Chromatin Organization Regulates Osteogenesis

总结 这种新的R 01建立在R37期间（2008-2018）的发现基础上，这些发现建立了表观遗传机制 （miRNAs，组蛋白修饰）调控成骨细胞分化。我们首次描述了 特定组蛋白修饰的“签名”，其与在基因表达过程中的动态变化相关。 骨生成的时间进程这些组蛋白修饰也预测了“增强子”， 关键的顺式调控元件，有助于局部基因表达。我们现在建议研究最近 公认的“超级增强子”结构域（SED），包括多种转录因子的调控元件 已经成为细胞表型的关键调节因子。SEDs通过远程在染色质组织中发挥作用 染色体内和染色体间相互作用，协调控制负责谱系的基因组 规范和独特的小区标识。我们的初步研究已经确定了一个子集的SED， 建议是公认的“骨必需的超级增强剂”和候选人的重要决定阶段， 致力于从MSC成骨。我们假设超级增强子结构域是不同的， 从未分化的MSC激活到成骨细胞定型阶段，并发挥作用， 成骨表型通过协调调节基因网络和促进更高级的染色质 支持细胞识别的组织。我们的研究将在：目的1-分析优先的SED的功能效果 我们已经通过定向抑制确定了与成骨细胞生成和成熟骨活性相关的， 在MSC中使用CRISPR/Cas9激活SED;目的2-确定相互作用的染色体域 与SED控制多个基因和网络，使MSC成为成骨细胞表型， 目的3-在小鼠模型中证明使用CRISPR激活MSC中的SED 会刺激骨骼形成 影响：这些研究开创了MSC谱系致力于成骨的基因调控的新水平， 基于对其他组织中SED功能的新认识，但在骨中的研究很少。 通过表征与MSC中染色质组织和稳定相关的SED机制，我们将发现 转录枢纽和蛋白质复合物的多维协调控制的新机制， 一种负责建立成骨细胞表型定型的SED。重要的是， 稳定成骨表型的染色质组织对未来新治疗策略的影响 治疗骨骼疾病

SUMMARY This new R01 builds on discoveries during the R37 period (2008-2018) that established epigenetic mechanisms (miRNAs, histone modifications) regulating osteoblast differentiation. We characterized for the first time a “signature” of specific histone modifications that are associated with dynamic changes in gene expression during the temporal progression of osteogenesis. These histone modifications also predicted “enhancers”, which are critical cis-regulatory elements that contribute to local gene expression. We now propose to examine the recently recognized “super enhancer” domains (SEDs) that include regulatory elements for multiple transcription factors that have emerged as key regulators of cell phenotypes. SEDs function in chromatin organization via long range intra- and inter-chromosomal interactions that coordinate control of gene cohorts responsible for lineage specification and distinct cell identity. Our preliminary studies have identified a subset of SEDs that we now propose are putative “bone-essential super-enhancers” and candidates for the important decision stage of commitment to osteogenesis from MSCs. We hypothesize that super-enhancer domains are differentially activated from the undifferentiated MSC to the osteoblast commitment stage, and function to establish the osteogenic phenotype by coordinately regulating gene networks and contributing to higher order chromatin organization that supports cell identity. Our studies will in: Aim1- analyze the functional effects of prioritized SEDs we have identified related to osteoblastogenesis and mature bone activities through directed inhibition and activation of SEDs using CRISPR/Cas9 in MSCs; Aim 2- determine the chromosomal domains that interact with SEDs to control multiple genes and networks that commit MSCs to the osteoblast phenotype through chromatin organization; and Aim 3- demonstrate in mouse models that using CRISPR activated SEDs in MSCs will stimulate bone formation. Impact: These studies pioneer a new level of gene regulation for MSC lineage commitment to osteogenesis, based on an emerging understanding of SED functions in other tissues but have been minimally studied in bone. By characterizing SED mechanisms related to chromatin organization and stabilization in MSCs, we will discover novel mechanisms of multi-dimensional coordinate control of transcriptional hubs and protein complexes within an SED that is responsible for establishing commitment to the osteoblast phenotype. Importantly, knowledge of the chromatin organization that stabilizes the osteogenic phenotype impacts on future novel treatment strategies for skeletal disorders.