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

项目总结/摘要 细胞身份在很大程度上取决于特定的表观遗传景观和转录网络。成釉细胞是 在发育过程中唯一可以产生钙化组织的上皮细胞，其中成纤维细胞（PABs）首先 分化为分泌性成釉细胞（SAB），其合成并存款釉基质蛋白（EMP）， 支架有机基质，然后成熟成釉细胞（MAB）水解，内吞EMP， 运输离子使釉质矿化。为了对非再生组织牙釉质进行生物工程改造，我们必须了解 成釉细胞的转录调控由于牙齿脱落后成釉细胞的丧失而受到限制 缺乏完全再现成釉细胞特征的细胞系。以前的资金允许我们 建立一个新的和全面的基因清单显着的每个发育阶段的成釉细胞 并探讨染色质组织者SATB 1的功能，以及釉基质建模 调节因子-肽酶KLK 4和主要的钙转运蛋白NCKX 4-在成釉细胞的背景下 分化这些努力导致发现SATB 1，KLK 4和NCKX 4都有助于 成釉蛋白（Ambn）和釉蛋白（Enam）的转录调控，编码主要的EMPs共 在SAB中上调，然后在MAB中共下调。我们发现，SATB 1的消融，高表达， 在PABs中，抑制Ambn & Enam转录和H3 K27 ac水平。我们的器官培养显示 组蛋白乙酰化上调Ambn & Enam.增强子和碱基不配对区（BUR，选择性 SATB 1 DNA结合位点）已被预测在Ambn和Enam附近。这些数据表明 SATB 1组织染色质构象并平衡转录复合物以上调Ambn & Enam， 将PAB提前到SAB。在缺乏Klk 4和Nckx 4的小鼠的情况下， 此外，我们发现保留脯氨酸/谷氨酰胺（P/G）丰富的EMP造成的缺陷水解。 这些Nckx 4-/-和Klk 4-/-MAB上调了Ambn和Enam，下调了Hif 1a。体外研究 结果表明，富含P/G的肽下调Ambn和Enam，上调Hif 1a。我们的RNA-seq分析 揭示了HIF 1A，一种调节细胞对氧化应激反应的转录因子，具有6倍的 MAB与SAB相比上调，反映了MAB强大的抗氧化能力， 用于离子运输和蛋白质降解。这些数据表明，重摄取富含P/G的肽上调 Hif 1a，反过来下调Ambn & Enam。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是协调的 由不同的因子染色质组织者SATB 1和转录因子HIF 1A调节。这一假设将是 具体目标1：确定SATB 1作为PAB中的先驱因素的作用， 用于激活SAB中Ambn和Enam基因转录的增强子的建立;以及具体目的2： 确定HIF 1A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.