The goal of this proposal is to understand the role of the transcription factor complex Core Binding Factor (CBF) in prostatic epithelial cell gene expression. The hypothesis that CBF regulates prostate gene transcription through pathways that may co-operate with the androgen receptor (AR) will be tested with the following specific aims: (1) Determine the components of CBF activity in prostate cell lines and determine the effect of these factors on transcription driven by the prostate specific antigen (PSA) and the p21 WAF1/CIP-1 regulatory regions and (2) Determine the physical and functional relationship between CBF and the AR in prostatic epithelial cell lines. CBF activity consists of one of three DNA-binding RUNX family members (RUNX1, RUNX2, and RUNX3) in combination with CBFb.
Specific aim 1 seeks to determine the components of CBF in prostate by using a combination of electrophoretic mobility shift assays employing RUNX specific antibodies and RT- PCR employing DNA oligomer pairs specific to each RUNX protein in reactions containing cDNA derived from prostate cells. The role of CBF in prostate gene transcription will be assessed using the regulatory regions of the PSA and the p21 WAF1/CIP-1 genes linked to firefly luciferase in transient transcription assays in combination with vectors expressing each of the RUNX proteins. The CBF responsive sites will be mapped and the role of the RUNX proteins in basal and activated expression of these promoters will be determined.
Specific aim 2 seeks to define the physical and functional interactions between CBF and the AR using a combination of biochemical and molecular techniques. Physical interactions between the RUNX proteins and AR will be dissected in vitro using bacterially expressed RUNX proteins and AR produced in vitro. The required interaction domains on the respective proteins will be mapped and the effect of the RUNX proteins on androgen responsiveness will be functionally assessed in transient transcription assays using the AR-regulated PSA and p21 WAF1/CIP-1 reporter constructs. Identification of a role for CBF in prostate gene transcription will enhance our understanding of prostate cell biology and could provide molecular markers or therapeutic targets for prostate cancer.
| Fowler, Marcie; Borazanci, Erkut; McGhee, Laura et al. (2006) RUNX1 (AML-1) and RUNX2 (AML-3) cooperate with prostate-derived Ets factor to activate transcription from the PSA upstream regulatory region. J Cell Biochem 97:1-17 |
