Epigenetic Control of Osteoblast Differentiation and Bone Formation

描述(申请人提供)：Osterix(OSX)是一种成骨细胞特异性转录因子，在成人生活中是骨形成和维持骨稳态所必需的。它激活了成骨细胞在骨形成过程中成熟和功能所必需的一系列基因。这项研究集中于在骨骼形成过程中控制骨特异基因的激活和Osterix转录活性的表观遗传现象。这项建议解决了资助机构NIAMS的核心使命，该机构旨在促进研究资助我们对骨骼和骨相关疾病的了解。该提案有三个具体目标；每个目标都解决了一个特定的问题，以更好地了解成骨细胞中主要调控因子OSX对骨特异基因的转录控制。具体目的1是验证我们的假设，即在成骨细胞分化过程中，OSX靶基因的染色质受到组蛋白甲基化和去甲基化的表观遗传控制组蛋白66去甲基化。使用特异性抗体的染色质免疫沉淀(ChIP)分析将确定OSX和NO66的相互作用位置，以及OSX靶基因上H3K4me3和H3K36me3的组蛋白甲基化水平。在胚胎时期E14.5和E18.5以及出生后第7天(P7)分离的小鼠颅骨成骨细胞将被用于抗体的免疫沉淀，然后将免疫沉淀的DNA杂交到包含几个成骨细胞基因的高密度定制DNA阵列。这种方法被称为芯片上芯片。接下来，将测量OSX靶基因在不同阶段的表达谱，并与OSX和NO66相互作用的动态以及这些OSX靶基因上活性的组蛋白甲基标记H3K4me3和H3K36me3的水平相关联。具体目的2是测试OSX靶基因的DNA在激活之前是否在CpG二核苷酸上甲基化。CpG甲基化位点将首先通过亚硫酸氢盐处理DNA来鉴定OSX靶基因BSP、Oc和SoST的启动子中的CpG甲基化位点，然后进行PCR扩增和测序。亚硫酸氢钠将DNA中所有未甲基化的胞嘧啶转化为尿嘧啶，但不能将甲基化的胞嘧啶转化为尿嘧啶。从OSX缺失的颅骨细胞中提取的基因组DNA将首先用于鉴定被OSX激活的OSX靶基因启动子DNA中的CpG甲基化。接下来，我们将研究OSX靶基因启动子的DNA甲基化与野生型小鼠骨形成不同阶段(E13.5、E15.5、E18.5和出生后第7天)各自基因的转录状态是否存在反向关系。最后，具体目标3是研究OSX甲基化在成骨细胞中的潜在生物学意义。利用OSX多肽的质谱分析，OSX的几个赖氨酸残基被鉴定为甲基化后的翻译后修饰。将进行生化和体外研究，以研究OSX甲基化在成骨细胞分化过程中激活OSX靶基因的潜在作用。

DESCRIPTION (provided by applicant): Osterix (Osx) is an osteoblast-specific transcription factor and is required for bone formation and maintenance of bone homeostasis in adult life. It activates a repertoire of genes essential for maturation and function of osteoblasts during bone formation. This study focuses on epigenetic phenomena that control the activation of bone-specific genes in parallel to the transcriptional activity of Osterix during skeletal formation. This proposal addresses the core mission of funding agency NIAMS which promotes research funding in our understanding of bone and bone related diseases. The proposal has three specific aims; each one addresses a specific question to better understand the transcriptional control of bone-specific genes by the master regulator Osx in osteoblasts. Specific aim 1 is to test our hypothesis that the chromatin of Osx-target genes is under epigenetic control of histone methylation and demethylation through NO66 demethylase during osteoblast differentiation. Chromatin immunoprecipitations (ChIP) assays using specific antibody will be performed to determine the interaction sites of Osx and NO66, and the levels of histone methylation of H3K4me3 and H3K36me3 at Osx target genes. Mouse calvarial osteoblasts, isolated at embryonic stages E14.5 and E18.5, and postnatal day 7 (P7) of bone formation, will be used for immunoprecipitation with antibodies followed by hybridization of immunoprecipitated DNA to a high density custom DNA array containing several osteoblast genes. This method is called ChIP-on-ChIP. Next, expression profiles of Osx target genes will be measured at different stages and correlated with the dynamics of the interactions of Osx and NO66, and the levels of active histone methyl marks H3K4me3 and H3K36me3 at those Osx target genes. Specific aim 2 is to test whether the DNA of Osx target genes are methylated on CpG dinucleotides prior to their activation. The CpG methylation sites will be first identified in the promoters of the Osx target genes including Bsp, Oc, and Sost by bisulfite treatment to DNA followed by PCR amplification and sequencing. Sodium bisulfite converts all unmethylated cytosine in DNA to uracil, but not the methylated cytosine. Genomic DNA isolated from Osx null calvarial cells will be used to first identify CpG methylation in the DNA of the promoter of Osx target genes which are activated by Osx. Next we will study whether there is an inverse relationship between the DNA methylation of Osx target promoters and the transcriptional state of the respective genes at different stages (E13.5, E15.5, E18.5 and postnatal day 7) during bone formation in wild type mice. Finally specific aim 3 is to study the potential biological significance of methylation of Osx in osteoblasts. Using a mass spectrometry analysis of Osx polypeptides, several lysine residues of Osx were identified as post-translationally modified by methylation. Biochemical and in vitro studies will be performed to study the potential role of Osx methylation in activation of Osx target genes during osteoblast differentiation.