ChIP DISPLAY OF RUNX2 TARGETS IN OSTEOBLASTS

描述（由申请人提供）：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Runx 2在生理和病理钙化中起关键作用。然而，有很少的知识Runx 2靶基因介导其生物活性。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方式识别转录因子靶基因的方法。CD需要用限制性内切酶消化通过ChIP获得的DNA，然后将沉淀的DNA扩增并分离成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ChIP过程中产生的压倒性背景。初步的CD实验，通过Avall消化的ChIP的凝胶电泳呈现潜在的Runx 2靶标，已经产生了四个新的Runx 2靶标。我们提出了三个具体目标：1）用AvaII完成Runx 2 CD，并使用不同的限制性内切酶Tfil进行类似的CD。我们预计，在活的成骨细胞中，Runx 2高度占据了大约100个Runx 2靶点。我们将比较AvaII和Tfil介导的CD分析的结果，以评估我们能够全面发现Runx 2靶基因的程度。2)比较Runx 2 CD分析结果与Runx 2 ChIP与CpG岛微阵列杂交所得结果。这将提供CD发现Runx 2靶基因的能力的独立测量。3)检测目标1和2中的每个潜在Runx 2靶标：（i）使用基因特异性引物的常规ChIP试验检测Runx 2的占用率;（ii）成骨细胞分化期间的基因表达模式;以及（iii）使用启动子-报告基因试验检测Runx 2靶标的直接调节。Runx 2靶基因的组装库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.