ChIP DISPLAY OF RUNX2 TARGETS IN OSTEOBLASTS

描述(申请人提供)：哺乳动物组织生物矿化的分子机制知之甚少。大量证据表明转录因子Runx2在生理性和病理性钙化中都起着关键作用。然而，对调节其生物学活性的Runx2靶基因知之甚少。我们发明了染色质免疫沉淀(CHIP)显示(CD)，这是一种允许以无偏见的方式识别转录因子的目标基因的方法。CD需要用限制性内切酶消化芯片获得的DNA，然后扩增沉淀的DNA并将其分离成36个不同的片段家族。这种方法使我们能够将真正的目标集中在凝胶上，同时分散芯片过程中产生的压倒性背景。初步的CD实验，通过Avall消化芯片的凝胶电泳法呈现潜在的Runx2靶点，已经产生了四个新的Runx2靶点。我们提出了三个具体目标：1)用AvaII完成Runx2 CD，并使用不同的限制性内切酶TFIL执行类似的CD。我们期望在活的成骨细胞中鉴定出大约100个最高被Runx2占据的靶点。我们将比较AvaII和TFIL介导的CD分析的结果，以评估我们能够在多大程度上全面发现Runx2靶基因。2)将Runx2 CD分析结果与Runx2芯片与CpG岛芯片杂交结果进行比较。这将为CD发现Runx2靶基因的能力提供一个独立的衡量标准。3)测试来自AIMS 1和2的每个潜在的Runx2靶点：(I)使用传统的基因特异性引物芯片分析Runx2的占有率；(Ii)成骨细胞分化过程中的基因表达模式；以及(Iii)Runx2对一组集中的Runx2靶点的启动子-报告基因分析的直接调节。Runx2靶基因的集合将为未来的研究奠定基础，在未来的研究中，将对选定的候选基因在成骨细胞分化、骨形成和生物矿化中的作用进行功能评估。

DESCRIPTION (provided by applicant): The molecular mechanisms of mammalian tissue biomineralization are poorly understood. Ample evidence suggests a key role for the transcription factor Runx2 in both physiological and pathological calcification. However, there is little knowledge of Runx2 target genes that mediate its biological activities. We invented chromatin immunoprecipitation (ChIP) Display (CD), a method that allows the identification of target genes for transcription factors in an unbiased fashion. CD entails digestion of DNA obtained by ChIP with a restriction enzyme, followed by amplification and segregation of the precipitated DNA into thirty-six distinct families of fragments. This approach allows us to concentrate true targets on gels, while scattering the overwhelming background generated during ChIP. Preliminary CD experiments, presenting potential Runx2 targets by gel electrophoresis of Avall-digested ChIP, have already yielded four novel Runx2 targets. We propose three specific aims: 1) To complete the Runx2 CD with AvaII and to perform a similar CD using a different restriction enzyme, Tfil. We anticipate the identification of approximately one hundred Runx2 targets most highly occupied by Runx2 in living osteoblasts. We will compare the results of the AvaII and Tfil mediated CD analyses to assess the degree to which we are able to comprehensively discover Runx2 target genes. 2) To compare the results of Runx2 CD analyses to the results obtained by hybridization of Runx2 ChIP to CpG island microarrays. This will provide an independent measure of the power of CD to discover Runx2 target genes. 3) To test each potential Runx2 target from Aims 1 and 2 for: (i) occupancy by Runx2 using conventional ChIP assay with gene-specific primers; (ii) pattern of gene expression during osteoblast differentiation; and (iii) direct regulation by Runx2, using promoter-reporter assays for a focused group of Runx2 targets. The assembled repertoire of Runx2 target genes will serve the basis for future studies, in which selected candidates will be evaluated functionally for their role in osteoblast differentiation, bone formation and biomineralization.