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.
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