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）：我们研究釉质形成过程中淀粉原蛋白表达的生理调控机制。在体内发育的小鼠门牙原位杂交发现了成釉原蛋白转录物积累的周期性，其中一个矢状行成釉细胞含有大量的成釉原蛋白转录物，而相邻平行行成釉细胞显示出逐渐减少的成釉原蛋白转录物数量。成釉细胞样细胞系LS8显示出一个昼夜节律周期，即成釉原蛋白转录丰度周期为昼夜节律的负分支。通过双基因消融，我们敲除了C/ ebp - α在成釉细胞中的表达，发现C/ ebp - δ补偿维持了成釉蛋白mRNA的丰度。在LS8细胞中，我们发现C/EBP家族成员与NF-Y配对，协同激活淀粉原启动子。我们发现转录因子YY1和Msx2与淀粉原蛋白反转激活有关。C/ ebp - α在LS8细胞中的过表达增加了选择性剪接的小鼠59个氨基酸（M59）长淀粉原蛋白mRNA亚型的表达。M59蛋白显示出有限的自组装成蛋白质纳米球的能力，但越来越多的证据表明M59可以向成釉细胞提供生理相关信号。我们假设淀粉原蛋白mRNA的周期性和异构体丰度是转录因子在启动子上相互作用的结果。淀粉原蛋白丰度周期性的生理作用是协调基质的产生和矿物质的替代。我们假设成釉细胞与蛋白基质之间的联系参与向成釉细胞提供反馈，以协调转录，包括在预定的牙釉质厚度上停止成釉原蛋白的表达。为了验证这些假设，我们提出了四个相互关联的具体目标。具体目的1：利用染色质免疫沉淀（ChIP）策略确定C/EBP-alpha、C/EBP-delta、NF-Y、YY1和Msx2在调节淀粉原启动子中的作用。具体目的2：确定C/ ebp - α和C/ ebp - δ对产生淀粉原蛋白mRNA亚型的贡献。具体目标3：确定选定的淀粉原异构体和淀粉原降解片段的作用，为成釉细胞提供改变转录物丰度和稳定性的反馈信号。具体目标4：研究昼夜节律转录因子在调节淀粉原蛋白转录丰度中的作用。通过探索调节独特的淀粉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，同时有助于我们对牙齿发育的一般理解