Epigenetic Control of Osteoblast Differentiation and Bone Formation

成骨细胞分化和骨形成的表观遗传控制

• 批准号: 8487371
• 负责人: Krishna M Sinha
• 金额: $ 7.51万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8487371
• 关键词: Address; Adult; Affect; Antibodies; Biochemical; Biological; Biological Assay; Birth; Bone Diseases; Calvaria; Cartilage; Cell Lineage; Cells; Child; Chromatin; CpG dinucleotide; Custom; Cytosine; DNA; DNA Methylation; Data; Defect; Deposition; Development; Disease; Embryo; Epigenetic Process; Funding; Funding Agency; Gene Activation; Gene Expression; Gene Targeting; Genes; Genomics; Histones; Homeostasis; Immunoprecipitation; In Vitro; Life; Lysine; Maintenance; Mass Spectrum Analysis; Measures; Methods; Methylation; Mission; Molecular Profiling; Molecular Target; Mus; Mutant Strains Mice; Mutation; National Institute of Arthritis and Musculoskeletal and Skin Diseases; Osteoblasts; Osteocytes; Osteogenesis; Osteoporosis; Phenotype; Phosphorylation; Physiological; Proteomics; Recruitment Activity; Regulation; Reporting; Research; Role; Serine; Site; Staging; Testing; Threonine; Transcriptional Regulation; Uracil; Wild Type Mouse; arginyllysine; bisulfite; bone; chromatin immunoprecipitation; chromatin protein; chromatin remodeling; demethylation; density; insight; mutant; osteoblast differentiation; osteosarcoma; polypeptide; postnatal; promoter; research study; skeletal; skeletal disorder; sodium bisulfite; transcription factor

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.

描述（由申请人提供）：Osterix (Osx) 是一种成骨细胞特异性转录因子，是成人骨形成和维持骨稳态所必需的。它在骨形成过程中激活成骨细胞成熟和功能所必需的一系列基因。这项研究的重点是在骨骼形成过程中控制骨特异性基因激活与 Osterix 转录活性平行的表观遗传现象。该提案解决了资助机构 NIAMS 的核心使命，该机构促进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，以确定 Osx 和 NO66 的相互作用位点，以及 Osx 靶基因上 H3K4me3 和 H3K36me3 的组蛋白甲基化水平。在胚胎阶段 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将用于用抗体进行免疫沉淀，然后将免疫沉淀的 DNA 与包含多个成骨细胞基因的高密度定制 DNA 阵列杂交。该方法称为 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NO66相互作用的动态以及这些Osx靶基因上的活性组蛋白甲基标记H3K4me3和H3K36me3的水平相关联。具体目标 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。首先通过对 DNA 进行亚硫酸氢盐处理，然后进行 PCR 扩增和测序，在 Osx 靶基因（包括 Bsp、Oc 和 Sost）的启动子中鉴定出 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，但不会将甲基化的胞嘧啶转化为尿嘧啶。从 Osx 无效颅骨细胞中分离的基因组 DNA 将用于首先鉴定被 Osx 激活的 Osx 靶基因启动子 DNA 中的 CpG 甲基化。接下来我们将研究野生型小鼠骨形成过程中Osx靶启动子的DNA甲基化与各个基因在不同阶段（E13.5、E15.5、E18.5和出生后第7天）的转录状态之间是否存在负相关关系。最后的具体目标 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。