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.

项目摘要/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。木材细胞是 唯一可以在发育过程中生成计算的组织的上皮细胞。分为两个主要阶段， 成成木（SABS）合成并沉积牙釉质基质蛋白（EMP）的秘密阶段 有机基质和成熟阶段的成熟阶段（mAb）水解，内吞EMP和运输 离子以矿物质化牙釉质。对于非再生组织的生物工程搪瓷，我们必须了解 成成木的转录调节。迄今为止，了解对独特的转录调节的理解 由于牙齿爆发后失去木质细胞和完全缺乏细胞系的木材细胞损失，因此成熟的细胞受到限制。 概括成成木的特征。以前的资金使我们能够建立小说， 对各种物种的成成细胞的每个发育阶段重要的基因综合列表 探索染色质组织者SATB1，Pepperidase KLK4和主要钙转运蛋白的功能 NCKX4在成成木分化的背景下。这些努力导致发现所有SATB1 KLK4， NCKX4有助于氨基蛋白酶（AMBN）和搪瓷蛋白（eNAM）的转录调节，编码 主要的EMP在SABS中共同升级，然后在mAb中共同调节。在SABS中，我们发现消融 SATB1，高度表达在前层细胞（PABS）中，大量复制了AMBN和ENAM转录 H3K27AC级别。我们的器官培养表明，升高的组蛋白乙酰化更新了AMBN＆ENAM。一个 增强剂和基础不属于区域（BUR，最好由SATB1选择）。 Ambn＆Enam附近。这些数据表明，SATB1在建立增强子方面起着重要作用 SABS中AMBN＆ENAM的上调。对于缺乏KLK4和NCKX4的小鼠 - 休闲基因 对于不完美的症状 - 我们发现水解缺陷导致脯氨酸富含EMP的保留。 响应搪瓷，NCKX4 - / - 和KLK4 - / - mabs更新的AMBN＆ enam并下调HIF1A。体外研究表明，脯氨酸下调了AMBN＆ENAM和 上调HIF1A。 HIF1A是调节细胞对氧化应激反应的转录因子，具有6倍 在我们的RNA-seq分析中，mAbs vs sabs的上调，反映了mab的强大抗氧化能力 连续提供离子运输和蛋白质降解的能量。这些数据表明重新夺回 Proline上调HIF1A，而HIF1A反过来下调Ambn＆Enam。我们的体内和体外研究使我们 假设AMBN＆ENAM在木材细胞的两个主要功能阶段的动态表达 由不同因子染色质组织者SATB1和转录因子HIF1A协调调节。这 假设将通过特定目标1：确定SATB1作为Pioneer因素的作用 在SABS中激活AMBN＆ENAM基因的转录，使增强剂建立;和具体 目标2：确定HIF1A在AMBN和ENAM表达和搪瓷形成中的调节作用。