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

• 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
• 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

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.

项目摘要 骨骼疾病是昂贵，痛苦且使人衰弱的。增强骨修复的疗法的有效性各不相同， 强调开发新治疗方法的必要性。斑马鱼不同于人类 通过称为 增生再生。对成人斑马鱼再生的机械理解可以为 在人体组织中解锁这种能力。斑马鱼鳍的独特骨射线的再生 当损伤部位附近成熟成骨细胞（obs）时，截肢会启动。 上调关键基因，包括转录因子Runx2和下调成熟基因，此类 作为转录因子SP7/Osterix。由此产生的祖细胞成骨细胞（POB）是 通过其精致平衡的增殖和重新分化的细胞，直到 再生完成。在整个再生过程中，POB不断保持 这样做会破坏再生。 POB如何保持祖细胞状态，同时面对潜在的分化 信号在很大程度上尚未解决。初步体内和体外数据表明，更新了EZH2， Hisstone H3赖氨酸27三甲基化（H3K27me3）Polycomb抑制剂的甲基转移酶成分 复杂2（PRC2），有助于维持POB。这些结果支持一个工作假设，即EZH2/H3K27ME3 通过代表驱动OB的基因转录的“表观遗传障碍”来促进POB状态 分化。该模型将通过两个具体目的测试：1）确定EZH2对的体内贡献 在再生斑马鱼鳍中维护POB，2）定义PRC2/EZH2如何维护POB。申请人 将使用体外和体内方法，包括CRISPR/CAS9生成的EZH2突变体斑马鱼，可诱导 转基因斑马鱼和EZH2小分子抑制剂可执行功能丧失和功能增益 研究。进一步的结果包括对EZH2调节的基因表达程序的全面描述 祖细胞中与分化成骨细胞中的H3K27me3染色质景观。奖学金期 此外，将纳入心理和课堂教学活动，广泛的科学沟通 经验和培训，并参加职业发展研讨会。