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

不同核因子对成釉素和釉质的阶段特异性调节

• 批准号: 10645781
• 负责人: Yan Zhang
• 金额: $ 32.3万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-08 至 2023-09-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10645781
• 关键词: ATAC-seq; Ablation; Acetylation; Address; Ameloblasts; Amelogenesis Imperfecta; Architecture; Biological Markers; Biology; Biomedical Engineering; CRISPR/Cas technology; Calcium; Cell Culture Techniques; Cell Line; Cells; Characteristics; Chromatin; Complex; Correlation Studies; DNA Binding; Data; Dental Enamel; Dental Pulp; Dentin; Deposition; Development; Enamel Formation; Endocytosis; Enhancers; Epigenetic Process; Epithelial Cells; Fracture; Funding; Gene Cluster; Genes; Genetic Transcription; Genome engineering; HIF1A gene; Hardness; Histone Acetylation; Hydrolysis; In Vitro; Ion Transport; Knockout Mice; Knowledge; Maturation-Stage Ameloblast; Minerals; Mus; Natural regeneration; Nuclear; Organ Culture Techniques; Oxidative Stress; Peptide Hydrolases; Peptides; Phenotype; Play; Proline; Protein Biochemistry; Rattus; Regulation; Resistance; Resolution; Role; Secretory-Stage Ameloblast; Site; Sodium-Calcium Exchanger; Testing; Tissues; Tooth eruption; Transcriptional Regulation; Up-Regulation; XCL1 gene; ameloblastin; amelogenin; base; calcification; conditional knockout; enamel matrix proteins; enamelin; genome-wide; in vivo; mouse model; novel; protein degradation; protein expression; repaired; resilience; response; scaffold; success; transcription factor; transcriptome sequencing

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.

项目概要/摘要 细胞身份很大程度上取决于特定的表观遗传景观和成釉细胞的转录网络。 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 (SAB) 的分泌阶段合成釉质基质蛋白 (EMP) 并将其沉积到支架上 有机基质、成釉细胞 (MAB) 水解、内吞 EMP 和运输的成熟阶段 为了对牙釉质（一种非再生组织）进行生物工程，我们必须了解 迄今为止，对成釉细胞独特转录调控的了解。 由于牙齿萌出后成釉细胞的损失以及完全缺乏细胞系，成釉细胞受到限制 回顾以前的资金使我们能够建立一种新颖的和成釉细胞的特征。 对跨物种成釉细胞每个发育阶段具有重要意义的基因的全面列表 探索染色质组织者 SATB1、肽酶 KLK4 和主要钙转运蛋白的功能 NCKX4 在成釉细胞分化的背景下，这些努力发现所有 SATB1、KLK4、 NCKX4 有助于成釉素 (Ambn) 和牙釉质 (Enam) 的转录调节，编码 主要的 EMP 在 SAB 中共同上调，然后在 MAB 中共同下调。在 SAB 中，我们发现消融。 SATB1 在前成釉细胞 (PAB) 中高度表达，极大地抑制 Ambn 和 Enam 转录， 我们的器官培养显示，组蛋白乙酰化水平升高，Ambn 和 Enam An 上调。 增强子和碱基不配对区域（BUR，优先由 SATB1 选择）已在 Ambn 和 Enam 附近的这些数据表明 SATB1 在建立增强子方面发挥着重要作用。 在缺乏 Klk4 和 Nckx4（致病基因）的小鼠中，SAB 中 Ambn 和 Enam 的上调。 对于釉质形成不全症——我们发现由于水解缺陷而导致富含脯氨酸的 EMP 的保留。 为了应对从牙釉质中重新摄取脯氨酸的减少，Nckx4-/- 和 Klk4-/- MAB 上调 Ambn & Enam 和下调 Hif1a 体外研究表明脯氨酸下调 Ambn 和 Enam 以及 HIF1A（一种调节细胞对氧化应激反应的转录因子）上调了 6 倍。 在我们的 RNA-seq 分析中，MAB 与 SAB 的上调，反映了 MAB 强大的抗氧化能力 这些数据表明，不断地为离子运输和蛋白质降解提供能量。 脯氨酸上调 Hif1a，进而下调 Ambn 和 Enam。我们的体内和体外研究使我们能够做到这一点。 扰乱Ambn和Enam在成釉细胞两个主要功能阶段的动态表达 由不同的染色质组织因子 SATB1 和转录因子 HIF1A 协调调节。 假设将通过具体目标 1 来解决：确定 SATB1 作为 PAB 中先锋因子的作用，以 准备好用于激活 SAB 中 Ambn 和 Enam 基因转录的增强子的建立； 目标 2：确定 HIF1A 对 Ambn 和 Enam 表达以及牙釉质形成的调节作用。