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Mechanistic basis of the role of Cbx3 in negatively regulating osteoclast differentiation through epigenetic modification

Cbx3通过表观遗传修饰负调节破骨细胞分化作用的机制基础

基本信息

批准号：
10463857
负责人：
YI-PING LI
金额：
$ 49.59万
依托单位：
TULANE UNIVERSITY OF LOUISIANA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-06 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10463857
关键词：
Address Binding Binding Proteins Binding Sites Biology Bone Diseases CCAAT-Enhancer-Binding Protein-alpha Cathepsin C ChIP-seq Chromatin Complex Cre lox recombination system Data Development Disease Epigenetic Process Exhibits FOS gene Female Foundations Gene Expression Gene Expression Regulation Gene Silencing Genes Genetic Transcription Goals Heterochromatin Homeostasis Homologous Gene Immunoprecipitation Knockout Mice Mass Spectrum Analysis Mediating Metabolic Bone Diseases Modeling Modification Molecular Mus Osteoclasts Osteolytic Osteoporosis Pathologic Pattern Phenotype Physiological Proteins Regulation Role Signal Transduction Skeletal Development Skeletal bone Testing Transgenic Organisms bone bone loss cathepsin K conditional knockout design epigenetic regulation gain of function genome-wide histone methylation insight loss of function male monocyte mouse model new therapeutic target novel therapeutic intervention osteoclastogenesis overexpression prevent promoter therapeutically effective transcription factor transcriptome sequencing

项目摘要

The long term goal of this study is to develop safer and effective therapeutic approach for bone loss caused by excessive osteoclast (OC) differentiation implicated in many metabolic bone diseases. The immediate goal of this application is to understand how OC differentiation is regulated through negative signaling mediated by transcription factors and epigenetic regulators, which may serve as novel therapeutic targets for bone diseases such as osteoporosis. Current therapies for osteolytic diseases are hindered by lack of understanding of the mechanisms underlying the negative regulation of OC function to prevent bone loss. Moreover, the mechanism(s) underlying how transcriptional factors and epigenetic factors co-regulate, especially negatively co-regulate, OC differentiation and function remain unclear. We have identified Cbx3/HP1γ. Our subsequent immunoprecipitation analysis confirmed that C/EBPα interacts with Cbx3/HP1γ in OCs. Notably, our preliminary data showed that Cbx3/HP1γ overexpression inhibited OC differentiation and activity, while Cbx3/HP1γ silencing enhanced OC lineage commitment and formation. Consistently, Cbx3/HP1γ-deficient mice were found to exhibit osteoporosis-like phenotype due to enhanced OC formation and activity. Interestingly, through ChIP analysis, we found several binding sites of Cbx3/HP1γ on the promoters of C/EBPα regulated OC genes, NFATC1 and C-FOS. Our RNA-seq analysis also revealed that Cbx3 deficiency in monocytes led to increased OC gene expression. Collectively, the preliminary data indicated that Cbx3 can restrict C/EBPα-mediated OC differentiation and activity. Based on our preliminary studies, we hypothesize that Cbx3 negatively regulates osteoclast differentiation through interacting with C/EBPα and epigenetic factors as a result of epigenetic modification and preventing osteoclast gene expression in bone homeostasis. We will test the hypothesis through three specific aims. In Aim 1, we examine the function of Cbx3 in OC differentiation, skeletal development and bone homeostasis under physiological and pathological conditions through characterization of the phenotypes and pathomechanism in two Cbx3 CKO mouse models through loss-of-function studies. In Aim 2, we determine the role of Cbx3 in OC differentiation, skeletal development and bone homeostasis under physiological and pathological conditions by characterizing the phenotypes and pathomechanism in Cbx3 conditional transgenic overexpression mice through gain-of-function approach. We define the molecular mechanism underlying how Cbx3 negatively regulates OC differentiation through interacting with C/EBPα and controlling epigenetic modification in Aim 3. The study will elucidate the mechanism(s) through which Cbx3 cooperates with transcriptional factors and other epigenetic factors to negatively regulate OC differentiation and activity. Insights gained from this study will not only address the basic scientific question about epigenetic regulation of gene expression in OC biology, but also will provide the foundation for the ultimate goal of facilitating the design of safer and novel therapeutic approach for osteolytic diseases (e.g. osteoporosis).

本研究的长期目标是开发更安全，有效的治疗方法，破骨细胞（OC）过度分化与许多代谢性骨疾病有关。这一近期目标本申请旨在了解OC分化是如何通过负信号调节的，转录因子和表观遗传调节因子，可能成为骨疾病的新治疗靶点如骨质疏松症。目前用于溶骨性疾病的疗法由于缺乏对溶骨性疾病的分子机制的了解而受到阻碍。负调节OC功能以防止骨丢失的潜在机制。而且转录因子和表观遗传因子如何共同调节的潜在机制，特别是负调节但是，OC的分化和功能尚不清楚。我们鉴定了Cbx 3/HP 1 γ。我们随后免疫沉淀分析证实C/EBPα与Cbx 3/HP 1 γ在OC中相互作用。值得注意的是，我们的初步 Cbx 3/HP 1 γ过表达抑制OC的分化和活性，而Cbx 3/HP 1 γ沉默抑制OC的分化和活性。增强OC谱系承诺和形成。同样，发现Cbx 3/HP 1 γ缺陷小鼠表现出由于OC的形成和活性增强而导致的类神经纤维瘤表型。有趣的是，通过ChIP分析，我们在C/EBPα调控的OC基因、NFATC 1和NFATC 2的启动子上发现了Cbx 3/HP 1 γ的几个结合位点， C-FOS我们的RNA-seq分析还显示，单核细胞中Cbx 3缺陷导致OC基因表达增加，表情总的来说，初步数据表明，Cbx 3可以限制C/EBPα介导的OC 分化和活动。基于我们的初步研究，我们假设Cbx 3负调节破骨细胞的分化是通过与C/EBPα和表观遗传因子相互作用，修饰和阻止破骨细胞基因表达的骨稳态。我们将检验这个假设通过三个具体目标。在目的1中，我们研究了Cbx 3在OC分化、骨骼肌分化和成纤维细胞分化中的作用。发育和骨稳态的生理和病理条件下，通过表征通过功能丧失研究，在两种Cbx 3 CKO小鼠模型中观察表型和病理机制。在Aim中 2.我们确定了Cbx 3在OC分化，骨骼发育和骨稳态中的作用，通过表征Cbx 3中的表型和病理机制，条件性转基因过表达小鼠通过获得功能的方法。我们定义分子 Cbx 3通过与C/EBPα相互作用负调控OC分化的机制，控制目标3中的表观遗传修饰。该研究将阐明Cbx 3 与转录因子和其他表观遗传因子合作，负调控OC分化，活动从这项研究中获得的见解不仅将解决关于表观遗传的基本科学问题， OC生物学中基因表达的调控，也将为最终目标的实现提供基础。促进设计更安全和新颖的溶骨性疾病（例如骨质疏松症）的治疗方法。