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尚不清楚。大量证据表明转录因子Runx2在生理和病理钙化中都起着关键作用。然而，对于Runx2介导其生物活性的靶基因知之甚少。我们发明了染色质免疫沉淀（ChIP）显示（CD），这是一种允许以公正的方式鉴定转录因子靶基因的方法。CD需要用限制性内切酶消化ChIP获得的DNA，然后将沉淀的DNA扩增和分离成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显示了潜在的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)使用AvaII完成Runx2的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鉴定出大约100个Runx2高度被Runx2占据的靶点。我们将比较AvaII和Tfil介导的CD分析结果，以评估我们能够全面发现Runx2靶基因的程度。2)将Runx2 CD分析结果与Runx2 ChIP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)检测目标1和目标2中每个潜在的Runx2靶点：(i)使用常规的带有基因特异性引物的ChIP法检测Runx2的占用；（ii）成骨细胞分化过程中的基因表达模式；（iii）通过对Runx2靶点进行启动子报告子测定，直接调控Runx2。Runx2靶基因的组装库将为未来的研究提供基础，其中选择的候选基因将在功能上评估其在成骨细胞分化，骨形成和生物矿化中的作用。

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.