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

摘要 我们的建议测试了一种新的想法，即转录因子-DNA结合选择性是如何在上下文中调节的 神经脊(NC)发育和细胞内pH(Phi)动态的关系。FOX、SOX、MITF中的转录因子 MYC和其他在NC发育和头面部谱系中具有既定角色的家族具有保守的 与DNA核苷酸形成氢键的组氨酸。具有在细胞内滴定组氨酸的能力 PH范围与细胞核和细胞质的pH值相似，我们的想法认为组氨酸-核苷酸结合 亲和力(Kd‘s)和启动子的选择性可以由phi动力学来调节。尽管这个想法是 它适用于多个家族的至少65个转录因子，在很大程度上没有引起 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 在较低的pH下质子化，它将是氢键供体和氢键受体核苷酸，例如 胸腺嘧啶，当组氨酸在较高的pH下去质子化时，它将与氢形成氢键受体 键合供体核苷酸，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结合选择性对神经脊发育的影响 来自不同家族的因子，FOXD3、SOX10和MITF，它们在NC开发和 头面部谱系的详细说明。我们的假说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鉴定，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上的一个关键差距。 在发展计划中反复出现。我们的提案之所以取得成功，是因为我们的工作开创了 PHI动力学如何通过桥接蛋白质静电学调节无数细胞行为的分子理解 和细胞生物学。在目标1中，我们将确定依赖于pH的DNA结合亲和力和基序偏好 FOXD3、SOX10和MITF。我们将测定重组DNA结合区的pH调节Kd‘s以 之前利用荧光各向异性识别的基序，得到了关于pH调节的初步数据的支持 FOXM1和FOXC2的结合亲和力，并确定pH依赖的全基因组结合偏好 指数富集法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来源的神经脊细胞转录因子-DNA结合中的作用 在斑马鱼模型中。细胞研究将通过使用双重荧光来确定Phi调控基序的偏好 记者：我们开发的，并由芯片级。斑马鱼研究，支持我们的数据显示空间差异 在斑马鱼胚胎发育期间的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，我们提议的结果将是第一次确定 转录因子可以作为pH传感器的时间，其中phi调节NC发育的启动子选择性， 我们的发现在分子上详细地展示了这种现象是如何发生的，并与颅面畸形有关。

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.