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

项目总结 破骨细胞是多核的骨吸收细胞，由单核前体细胞融合而成 单核细胞-巨噬细胞谱系。破骨细胞在维持骨重建中起着至关重要的作用 和再生。一组调控破骨细胞分化的基因已经被发现，并且 这些基因的失控表达已被证明会导致各种骨骼疾病。给定 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，染色质是一种基本的 破骨细胞分化的机制应该涉及染色质调节通路。的研究 因此，染色质重组的转录调控机制可能有助于理解和 基因表达异常引起的骨病的治疗。基质金属蛋白酶-9是基质金属蛋白酶家族的成员， 主要针对其在细胞外基质重塑中的作用进行了研究。出乎意料的是，我们最近 研究表明，基质金属蛋白酶-9进入细胞核，并介导组蛋白H3N端尾(NT)。 RANKL诱导的破骨细胞前体(OCP)细胞中破骨细胞基因的蛋白分解。此外，我们 发现p300/CBP介导的H3K18乙酰化可刺激OCP中MMP9对H3NT的催化活性 诱导细胞。我们实验室最近的工作也证明了基质金属蛋白酶-9与靶核小体结合 依赖于G9a介导的H3K27me1，这种结合对基质金属蛋白酶-9的募集至关重要 破骨细胞基因的H3NT蛋白降解。根据这些发现，我们已经产生了细胞渗透性 H3NTK27me1模拟作为一种细胞工具来定义对基质金属蛋白酶-9的招募和功能重要的H3NT残基。 这项拟议研究的长期目标是了解由 H3NT蛋白分解及其作为破骨细胞发生的重要介体的分子基础。这个 总体目标是研究依赖于基质金属蛋白酶-9的H3NT蛋白分解作为破骨细胞形成信号，并 确定H3NT蛋白分解激活编码MASTER基因的分子机制 破骨细胞生成的调节剂。我们的假设是，基质金属蛋白酶-9建立并维持了血管紧张素转换酶活性状态。 通过两步机制获得破骨细胞基因，其中它通过感知局部H3K27me1来靶向基因 以K18ac依赖的方式状态和切割H3NT。在目标1中，我们将采用CRISPR-CAS9系统 在这个过程中，我们可以在特定的位置操纵H3NT蛋白的降解，并识别直接激活的基因 通过依赖于基质金属蛋白酶-9的H3NT蛋白分解，是熟练的破骨细胞分化所必需的。在目标2中，我们将 采用功能和结构相结合的方法，研究G9a介导的H3K27me1在 基质金属蛋白酶-9在靶基因的募集和破骨作用。在目标3中，我们将研究一种可能的 基质金属蛋白酶-9依赖的H3NT蛋白分解参与破坏核小体/染色质结构，以及 为H3NT蛋白分解在OCP诱导的细胞中介导的反式激活生成一张机制图。在目标4中， 我们将确定一组与H3NT相互作用并参与破骨细胞前基因的因子 沉默，并建立依赖于基质金属蛋白酶-9的H3NT蛋白水解酶是去除这些阻遏物的关键过程 以及在破骨细胞形成过程中激活转录。

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.