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。分为两个主要阶段， 成釉细胞(SAB)的分泌阶段合成并沉积釉基质蛋白(EMPs)以形成支架 有机基质和成釉细胞(MAB)成熟阶段的水解、内吞EMPs和运输 使牙釉质矿化的离子。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。到目前为止，对蛋白的独特转录调控的理解 由于牙萌出后成釉细胞的丧失和缺乏完整的细胞系，成釉细胞受到限制。 概括了成釉细胞的特征。之前的资金使我们能够建立一部小说和 对不同物种的成釉细胞每个发育阶段具有重要意义的基因的综合清单 探讨染色质组织者SATB1、多肽酶KLK4和主要钙转运蛋白的功能 NCKX4在成釉细胞分化中的作用。这些努力的结果是发现所有SATB1，KLK4， 和NCKX4参与成釉蛋白(AmBN)和成釉蛋白(Enam)的转录调控，编码 主要的EMP在SAB中协同上调，然后在MAB中协同下调。在SABS中，我们发现消融 SATB1在前成釉细胞(Pabs)中高表达，显著抑制Ambn和Enam的转录和 H3K27ac水平。我们的器官培养显示，组蛋白乙酰化升高上调了AMBN和ENAM的表达。一个 增强子和碱基非配对区(BUR，由SATB1优先选择)已在 在安本和伊纳姆附近。这些数据表明，SATB1在建立增强子的过程中起着重要作用 AMBN和ENAM在SABS中的上调。在缺乏Klk4和Nockx4致病基因的小鼠中 对于釉质形成不完善-我们发现有缺陷的水解导致富含脯氨酸的EMPs的保留。 作为对牙釉质中脯氨酸重摄取量减少的反应，Nck x4-/-和Klk4-/-MAB上调了Ambn& ENAM和下调的HIF1a。体外研究表明，Pro下调AMBN和ENAM的表达 上调HIF1a的表达。HIF1a，一种调节细胞对氧化应激反应的转录因子，有6倍于 在我们的RNA-SEQ分析中，MAB和SAB的表达上调，反映了MAB强大的抗氧化能力 持续为离子运输和蛋白质降解提供能量。这些数据表明， Proline上调HIF1a，HIF1a进而下调AMBN和ENAM。我们的体内和体外研究使我们能够 推测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的协调调节。这 假说将通过具体目标1来解决：确定SATB1作为Pabs to的先驱因子的作用 稳定激活SABS中ambn和enam基因转录的增强子的建立； 目的2：确定HIF1a对Ambn和Enam表达及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. Classified into two major stages, the secretory stage of ameloblasts (SABs) synthesize and deposit enamel matrix proteins (EMPs) to scaffold organic matrix, and the maturation stage of ameloblasts (MABs) hydrolyze, endocytose EMPs, and transport ions to mineralize enamel. To bioengineer enamel, a nonregenerative tissue, we must understand the transcriptional regulation of ameloblasts. To date, understanding of the unique transcriptional regulation of ameloblasts 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 funding allows 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, peptidase KLK4, and the major calcium transporter NCKX4 in the context of ameloblast differentiation. These efforts resulted in a discovery that all SATB1, KLK4, and NCKX4 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. In SABs, we found that ablation of SATB1, highly expressed in preameloblasts (PABs), greatly 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, preferentially selected by SATB1) have been predicted in the vicinity of Ambn & Enam. These data suggest that SATB1 plays an important role in establishing enhancer for the upregulation of Ambn & Enam in SABs. In the case of mice lacking Klk4 and Nckx4—the causative genes for amelogenesis imperfecta—we found a retention of proline-rich EMPs resulting from defective hydrolysis. Responding to the reduced proline retake from enamel, the Nckx4-/- and Klk4-/- MABs upregulated Ambn & Enam and downregulated Hif1a. In vitro studies showed that proline downregulated Ambn & Enam and upregulated Hif1a. HIF1A, a transcription factor regulating cell responses to oxidative stress, had a 6-fold upregulation in MABs vs SABs in our RNA-seq analyses, reflecting MAB’s robust anti-oxidative capacity to continuously provide energy for ion transport and protein degradation. These data suggest that retake of proline upregulates Hif1a, which in turn downregulats 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.