Epigenetic Regulation of Osteoclastogenic Gene Expression: Factors, Targets, and Mechanisms

破骨细胞基因表达的表观遗传调控：因素、靶点和机制

• 批准号: 10531536
• 负责人: WOOJIN AN
• 金额: $ 36.3万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10531536
• 关键词: Acetylation; Binding; Biochemical; Biological Assay; Biological Process; Bone Development; Bone Diseases; Bone Regeneration; Bone Resorption; Bone remodeling; CRISPR/Cas technology; Catalytic Domain; Cell Differentiation process; Cell Nucleus; Cells; Chromatin; Chromatin Structure; Chromatin Structure Alteration; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Data; Deoxyribonuclease I; Digestion; Disulfides; EP300 gene; Epigenetic Process; Event; Excision; Extracellular Matrix; Family; Gelatinase B; Gene Abnormality; Gene Activation; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Goals; Guide RNA; Histone H3; Histones; Hydroxyl Radical; In Vitro; Laboratories; Light; Link; Macrophage; Maintenance; Maps; Matrix Metalloproteinases; Mediating; Mediator; Methylation; Modeling; Molecular; Molecular Conformation; Mononuclear; N-terminal; Nucleosomes; Osteoclasts; Pathway interactions; Peptide Hydrolases; Peptides; Permeability; Play; Process; Property; Proteolysis; Regulatory Pathway; Research; Role; Secure; Series; Signal Pathway; Signal Transduction; Site; System; TNFSF11 gene; Tail; Techniques; Therapeutic; Transactivation; Transcription Repressor; Transcriptional Activation; Transcriptional Regulation; Work; collaborative environment; crosslink; domain mapping; epigenetic regulation; epigenome; experimental study; genome-wide; hematopoietic differentiation; in vivo; insight; member; monocyte; osteoclastogenesis; precursor cell; programs; reconstitution; recruit; skeletal; skeletal disorder; tool; transcriptome sequencing

PROJECT SUMMARY Osteoclasts are multinucleated bone-resorbing cells and formed by the fusion of mononuclear precursor cells of the monocyte-macrophage lineage. Osteoclasts play a crucial role in the maintenance of bone remodeling and regeneration. A group of genes regulating osteoclast differentiation have been identified, and the deregulated expression of these genes has been documented to cause various skeletal diseases. Given the fact that all genes encoding osteoclastogenic factors are expressed in the context of chromatin, a fundamental mechanism underlying osteoclast differentiation should involve chromatin regulatory pathways. Studies of transcription regulation mechanisms by chromatin reorganization may thus aid in the understanding and treatment of bone disorders caused by abnormal gene expression. MMP-9 is a member of MMP family that has been studied mainly with respect to its role in extracellular matrix remodeling. Unexpectedly, our recent studies have revealed that MMP-9 moves into the nucleus and mediates histone H3 N-terminal tail (NT) proteolysis at osteoclastogenic genes in RANKL-induced osteoclast precursor (OCP) cells. Furthermore, we found that p300/CBP-mediated H3K18 acetylation stimulates MMP-9 enzymatic activity toward H3NT in OCP- induced cells. More recent work from our laboratory also demonstrated that MMP-9 binds target nucleosomes in a manner dependent upon G9a-mediated H3K27me1 and that this binding is critical for MMP-9 recruitment and H3NT proteolysis at osteoclastogenic genes. In light of these findings, we have generated cell-permeable H3NTK27me1 mimics as a cellular tool to define H3NT residues important for MMP-9 recruitment and function. The long-term goal of the proposed research is to understand the biological processes that are controlled by H3NT proteolysis and the molecular basis of its action as an essential mediator of osteoclastogenesis. The overall objectives are to investigate MMP-9-dependent H3NT proteolysis as an osteoclastogenic signal, and to determine the molecular mechanisms whereby H3NT proteolysis activates the genes encoding master regulators of osteoclastogenesis. Our hypothesis is that MMP-9 establishes and maintains the active state of osteoclastogenic genes by a two-step mechanism wherein it gets to target genes by sensing local H3K27me1 states and cleaving H3NT in a K18ac-dependent manner. In Aim 1, we will employ the CRISPR-Cas9 system in which we can manipulate H3NT proteolysis at specific loci, and identify the genes that are directly activated by MMP-9-dependent H3NT proteolysis and necessary for proficient osteoclast differentiation. In Aim 2, we will use combined functional and structural approaches, and investigate the role of G9a-mediated H3K27me1 in the recruitment and osteoclastogenic function of MMP-9 at target genes. In Aim 3, we will examine a possible involvement of MMP-9-dependent H3NT proteolysis in disrupting nucleosome/chromatin structure, and generate a mechanistic picture for H3NT proteolysis-mediated transactivation in OCP-induced cells. In Aim 4, we will identify a group of factors that interact with H3NT and participate in pre-osteoclastogenic gene silencing, and establish MMP-9-dependent H3NT proteolysis as a key process in removing these repressors and activating transcription during osteoclastogenesis.

项目概要 破骨细胞是多核骨吸收细胞，由单核前体细胞融合形成 单核细胞-巨噬细胞谱系。破骨细胞在维持骨重塑中发挥着至关重要的作用 和再生。一组调节破骨细胞分化的基因已被鉴定，并且 据记录，这些基因的表达失调会导致各种骨骼疾病。鉴于 事实上，所有编码破骨细胞因子的基因都在染色质的背景下表达，这是一个基本的 破骨细胞分化的机制应涉及染色质调控途径。研究 因此，染色质重组的转录调控机制可能有助于理解和 治疗由基因表达异常引起的骨骼疾病。 MMP-9 是 MMP 家族的成员 主要研究其在细胞外基质重塑中的作用。没想到我们最近 研究表明，MMP-9 进入细胞核并介导组蛋白 H3 N 末端尾部 (NT) RANKL 诱导的破骨细胞前体 (OCP) 细胞中破骨细胞生成基因的蛋白水解。此外，我们 发现 p300/CBP 介导的 H3K18 乙酰化可刺激 MMP-9 对 OCP-H3NT 的酶活性 诱导细胞。我们实验室最近的工作还表明 MMP-9 结合靶核小体 以一种依赖于 G9a 介导的 H3K27me1 的方式，并且这种结合对于 MMP-9 招募至关重要 和 H3NT 破骨细胞基因的蛋白水解作用。根据这些发现，我们产生了细胞渗透性 H3NTK27me1 模拟作为细胞工具来定义对 MMP-9 募集和功能重要的 H3NT 残基。 拟议研究的长期目标是了解受控制的生物过程 H3NT 蛋白水解及其作为破骨细胞生成的重要介质的分子基础。这 总体目标是研究 MMP-9 依赖性 H3NT 蛋白水解作为破骨细胞信号，并 确定 H3NT 蛋白水解激活主编码基因的分子机制 破骨细胞生成的调节剂。我们的假设是 MMP-9 建立并维持了 通过两步机制产生破骨细胞基因，其中通过感测局部 H3K27me1 到达目标基因 状态并以 K18ac 依赖性方式裂解 H3NT。在目标 1 中，我们将采用 CRISPR-Cas9 系统 我们可以在特定位点操纵 H3NT 蛋白水解，并识别直接激活的基因 通过 MMP-9 依赖性 H3NT 蛋白水解作用，是破骨细胞熟练分化所必需的。在目标 2 中，我们将 使用功能和结构相结合的方法，并研究 G9a 介导的 H3K27me1 在 MMP-9 在靶基因上的募集和破骨细胞功能。在目标 3 中，我们将研究一种可能的 MMP-9 依赖性 H3NT 蛋白水解参与破坏核小体/染色质结构，以及 生成 OCP 诱导细胞中 H3NT 蛋白水解介导的反式激活的机制图。在目标 4 中， 我们将鉴定一组与H3NT相互作用并参与前破骨细胞基因的因子 沉默，并建立 MMP-9 依赖性 H3NT 蛋白水解作为去除这些阻遏物的关键过程 并在破骨细胞生成过程中激活转录。