法，如何转录因子-DNA结合的选择性是调节的背景下， 神经嵴（NC）的发展和细胞内pH值（pHi）动态。FOX，SOX，MITF， 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 与DNA核苷酸形成氢键的组氨酸。由于组氨酸能够在细胞内滴定， pH范围和细胞核和胞质的pH值是相似的，我们的想法提出， 亲和力（Kd）和因此的启动子选择性可以通过pHi动力学来调节。尽管这个想法 它适用于多个家族中的至少65种转录因子，但它在很大程度上没有引起人们的注意。 不同领域的调查员。我们预测的生物物理学原理是，当组氨酸 如果在较低pH下质子化，则它将是具有氢键受体核苷酸的氢键供体，例如 当组氨酸在较高pH下去质子化时，它将是具有氢原子的氢键受体。 键供体核苷酸，如腺嘌呤。因此，我们将检验pHi动力学调节 通过聚焦于三种转录因子对神经嵴发育的DNA结合选择性 来自不同家族的因子，FOXD 3，SOX 10和MITF，它们在NC发展中发挥了作用， 颅面谱系的详细说明。我们的假设得到了pHi动力学调控基因表达的支持 干细胞分化和谱系特化，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空白 在发展计划中的重要性。我们的提案之所以取得成功，是因为我们的工作是开创性的， 对pHi动力学如何通过桥接蛋白质静电来调节无数细胞行为的分子理解 和细胞生物学。在目标1中，我们将确定pH依赖的DNA结合亲和力和基序偏好， FOXD 3、SOX 10和MITF。我们将确定重组DNA结合结构域的pH调节的Kd， 先前通过使用荧光各向异性识别的基序，由pH调节的初步数据支持 FOXM 1和FOXC 2的结合亲和力，并确定pH依赖性全基因组结合偏好， 无偏配体系统进化指数富集法（SELEX）。在目标2中， 确定pHi动力学在iPSC衍生的神经嵴细胞中转录因子-DNA结合中的作用 和斑马鱼模型。细胞研究将通过使用双荧光标记识别pHi调节的基序偏好。 我们开发的报告基因和ChIP-seq。斑马鱼研究，我们的数据支持显示空间差异 在斑马鱼胚胎中的pHi在NC发育期间，将测试纯合缺陷的拯救- null sox 10和mitf。如果我们的预测是正确的，我们的建议的结果将是确定第一个 时间，转录因子可以是pH传感器与pHi调节启动子选择性为NC发展， 我们的研究结果从分子上详细说明了这是如何发生的，以及与颅面异常的相关性。

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.