The stage-specific regulation of ameloblastin and enamelin by the distinct nuclear factors

项目摘要/摘要 细胞特性在很大程度上由特定的表观遗传环境和转录网络决定。成釉细胞是 唯一可以在发育过程中产生钙化组织的上皮细胞，其中前成釉细胞(PAB)最先 分化为分泌性成釉细胞(SAB)，合成并沉积釉质基质蛋白(EMPs)以 支架有机基质，然后到成熟的成釉细胞(MAB)，内吞EMPs，和 运输离子使牙釉质矿化。为了生物工程牙釉质，一种不可再生的组织，我们必须了解 成釉细胞的转录调控由于牙齿后成釉细胞的丢失而受到限制 出疹和缺乏细胞系完全概括了成釉细胞的特征。之前的资金允许我们 建立与成釉细胞各个发育阶段相关的新的、全面的基因清单 跨物种和探索染色质组织者SATB1的功能，以及釉质基质建模 成釉细胞中的调节因子--多肽酶KLK4和主要钙转运蛋白NCKX4 差异化。这些努力的结果是发现SATB1、KLK4和NCKX4都对 成釉蛋白(AmBN)和成釉蛋白(ENAM)的转录调控，编码主要的EMPs共同- 在SAB中上调，然后在MAB中共同下调。我们发现SATB1的消融，高表达 在PABS中，抑制AMBN和ENAM转录和H3K27ac水平。我们的器官培养显示 组蛋白乙酰化上调AMBN和ENAM。增强子和碱基不配对区域(BUR，选择性 在Ambn和Enam附近预测了SATB1 DNA结合位点)。这些数据表明 SATB1组织染色质构象，平衡转录复合体，上调Ambn和Enam TO 将Pabs提升到SAB。在缺乏导致釉质发生的Klk4和Nockx4基因的小鼠中 不完美-我们发现由于有缺陷的水解导致富含脯氨酸/谷氨酰胺(P/G)的EMPs保留。 这些NCKX4-/-和KLK4-/-MAB上调了AMBN和ENAM，下调了HIF1a的表达。体外研究 富含P/G的多肽可下调AMBN和ENAM的表达，上调HIF1a的表达。我们的RNA-SEQ分析 HIF1a，一种调节细胞对氧化应激反应的转录因子，有6倍于 MAB与SAB的表达上调，反映MAB持续提供能量的强大抗氧化能力 用于离子传输和蛋白质降解。这些数据表明，重新摄取富含P/G的多肽可以上调 HIF1a，这反过来又下调了Ambn&Enam的监管。我们的体内和体外研究使我们能够假设 AMBN和ENAM在成釉细胞两个主要功能阶段的动态表达 受染色质组织者SATB1和转录因子HIF1a的不同因子调控。这一假设将是 具体目标1：确定SATB1在PABS中作为先锋因素的作用，以平衡 激活SABS中ambn和enam基因转录的增强子的建立及其特异性目标2： 确定HIF1a对Ambn和Enam表达和釉质形成的调节作用。

Project Summary/Abstract Cell identity is largely determined by specific epigenetic landscapes and transcriptional networks. Ameloblast is the only epithelial cell that can generate calcified tissue during development, where preameloblasts (PABs) first differentiate to the secretory ameloblasts (SABs) that synthesize and deposit enamel matrix proteins (EMPs) to scaffold organic matrix, and then to the maturation ameloblasts (MABs) that hydrolyze, endocytose EMPs, and transport ions to mineralize enamel. To bioengineer enamel, a nonregenerative tissue, we must understand the transcriptional regulation of ameloblasts that has been limited due to a loss of ameloblasts after the tooth eruption and a lack of cell line fully recapitulating the characteristics of ameloblasts. Previous fundings allow us to establish a novel and comprehensive list of genes significant to each developmental stage of ameloblasts across species and to explore the functions of chromatin organizer SATB1, and enamel matrix modeling regulators -peptidase KLK4 and the major calcium transporter NCKX4- in the context of ameloblast differentiation. These efforts resulted in a discovery that SATB1, KLK4, and NCKX4 all contribute to the transcriptional regulation of ameloblastin (Ambn) and enamelin (Enam), encoding the major EMPs co- upregulated in SABs and then co-downregulated in MABs. We found that ablation of SATB1, highly expressed in PABs, repressed Ambn & Enam transcription and H3K27ac level. Our organ culture showed that elevated histone acetylation upregulated Ambn & Enam. An enhancer and base unpairing region (BUR, selective SATB1 DNA binding site) have been predicted in the vicinity of Ambn & Enam. These data suggest that SATB1 organizes chromatin conformation and poises a transcriptional complex to upregulate Ambn & Enam to advance PABs to SABs. In the case of mice lacking Klk4 and Nckx4—the causative genes for amelogenesis imperfecta—we found a retention of proline/glutamine (P/G)-rich EMPs resulting from defective hydrolysis. These Nckx4-/- and Klk4-/- MABs had upregulated Ambn & Enam and downregulated Hif1a. In vitro studies showed that P/G-rich peptides downregulated Ambn & Enam and upregulated Hif1a. Our RNA-seq analyses revealed that HIF1A, a transcription factor regulating cell responses to oxidative stress, had a 6-fold upregulation in MABs vs SABs, reflecting MAB’s robust anti-oxidative capacity to continuously provide energy for ion transport and protein degradation. These data suggest that retake of P/G-rich peptides upregulate Hif1a, which in turn downregulate Ambn & Enam. Our in vivo and in vitro studies allow us to hypothesize that the dynamic expression of Ambn & Enam in the two major functional stages of ameloblasts is coordinately regulated by distinct factors chromatin organizer SATB1 and transcription factor HIF1A. This hypothesis will be addressed by specific aim 1: To determine the roles of SATB1 as a pioneer factor in PABs to poise the enhancer establishment for activating the transcription of Ambn & Enam gene in SABs; and specific aim 2: To determine the regulatory roles of HIF1A on Ambn & Enam expression and enamel formation.