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

这项研究的长期目标是开发更安全有效的治疗方法来治疗由骨质疏松引起的骨质流失 破骨细胞（OC）过度分化与许多代谢性骨疾病有关。近期目标是 该应用旨在了解 OC 分化是如何通过负信号介导的进行调节的 转录因子和表观遗传调节因子，可能作为骨疾病的新治疗靶点 例如骨质疏松症。目前对溶骨性疾病的治疗因缺乏对溶骨性疾病的了解而受到阻碍。 OC 功能负调控防止骨丢失的潜在机制。此外， 转录因子和表观遗传因子如何共同调节（尤其是负面调节）的机制 共同调节，OC 分化和功能仍不清楚。我们已经鉴定出 Cbx3/HP1γ。我们后续的 免疫沉淀分析证实，OC 中 C/EBPα 与 Cbx3/HP1γ 相互作用。值得注意的是，我们初步 数据显示，Cbx3/HP1γ过表达抑制OC分化和活性，而Cbx3/HP1γ沉默 增强 OC 血统承诺和形成。一致地，Cbx3/HP1γ 缺陷小鼠被发现表现出 由于 OC 形成和活性增强而导致骨质疏松症样表型。有趣的是，通过 ChIP 分析，我们 在 C/EBPα 调节的 OC 基因 NFATC1 和 NFATC1 的启动子上发现了 Cbx3/HP1γ 的几个结合位点 C-FOS。我们的 RNA-seq 分析还表明，单核细胞中的 Cbx3 缺陷导致 OC 基因增加 表达。总的来说，初步数据表明 Cbx3 可以限制 C/EBPα 介导的 OC 分化和活性。根据我们的初步研究，我们假设 Cbx3 负向调节 破骨细胞通过与 C/EBPα 和表观遗传因子相互作用而分化 修饰和防止骨稳态中破骨细胞基因表达。我们将检验假设 通过三个具体目标。在目标 1 中，我们检查了 Cbx3 在 OC 分化、骨骼 通过表征生理和病理条件下的发育和骨稳态 通过功能丧失研究研究两种 Cbx3 CKO 小鼠模型的表型和病理机制。瞄准 2、我们确定了Cbx3在OC分化、骨骼发育和骨稳态中的作用 通过表征 Cbx3 的表型和病理机制来了解生理和病理条件 通过功能获得方法有条件转基因过度表达小鼠。我们定义分子 Cbx3 通过与 C/EBPα 相互作用负向调节 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 Cbx3 的机制 与转录因子和其他表观遗传因子合作负向调节 OC 分化和 活动。从这项研究中获得的见解不仅将解决有关表观遗传的基本科学问题 OC生物学中基因表达的调控，也将为最终目标提供基础 促进针对溶骨性疾病（例如骨质疏松症）设计更安全、新颖的治疗方法。

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).