Control of Osteoblast Proliferation and Differentiation

 描述(由申请人提供)：这项修订后的提案是我们25年计划的更新应用，该计划为理解调控成骨细胞承诺、生长和分化的分子机制贡献了范式转换概念。我们通过体外和体内动物模型的遗传和表观遗传机制，建立了成骨细胞分化的阶段和成骨细胞表型的时间控制。该计划的贡献包括建立骨组织特异性转录因子的属性，染色质重塑和组蛋白修饰，与控制成骨细胞表达的mRNAs和非编码RNA有关。我们目前的计划发现，短的非编码microRNAs是骨形成所必需的信号通路的关键调节者。然而，对参与成骨的长非编码(LncRNAs)的身份和功能知之甚少。新的研究方向是鉴定和鉴定成骨细胞形成过程中的lncRNAs，并发现其在成骨细胞形成中的分子机制和功能相关性。最近，lncRNAs在发育和谱系承诺中作为一种新的基因调控水平而出现。它们通过复杂的机制发挥作用，包括：(I)引导招募染色质修饰酶来激活或抑制转录；(Ii)支架使蛋白质接近以形成激活或抑制复合体；(Iii)诱饵来隔离转录或其他调节因子，如microRNAs；以及(Iv)增强剂来增加邻近基因的表达。LncRNAs已被证明在许多生物学环境中发挥作用，包括脂肪生成、造血和肌肉生成。同样，我们假设lncRNAs是成骨所必需的，并调节正常骨形成所必需的关键发育阶段。我们用RNA-Seq分析的初步结果确定了间充质干细胞(MSCs)向成骨细胞分化过程中小鼠和人的lncRNA转录本。我们已经确定了人-鼠LncRNAs的保守表达，并选择了一个亚集用于体外和体内的机制和功能表征。对lncRNA基因敲除小鼠的初步分析揭示了骨表型。我们提出的目标将为lncRNAs如何参与调控成骨的复杂机制提供变革性的见解。目的1将在体外确定人-鼠同源LncRNAs在成骨细胞中的功能；Aim 2将通过表征LncRNA相互作用来发现机制；Aim 3将在小鼠模型中研究部分LncRNAs的体内活性。这项对成骨细胞lncRNAs的创新性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.