| Interaction of transcription factors (TFs) with DNA determines patterns of gene transcription. The first part of this thesis describes the development of ChIP Display (CD), a method for unbiased identification of novel genes regulated by a given TF, and application of CD to Runx2, a master TF in osteoblasts; the second part describes attempts to identify a TF that regulates a bone marker gene.;For Runx2 CD, osteoblasts were subjected to ChIP with Runx2 antibodies, resulting in a precipitate containing Runx2 targets, but also the inevitable vast excess of non-specifically co-precipitated DNA. This background makes identification of Runx2 targets problematic. To overcome this obstacle, true Runx2 targets were separated from the background using restriction digestion with AvaII, which brought all the fragments representing any one target to a unique size. Following linker ligation, the immunoprecipitated DNA fragments were PCR amplified in distinct families, segregated based on the identity of two nucleotides in the vicinity of the AvaII sites at the ends of each fragment. This results in improved signal-to-background ratio because each single target is wholly amplified within its family, while only a fraction of the background fragments is amplified in the same reaction. Indeed, upon gel electrophoresis, fragments representing true Runx2 target genes formed distinct bands. Bands of interest were excised, and the DNA fragments were identified by sequencing and comparison to public genomic databases. These include genes encoding the transcription factors Fli1 and Runx3, the less well-known gene ORP8 and a novel EST with homology to the DYRK1 kinase. Further evidence establishing ORP8 as a Runx2 target is provided. In principle, CD can facilitate the discovery of novel target genes for any DNA binding protein.;The second part deals with the reverse problem - identification of a TF that binds a novel cis-acting element at position -145/-175 of the osteocalcin gene. This element is an osteoblast-specific enhancer, whose activity is inhibited by glucorticoids - drugs widely used for immunosuppression, which cause osteoporosis as a side effect. I describe attempts to identify the -145/-175 binding protein by both biochemical purification and the candidate approach. |
