Chromatin Organization Regulates Osteogenesis

摘要 这一新的R01建立在R37时期(2008-2018)建立表观遗传机制的发现的基础上 (miRNAs，组蛋白修饰)调节成骨细胞分化。我们第一次描述了一个 与基因表达的动态变化相关的特异组蛋白修饰的“签名” 成骨的时间进程。这些组蛋白修饰也预示着“增强剂”，它们是 影响局部基因表达的关键顺式调控元件。我们现在建议研究一下最近的 公认的包含多种转录因子调控元件的“超级增强子”结构域(SED) 它们已经成为细胞表型的关键调节因子。SEDS通过远距离在染色质组织中发挥作用 染色体内和染色体间的相互作用，协调负责谱系的基因队列的控制 规格和独特的细胞身份。我们的初步研究已经确定了SED的子集，我们现在 被推定为“骨必需的超级增强剂”，也是重要决策阶段的候选人 致力于骨髓间充质干细胞的成骨。我们假设超级增强子域是不同的 从未分化的MSC激活到成骨细胞承诺阶段，并功能建立 协调调节基因网络和促进高阶染色质的成骨表型 支持小区身份的组织。我们的研究将在以下方面：AIM1-分析优先SED的功能效应 我们已经确定了与成骨细胞形成和成熟骨活动有关的直接抑制和 在MSCs中使用CRISPR/Cas9激活SED；目标2-确定相互作用的染色体结构域 通过SEDS控制多个基因和网络，使MSCs通过 染色质组织；以及目标3-在小鼠模型中演示使用CRISPR激活MSCs中的SED 会刺激骨骼的形成。 影响：这些研究开创了对MSC谱系承诺成骨的基因调控的新水平， 基于对SED在其他组织中的功能的新兴理解，但在骨骼中的研究很少。 通过刻画MSCs中与染色质组织和稳定相关的SED机制，我们将发现 转录中心和蛋白质复合体的多维协调控制新机制 一种SED，负责建立对成骨细胞表型的承诺。重要的是，了解 稳定成骨表型的染色质组织对未来新的治疗策略产生影响 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.