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

项目摘要 破骨细胞是由单核前体细胞融合而成的多核骨吸收细胞 单核细胞-巨噬细胞谱系的一部分。破骨细胞在维持骨重建中起着至关重要的作用 和再生。已经鉴定了一组调节破骨细胞分化的基因， 这些基因的失调表达已被证明可引起各种骨骼疾病。鉴于 事实上，所有编码破骨细胞因子的基因都在染色质中表达，这是一个基本的 破骨细胞分化的潜在机制应涉及染色质调节途径。研究 因此，通过染色质重组的转录调节机制可能有助于理解和 治疗由异常基因表达引起的骨疾病。MMP-9是MMP家族的一员， 主要研究了其在细胞外基质重塑中的作用。出乎意料的是，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（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。而且我们 发现p300/CBP介导的H3 K18乙酰化刺激OCP中MMP-9对H3 NT的酶活性。 诱导细胞我们实验室最近的工作也表明MMP-9结合靶核小体 以依赖于G9 a介导的H3 K27 me 1的方式结合，并且这种结合对于MMP-9募集是关键的 和H3 NT蛋白水解在破骨细胞基因。根据这些发现，我们已经产生了细胞可渗透的 H3 NTK 27 me 1模拟物作为细胞工具来定义对MMP-9募集和功能重要的H3 NT残基。 拟议研究的长期目标是了解由 H3 NT蛋白水解及其作为破骨细胞生成重要介质的分子基础。的 总体目标是研究MMP-9依赖的H3 NT蛋白水解作为破骨细胞生成信号， 确定H3 NT蛋白水解激活编码主基因的分子机制 破骨细胞生成的调节因子。我们的假设是MMP-9建立并维持了 破骨细胞生成基因通过两步机制，其中它通过感测局部H3 K27 me 1 状态并以K18 ac依赖性方式切割H3 NT。在目标1中，我们将使用CRISPR-Cas9系统 我们可以在特定位点操纵H3 NT蛋白水解，并鉴定直接激活的基因， 通过MMP-9依赖的H3 NT蛋白水解，并且是熟练的破骨细胞分化所必需的。在目标2中，我们将 使用功能和结构相结合的方法，并研究G9 a介导的H3 K27 me 1在 MMP-9在靶基因上的募集和破骨细胞生成功能。在目标3中，我们将研究一种可能的 MMP-9依赖性H3 NT蛋白水解参与破坏核小体/染色质结构， 生成OCP诱导的细胞中H3 NT蛋白水解介导的反式激活的机制图片。在目标4中， 我们将鉴定一组与H3 NT相互作用并参与破骨细胞前基因表达的因子， 沉默，并建立MMP-9依赖性H3 NT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。

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.