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: 10804126
• 负责人: Yan Zhang
• 金额: $ 48万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-06 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10804126
• 关键词: AT Rich Sequence; ATAC-seq; Ablation; Acetylation; Address; Affect; Ameloblasts; Amelogenesis; Amelogenesis Imperfecta; Architecture; Binding; Binding Proteins; Binding Sites; Biological Markers; Biology; Biomedical Engineering; CRISPR/Cas technology; Calcium; Cell Line; Cells; Characteristics; Chromatin; Complex; DNA Binding; Data; Dental Enamel; Deposition; Development; Down-Regulation; Enamel Formation; Enamel Organ; Endocytosis; Enhancers; Environmental Risk Factor; Epigenetic Process; Epithelial Cells; Fracture; Funding; Gene Cluster; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Genome engineering; Genomics; Glutamine; Grant; HIF1A gene; Hardness; High Pressure Liquid Chromatography; Histone Acetylation; Hydrolysis; Hypoxia; In Vitro; Ion Transport; Knowledge; Mediating; Modeling; Molecular Conformation; Mus; Natural regeneration; Nuclear; Organ Culture Techniques; Oxidative Stress; Peptide Hydrolases; Peptides; Proline; Protein Biochemistry; Rattus; Regulation; Reporter; Repression; Resistance; Resolution; Role; Site; Sodium; Testing; Tissues; Tooth eruption; Tooth structure; Transcriptional Regulation; Up-Regulation; Vertebrates; ameloblastin; amelogenin; base; calcification; cell regeneration; enamel matrix proteins; enamelin; extracellular; genetic signature; genome-wide; genomic locus; in vivo; mineralization; novel; protein degradation; repaired; resilience; response; scaffold; transcription factor; transcriptome sequencing; uptake

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.

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，首先是前素细胞（PAB） 将合成并沉积牙釉质基质蛋白（EMP）的秘密成成布（SAB）区分开 脚手架有机基质，然后到成熟的成熟成成木细胞（mAb），水解，内吞EMP和 运输离子以矿化搪瓷。对于非再生组织的生物工程搪瓷，我们必须了解 由于牙齿后的成成木损失而受到限制的成成木细胞的转录调节 喷发和缺乏细胞系充分概括了成成木的特征。以前的资金允许我们 建立针对成成木的每个发育阶段重要的基因的新颖而全面的列表 跨物种并探索染色质组织者SATB1和搪瓷基质建模的功能 调节剂 - 肽酶KLK4和主要钙转运蛋白NCKX4-在Ameloblast的背景下 分化。这些努力导致发现SATB1，KLK4和NCKX4都有助于 氨基蛋白细胞素（AMBN）和搪瓷（eNAM）的转录调节，编码主要的EMPS共同 在SABS中进行更新，然后在mAb中共同进行。我们发现Satb1的消融高度表达 在PAB中，复制AMBN和ENAM转录和H3K27AC水平。我们的器官文化表明升高 组蛋白乙酰化上调AMBN＆ENAM。增强剂和基础不纪念区域（bur，选择性 SATB1 DNA结合位点已在AMBN＆Enam附近预测。这些数据表明 SATB1组织染色质构象，并命令转录复合体，以上调AMBN＆ENAM至 前进到sabs。对于缺乏KLK4和NCKX4的小鼠 - 阿美酮的休闲基因 Imperfecta-我们发现缺乏水解导致的脯氨酸/谷氨酰胺（P/G） - 富含EMP。 这些NCKX4 - / - 和KLK4 - / - mabs上调了AMBN＆ENAM，并下调HIF1A。体外研究 表明P/G富含肽下调AMBN＆ENAM和更新的HIF1A。我们的RNA-seq分析 表明HIF1A是控制细胞对氧化应激反应的转录因子，其具有6倍 mabs vs sabs的上调，反映了mab的强大抗氧化能力，可连续提供能量 用于离子运输和蛋白质降解。这些数据表明，富含P/G的肽上调的重新上调 HIF1A，进而下调AMBN＆ENAM。我们的体内和体外研究使我们可以假设 AMBN＆ENAM在木材细胞的两个主要功能阶段的动态表达是协调的 由不同因素染色质组织者SATB1和转录因子HIF1A调节。这个假设将是 通过特定目的1：确定satb1作为先驱因素的作用 增强剂建立，用于激活SABS中AMBN和ENAM基因的转录；和特定目标2： 确定HIF1A在AMBN和ENAM表达和搪瓷形成中的调节作用。

项目成果

TRPM7-Mediated Calcium Transport in HAT-7 Ameloblasts.

• DOI: 10.3390/ijms22083992
• 发表时间: 2021-04-13
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Kádár K;Juhász V;Földes A;Rácz R;Zhang Y;Löchli H;Kató E;Köles L;Steward MC;DenBesten P;Varga G;Zsembery Á
• 通讯作者: Zsembery Á

Sodium/(calcium + potassium) exchanger NCKX4 optimizes KLK4 activity in the enamel matrix microenvironment to regulate ECM modeling.

• DOI: 10.3389/fphys.2023.1116091
• 发表时间: 2023
• 期刊: Frontiers in physiology
• 影响因子: 4

WDR72 regulates vesicle trafficking in ameloblasts.

• DOI: 10.1038/s41598-022-06751-1
• 发表时间: 2022-02-18
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Katsura K;Nakano Y;Zhang Y;Shemirani R;Li W;Den Besten P
• 通讯作者: Den Besten P

A N-Terminus Domain Determines Amelogenin's Stability to Guide the Development of Mouse Enamel Matrix.

• DOI: 10.1002/jbmr.4329
• 发表时间: 2021-09
• 期刊: JOURNAL OF BONE AND MINERAL RESEARCH
• 影响因子: 6.2
• 作者: Huang, Yulei;Bai, Yushi;Chang, Chih;Bacino, Margot;Cheng, Ieong Cheng;Li, Li;Habelitz, Stefan;Li, Wu;Zhang, Yan
• 通讯作者: Zhang, Yan