Epigenetic Control of Osteoblast Differentiation and Bone Formation

描述（由申请人提供）：Osterix （Osx）是一种成骨细胞特异性转录因子，是成人生活中骨形成和维持骨稳态所必需的。它激活了骨形成过程中成骨细胞成熟和功能所必需的一系列基因。本研究的重点是在骨骼形成过程中控制骨特异性基因激活的表观遗传现象，与Osterix的转录活性平行。该提案解决了资助机构NIAMS的核心任务，该机构促进了我们对骨骼和骨骼相关疾病的理解的研究资助。该提案有三个具体目标；每一个都解决了一个特定的问题，以更好地理解骨特异性基因的转录控制的主调控Osx成骨细胞。具体目的1是验证我们的假设，即osx靶基因的染色质在成骨细胞分化过程中受到组蛋白甲基化和通过NO66去甲基化酶去甲基化的表观遗传控制。使用特异性抗体进行染色质免疫沉淀（ChIP）测定Osx和NO66的相互作用位点，以及Osx靶基因上H3K4me3和H3K36me3组蛋白甲基化水平。在胚胎期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用于抗体免疫沉淀，然后将免疫沉淀的DNA与含有多个成骨细胞基因的高密度定制DNA阵列杂交。这种方法被称为ChIP-on-ChIP。接下来，我们将测量Osx靶基因在不同阶段的表达谱，并与Osx与NO66的相互作用动态进行关联，以及Osx靶基因上活性组蛋白甲基标记H3K4me3和H3K36me3的水平。具体目的2是测试Osx靶基因的DNA在CpG二核苷酸激活之前是否被甲基化。CpG甲基化位点将首先在Osx靶基因（包括Bsp， Oc和Sost）的启动子中通过亚硫酸盐处理DNA进行鉴定，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空头颅细胞中分离的基因组DNA将首先用于鉴定Osx靶基因启动子DNA中的CpG甲基化，这些基因被Osx激活。接下来我们将研究野生型小鼠骨形成过程中不同阶段（E13.5、E15.5、E18.5和出生后第7天）Osx靶启动子DNA甲基化与相关基因转录状态之间是否存在反比关系。最后，具体目的3是研究Osx甲基化在成骨细胞中的潜在生物学意义。通过对Osx多肽的质谱分析，鉴定出Osx的几个赖氨酸残基被甲基化修饰。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Osx靶基因激活中的潜在作用。

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.