Control of Osteoblast Proliferation and Differentiation

 描述（由申请人提供）：本修订提案是我们25年计划的更新申请，该计划为理解调节成骨细胞承诺、生长和分化的分子机制提供了范式转变概念。我们已经建立了成骨细胞分化的阶段和时间控制成骨细胞表型的遗传和表观遗传机制在体外和使用体内动物模型。该项目的贡献包括建立与成骨细胞表达的mRNA和非编码RNA控制相关的转录因子、染色质重塑和组蛋白修饰的骨组织特异性特性。我们目前的项目发现，短的非编码microRNA是骨形成所必需的信号通路的关键调节因子。然而，很少有人知道的身份和功能的长链非编码（lncRNA）参与成骨。这一更新的新方向是鉴定和表征成骨细胞发生过程中的lncRNA，并发现它们在成骨中的分子机制和功能相关性。lncRNA最近已经成为发育和谱系定型中基因调控的新水平。它们通过复杂的机制发挥作用，包括充当：（i）募集染色质修饰酶以激活或抑制转录的向导;（ii）使蛋白质紧密接近以形成激活或抑制复合物的支架;（iii）隔离转录或其他调控因子如microRNA的诱饵;以及（iv）增加相邻基因表达的增强子。lncRNA已被证明在许多生物学背景中发挥作用，包括脂肪生成、造血和肌生成。同样，我们假设lncRNA是骨生成所必需的，并调节正常骨形成所必需的关键发育阶段。我们使用RNA-Seq分析的初步结果已经确定了小鼠和人的lncRNA转录组在间充质干细胞（MSC）分化成骨细胞。我们已经确定了人-小鼠lncRNA的保守表达，并选择了一个子集，在体外和体内的机制和功能表征。对lncRNA敲除小鼠的初步分析揭示了骨表型。我们提出的目标将为lncRNA如何促进调节骨生成的复杂机制提供变革性的见解。目标1将确定人-小鼠同源lncRNA在体外成骨细胞中的功能;目标2将通过表征lncRNA相互作用组来发现机制;目标3将解决选定lncRNA在小鼠模型中的体内活性。这种对成骨细胞lncRNA的创新研究将促进对骨形成的表观遗传控制的理解。我们的发现将影响与人类骨骼疾病相关的研究的新维度的发展。

 DESCRIPTION (provided by applicant): This revised proposal is a renewal application of our 25 year program that has contributed paradigm- shifting concepts for understanding molecular mechanisms regulating osteoblast commitment, growth and differentiation. We have established the stages of osteoblast differentiation and temporal control of the osteoblast phenotype by genetic and epigenetic mechanisms in vitro and using in vivo animal models. The contributions of this program include establishing bone tissue-specific properties of transcription factors, chromatin remodeling and histone modifications related to control of osteoblast-expressed mRNAs and noncoding RNAs. Our current program discovered that short noncoding microRNAs are key regulators of signaling pathways essential for bone formation. Little is known, however, about the identity and function of long noncoding (lncRNAs) involved in osteogenesis. The new direction of this renewal is to identify and characterize lncRNAs during osteoblastogenesis, and discover their molecular mechanisms and functional relevance in osteogenesis. lncRNAs have recently emerged as a novel level of gene regulation in development and lineage commitment. They function through complex mechanisms including acting as: (i) guides to recruit chromatin modifying enzymes to activate or repress transcription; (ii) scaffolds bringing proteins into close proximity to form activation or repression complexes; (iii) decoys to sequester transcription or other regulatory factors such as microRNAs; and (iv) enhancers to increase expression of neighboring genes. lncRNAs have been demonstrated to play roles in numerous biological contexts including adipogenesis, hematopoiesis and myogenesis. Likewise, we hypothesize that lncRNAs are essential for osteogenesis and regulate key developmental stages necessary for normal bone formation. Our preliminary results using RNA-Seq analysis have defined the mouse and human lncRNA transcriptomes during differentiation of mesenchymal stem cell (MSCs) to osteoblasts. We have identified conserved expression of human-mouse lncRNAs and selected a subset for mechanistic and functional characterization in vitro and in vivo. Preliminary analysis of lncRNA-knockout mice has revealed bone phenotypes. Our proposed aims will provide transformative insight into how lncRNAs contribute to the complex machinery regulating osteogenesis. Aim 1 will determine the function of human-mouse homologous lncRNAs in osteoblasts in vitro; Aim 2 will discover mechanisms by characterizing lncRNA interactomes; and Aim 3 will address the in vivo activity of select lncRNAs in mouse models. This innovative investigation of osteoblast lncRNAs will advance understanding of epigenetic control for bone formation. Our findings will impact the development of new dimensions for investigations that are relevant to human skeletal disease.