DETERMINATION AND EXPRESSION OF AMELOGENIN GENE PRODUCTS

釉原蛋白基因产物的测定和表达

• 批准号: 7841082
• 负责人: Malcolm L. Snead
• 金额: $ 0.82万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-02 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7841082
• 关键词: Ablation; Accounting; Ameloblasts; Amino Acids; Animals; CCAAT-Enhancer-Binding Protein-alpha; CCAAT-Enhancer-Binding Proteins; CCAAT-enhancer-binding protein-delta; CEBPA gene; Cell Line; Cells; Circadian Rhythms; Collagen; Communicable Diseases; Data; Dental Enamel; Dental caries; Development; Disease; Ectoderm; Enamel Formation; Extracellular Space; Family member; Feedback; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Growth; Habits; Helix-Turn-Helix Motifs; Housing; In Situ Hybridization; In Vitro; Incisor; Intervention; Knock-out; Knowledge; Left; Light; Limb structure; Link; Messenger RNA; Minerals; Molecular; Mus; Nanosphere; Natural regeneration; Outcome; Pathway interactions; Pattern; Periodicity; Phase; Physiological; Production; Protein Isoforms; Proteins; Quality Control; RNA Splicing; Reagent; Recording of previous events; Regulatory Pathway; Role; Signal Transduction; Testing; Thick; Tissues; Tooth structure; Transactivation; Transcript; Transcriptional Regulation; Trauma; YY1 Transcription Factor; amelogenin; base; biomineralization; chromatin immunoprecipitation; experience; feeding; in vivo; man; postnatal; promoter; public health relevance; repaired; response; self assembly; transcription factor

DESCRIPTION (provided by applicant): We investigate the physiologic regulatory mechanisms for amelogenin expression during enamel formation. In situ hybridization of in vivo developing mouse incisors identified a periodicity for amelogenin transcript accumulation wherein one sagittal row of ameloblasts contained numerous amelogenin transcripts while the adjacent parallel rows of ameloblasts showed progressively diminishing numbers of amelogenin transcripts. Ameloblast-like cell line LS8 shows a circadian cycle for amelogenin transcript abundance that cycles out of phase to the negative limb of the circadian rhythm. By binary gene ablation, we knocked-out C/EBP-alpha expression in ameloblasts and found that C/EBP-delta compensated to maintain amelogenin mRNA abundance. In LS8 cells we showed that C/EBP family members pair with NF-Y to synergistically activate the amelogenin promoter. We identified transcription factors YY1 and Msx2 as opposing amelogenin transactivation. C/EBP-alpha over expression in LS8 cells increases the expression of the alternatively spliced mouse 59 amino acid (M59) long amelogenin mRNA isoforms. M59 protein shows limited ability to self-assemble into protein nanospheres while growing evidence suggests that M59 can provide physiologically relevant signals to ameloblasts. We hypothesize that amelogenin mRNA periodicity and isoform abundance is a consequence of transcription factor interactions on the promoter. The physiologic role for periodicity in amelogenin abundance is to coordinate matrix production with mineral replacement. We hypothesize that linkage between the ameloblasts with the protein matrix participates in providing feedback to ameloblasts serving to coordinate transcription, including to stop amelogenin expression at a predetermined enamel thickness. To test these hypotheses we propose four interrelated specific aims. Specific aim 1: Use chromatin immunoprecipitation (ChIP) strategies to determine the contribution of C/EBP-alpha, C/EBP-delta, NF-Y, YY1 and Msx2 to regulating the amelogenin promoter. Specific aim 2: Determine the contribution of C/EBP-alpha and C/EBP-delta to generate amelogenin mRNA isoforms. Specific aim 3: Determine the role of selected amelogenin isoforms and amelogenin degradation fragments to provide feedback signals to ameloblasts altering transcript abundance and stability. Specific Aim 4: Examine the role of circadian transcription factors to regulate amelogenin transcript abundance. By exploring the physiologic pathways responsible for regulating the unique amelogenin gene, these studies will provide a better understanding of tooth development while contributing to our general understanding of gene regulation. Knowledge of these pathways will contribute to enamel regeneration strategies. Public Health Relevance: Enamel caries is the most prevalent infectious disease of man. Correct orchestration of enamel gene expression is essential to the formation of the enamel organic matrix that must be created first in order to guide enamel biomineralization. We are studying the transcription factors responsible for expression of the dominant protein of forming mammalian enamel, amelogenin, and the circadian rhythms that influence cyclic oscillation in the abundance of amelogenin transcripts.

描述（由申请人提供）：我们研究了釉质形成过程中釉原蛋白表达的生理调节机制。在体内发育的小鼠切牙的原位杂交确定了釉原蛋白转录积累的周期性，其中一个矢状行的成釉细胞含有大量的釉原蛋白转录，而相邻的平行行的成釉细胞表现出数量逐渐减少的釉原蛋白转录。成釉细胞样细胞系LS 8显示了釉原蛋白转录物丰度的昼夜节律周期，其周期与昼夜节律的负肢异相。通过二元基因切除，我们敲除成釉细胞中的C/EBP-α表达，发现C/EBP-δ补偿维持釉原蛋白mRNA丰度。在LS 8细胞中，我们发现C/EBP家族成员与NF-Y配对协同激活釉原蛋白启动子。我们确定转录因子YY 1和Msx 2作为对釉原蛋白反式激活。C/EBP-α在LS 8细胞中的过表达增加了可变剪接的小鼠59个氨基酸（M59）长釉原蛋白mRNA同种型的表达。M59蛋白显示出有限的自组装成蛋白纳米球的能力，而越来越多的证据表明，M59可以提供成釉细胞生理相关的信号。我们推测釉原蛋白mRNA的周期性和异构体丰度是转录因子相互作用的结果。釉原蛋白丰度的周期性的生理作用是协调基质生产与矿物质替代。我们假设成釉细胞与蛋白质基质之间的联系参与向成釉细胞提供反馈，以协调转录，包括在预定的釉质厚度下停止釉原蛋白表达。为了验证这些假设，我们提出了四个相互关联的具体目标。具体目标1：使用染色质免疫沉淀（ChIP）策略来确定C/EBP-α、C/EBP-δ、NF-Y、YY 1和Msx 2对调节釉原蛋白启动子的贡献。具体目标2：确定C/EBP-α和C/EBP-δ对生成釉原蛋白mRNA亚型的贡献。具体目标3：确定选定的釉原蛋白异构体和釉原蛋白降解片段的作用，以提供反馈信号，改变成釉细胞转录丰度和稳定性。具体目标4：检测昼夜节律转录因子在调节釉原蛋白转录丰度中的作用。通过探索负责调节独特的釉原蛋白基因的生理途径，这些研究将提供对牙齿发育的更好理解，同时有助于我们对 基因调控这些途径的知识将有助于牙釉质再生策略。 公共卫生相关性：釉质龋是人类最常见的传染性疾病，正确调控釉质基因的表达对釉质有机基质的形成至关重要，而釉质有机基质的形成是引导釉质生物矿化的前提。我们正在研究的转录因子负责形成哺乳动物釉质，釉原蛋白的优势蛋白质的表达，和昼夜节律的影响周期振荡的釉原蛋白转录丰度。