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

项目概要/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 唯一能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， 成釉细胞分泌期合成釉基质蛋白并将其存款到支架上 有机基质，成釉细胞（MABs）的成熟阶段水解，内吞EMPs， 离子来矿化釉质。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。到目前为止，了解独特的转录调控， 由于成釉细胞在牙齿萌出后的丢失和缺乏完全分化的细胞系， 概括了成釉细胞的特征。以前的资金使我们能够建立一个新的， 对不同物种的成釉细胞的每个发育阶段具有重要意义的基因的综合列表， 探索染色质组织者SATB 1、肽酶KLK 4和主要钙转运蛋白的功能 NCKX 4在成釉细胞分化中的作用这些努力导致发现所有SATB 1，KLK 4， 和NCKX 4有助于成釉蛋白（Ambn）和釉蛋白（Enam）的转录调节，编码 主要的EMP在SAB中共上调，然后在MAB中共下调。在SABs中，我们发现消融 SATB 1在成纤维细胞（PABs）中高度表达，极大地抑制了Ambn和Enam的转录， H3 K27 ac级别。我们的器官培养表明，组蛋白乙酰化水平升高上调Ambn和Enam。一个 增强子和碱基不配对区（BUR，优先由SATB 1选择）已在 Ambn & Enam附近的景点这些数据表明，SATB 1在建立增强子中起重要作用， SAB中Ambn和Enam的上调。在缺乏Klk 4和Nckx 4的小鼠的情况下， 对于成釉不全症，我们发现由于水解缺陷而导致富含脯氨酸的EMP保留。 响应于减少的脯氨酸从釉质的再摄取，Nckx 4-/-和Klk 4-/-MAB上调Ambn & Enam和下调的Hif 1a。体外研究表明，脯氨酸下调Ambn和Enam， Hif 1a的表达上调。HIF 1A是一种调节细胞对氧化应激反应的转录因子， 在我们的RNA-seq分析中，MAB相对于SAB的上调，反映了MAB强大的抗氧化能力， 为离子运输和蛋白质降解提供能量。这些数据表明， 脯氨酸上调Hif 1a，Hif 1a反过来下调Ambn和Enam。我们的体内和体外研究让我们 推测Ambn和Enam在成釉细胞两个主要功能阶段的动态表达， 由不同的因子染色质组织者SATB 1和转录因子HIF 1A协同调节。这 假设将通过具体目标1来解决：确定SATB 1作为PAB中的先驱因子的作用， 平衡增强子建立以激活SABs中Ambn和Enam基因的转录;并且特异性地 目的2：探讨HIF 1A对Ambn和Enam基因表达及成釉的调控作用。

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.