Epigenetic maintenance of progenitor osteoblasts by Ezh2 during bone regeneration

Ezh2在骨再生过程中对祖成骨细胞的表观遗传维持

基本信息

批准号：
9401996
负责人：
Gabriel Yette
金额：
$ 3.79万
依托单位：
UNIVERSITY OF OREGON
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-16 至 2020-08-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9401996
关键词：
Acute Adult Aging Alleles Amputation Bone Diseases Bone Regeneration CRISPR/Cas technology Cell Culture Techniques Cells ChIP-seq Chromatin Chronic Collection Communication Complex Data Degenerative Disorder Deposition Development Disease Educational process of instructing Educational workshop Effectiveness Environment Epigenetic Process Event Failure Fellowship Fishes Fracture Gene Expression Gene Targeting Genes Genetic Genetic Transcription Growth Histone H3 Histones Human Human body Humpback Dolphins In Vitro Injury Innovative Therapy Lysine Maintenance Mentors Methodology Methods Methylation Methyltransferase Modeling Molecular Monitor Natural regeneration Osteoblasts Outcome Outcome Study Pain Pharmacology Plasticizers Polycomb Process Property Repression Role Signal Transduction Site Source Stem cells Testing Therapeutic Tissues Training Transgenic Organisms Work Zebrafish bone bone healing career development chemical genetics cost cost efficient design embryonic stem cell epigenomics experience gain of function genome-wide histone modification human tissue in vivo innovation loss of function mutant novel organ regeneration osteoblast differentiation overexpression progenitor programs promoter regenerative repaired response self-renewal small molecule inhibitor tool trait transcription factor transcriptome sequencing

项目摘要

PROJECT SUMMARY Bone ailments are costly, painful, and debilitating. Therapies to augment bone repair vary in effectiveness, highlighting the need to develop new treatment approaches. Zebrafish, unlike humans, possess the remarkable capability to completely and robustly regenerate multiple tissues through a process called epimorphic regeneration. A mechanistic understanding of adult zebrafish regeneration could provide clues to unlock this capability in human tissues. Regeneration of the distinct bony rays of zebrafish fins following amputation initiates when mature osteoblasts (Obs) near the site of damage acquire progenitor-like traits by up-regulating key genes, including the transcription factor Runx2, and down-regulating maturation genes, such as the transcription factor sp7/Osterix. The resulting progenitor osteoblasts (pObs) are the singular source of cells from which new bone is derived through their delicately balanced proliferation and re-differentiation until regeneration is complete. pObs are continuously maintained throughout the regeneration process and failure to do so disrupts regeneration. How pObs are maintained in a progenitor state while facing potent differentiation signals remains largely unresolved. Preliminary in vivo and in vitro data suggests that upregulated Ezh2, the histone H3 lysine 27 tri-methylation (H3K27me3) methyltransferase component of the Polycomb Repressive Complex 2 (PRC2), helps sustain pObs. These results support a working hypothesis that Ezh2/H3K27me3 promotes the pOb state through an “epigenetic barrier” repressing the transcription of genes that drive Ob differentiation. This model will be tested by two Specific Aims: 1) Determine the in vivo contributions of Ezh2 to pOb maintenance in regenerating zebrafish fins and 2) Define how PRC2/Ezh2 maintains pObs. The applicant will use in vitro and in vivo approaches, including CRISPR/Cas9-generated ezh2 mutant zebrafish, inducible transgenic zebrafish and an Ezh2 small molecule inhibitor to perform loss of function and gain of function studies. Further outcomes include a comprehensive description of Ezh2-regulated gene expression programs and H3K27me3 chromatin landscapes in progenitor vs. differentiated osteoblasts. The fellowship period additionally will incorporate mentoring and classroom teaching activities, extensive scientific communication experience and training, and participation in career development workshops.

项目概要骨疾病的治疗费用昂贵、痛苦且使人衰弱，但增强骨修复的效果各不相同。强调需要开发新的治疗方法，与人类不同，斑马鱼拥有这种能力。通过称为“再生”的过程完全、稳健地再生多个组织的卓越能力对成年斑马鱼再生的机制理解可以提供线索。解锁人体组织中斑马鱼鳍独特骨射线的再生能力。当损伤部位附近的成熟成骨细胞 (Obs) 获得祖细胞样特征时，截肢就会开始上调关键基因，包括转录因子 Runx2，并下调成熟基因，例如作为转录因子 sp7/Osterix，产生的祖成骨细胞 (pObs) 是唯一的来源。通过微妙平衡的增殖和再分化产生新骨的细胞，直到再生完成后，pOb 在整个再生过程中持续保持。这样做会破坏 pOb 在面临有效分化时如何维持祖细胞状态。初步的体内和体外数据表明，Ezh2 信号在很大程度上尚未得到解决。 Polycomb 抑制的组蛋白 H3 赖氨酸 27 三甲基化 (H3K27me3) 甲基转移酶成分复合物 2 (PRC2) 有助于维持 pObs 这些结果支持 Ezh2/H3K27me3 的工作假设。通过抑制驱动 Ob 的基因转录的“表观遗传屏障”促进 pOb 状态该模型将通过两个具体目标进行测试：1) 确定 Ezh2 对分化的体内贡献。斑马鱼鳍再生中的 pOb 维持以及 2) 定义 PRC2/Ezh2 如何维持 pOb 申请人。将使用体外和体内方法，包括 CRISPR/Cas9 生成的 ezh2 突变斑马鱼、诱导型转基因斑马鱼和 Ezh2 小分子抑制剂执行功能丧失和功能获得进一步的研究结果包括 Ezh2 调控基因表达程序的全面描述。祖细胞与分化成骨细胞中的 H3K27me3 染色质景观。此外还将纳入指导和课堂教学活动、广泛的科学交流经验和培训，以及参加职业发展研讨会。