Epigenetic Control of Osteoblast Differentiation and Bone Formation

描述（由申请方提供）：Osterix（Osx）是一种成骨细胞特异性转录因子，是成年期骨形成和维持骨稳态所必需的。在骨形成过程中，它激活成骨细胞成熟和功能所必需的基因库。这项研究的重点是表观遗传现象，控制骨特异性基因的激活，在骨骼形成过程中的转录活性的Osterix平行。该提案涉及资助机构NIAMS的核心使命，该机构促进我们对骨骼和骨骼相关疾病的理解的研究资金。该提案有三个具体目标;每个目标都解决了一个具体问题，以更好地了解成骨细胞中主调节因子Osx对骨特异性基因的转录控制。具体目标1是检验我们的假设，即Osx靶基因的染色质在成骨细胞分化过程中通过NO 66去甲基化酶受到组蛋白甲基化和去甲基化的表观遗传控制。将使用特异性抗体进行染色质免疫沉淀（ChIP）测定，以确定Osx和NO 66的相互作用位点，以及Osx靶基因处H3 K4 me 3和H3 K36 me 3的组蛋白甲基化水平。将在胚胎期E14.5和E18.5以及出生后骨形成第7天（P7）分离的小鼠颅骨成骨细胞用于抗体免疫沉淀，然后将免疫沉淀的DNA与含有几种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，将在不同阶段测量Osx靶基因的表达谱，并将其与Osx和NO 66相互作用的动力学以及这些Osx靶基因上的活性组蛋白甲基标记H3 K4 me 3和H3 K36 me 3的水平相关联。具体目标2是测试Osx靶基因的DNA在其活化之前是否在CpG二核苷酸上甲基化。首先通过亚硫酸氢盐处理DNA，然后进行PCR扩增和测序，在Osx靶基因（包括Bsp、Oc和Sost）的启动子中鉴定CpG甲基化位点。亚硫酸氢钠将DNA中所有未甲基化的胞嘧啶转化为尿嘧啶，但不转化甲基化的胞嘧啶。从Osx缺失的颅骨细胞分离的基因组DNA将用于首先鉴定Osx靶基因启动子DNA中的CpG甲基化，所述Osx靶基因被Osx激活。接下来，我们将研究在野生型小鼠骨形成的不同阶段（E13.5，E15.5，E18.5和出生后第7天），Osx靶启动子的DNA甲基化与相应基因的转录状态之间是否存在反比关系。具体目的3是研究成骨细胞中Osx甲基化的潜在生物学意义。使用Osx多肽的质谱分析，Osx的几个赖氨酸残基被鉴定为通过甲基化进行翻译后修饰。将进行生物化学和体外研究以研究成骨细胞分化过程中Osx甲基化在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.