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）：我们研究了牙釉质形成过程中阿米他素表达的生理调节机制。体内发育中的小鼠牙的原位杂交确定了氨基蛋白酶转录本的周期性，其中一组矢状成amelogen蛋白转录本包含大量的amelogen蛋白转录本，而相邻的阿素细胞平行行则逐渐减少amelegoentin转录物的数量。成酰胺细胞样细胞系LS8显示了肌蛋白蛋白转录物丰度的昼夜节律循环，该循环循环循环到昼夜节律的负肢。通过二进制基因消融，我们在成成布中敲出了C/EBP-Alpha表达，发现C/EBP-delta补偿以维持氨基蛋白蛋白mRNA的丰度。在LS8细胞中，我们表明C/EBP家族成员与NF-Y配对，以协同激活Amelogen蛋白启动子。我们确定了转录因子YY1和MSX2是相对的氨基蛋白蛋白反式激活。 C/EBP-Alpha在LS8细胞中的表达上增加了剪接的小鼠59氨基酸（M59）长氨基蛋白mRNA同工型的表达。 M59蛋白表现出有限的自我组装成蛋白质纳米球的能力，而越来越多的证据表明M59可以为成成成成组织提供与生理相关的信号。我们假设氨基蛋白蛋白mRNA的周期性和同工型丰度是启动子上转录因子相互作用的结果。周期性的生理性作用在氨基蛋白蛋白丰度中的生理作用是将基质的产生与矿物替代配合。我们假设蛋白质基质之间的成核细胞之间的联系参与提供对用于协调转录的成核细胞的反馈，包括在预定的搪瓷厚度下停止amelegen蛋白表达。为了检验这些假设，我们提出了四个相互关联的特定目标。具体目标1：使用染色质免疫沉淀（CHIP）策略来确定C/EBP-Alpha，C/EBP-DELTA，NF-Y，YY1和MSX2的贡献来调节Amelegenin启动子。具体目标2：确定C/EBP-Alpha和C/EBP-DELTA对产生氨基蛋白蛋白mRNA同工型的贡献。具体目标3：确定选定的氨基蛋白蛋白同工型和氨基蛋白蛋白降解片段的作用，以提供反馈信号，以改变转录本丰度和稳定性的成成木。特定目的4：检查昼夜节律转录因子在调节氨基蛋白蛋白转录丰度方面的作用。通过探索负责调节独特蛋白质蛋白基因的生理途径，这些研究将为我们对牙齿发育提供更好的了解，同时有助于我们对我们对 基因调节。这些途径的知识将有助于搪瓷再生策略。 公共卫生相关性：搪瓷龋齿是人类最普遍的传染病。牙釉质基因表达的正确编排对于必须首先创建的搪瓷有机基质的形成至关重要，以指导搪瓷生物矿化。我们正在研究负责形成哺乳动物搪瓷，氨基蛋白蛋白和昼夜节律的转录因子，这些蛋白质在丰富的蛋白酶转录本中影响环状振荡。