Transcriptional Regulation of the Prostate-Restricted Gene Hoxb13

The prostate gland is highly dependent upon androgens for development and homeostasis. Prostate cancer also shares this dependence for growth and survival, however it can progress to a state of androgen-independence. Currently, there is a dearth of treatment options for advanced prostate cancer. Gene therapy has been proposed, however that approach requires specificity either at the cellular level for delivery or the genetic level for expression. There has been great interest in achieving genetic specificity through the characterization of prostate-specific promoters, yet paradoxically, all currently identified promoters require androgens.. Hoxb13 expression was previously reported by our laboratory to be restricted to the prostate and colon of adult mice. Remarkably, this expression persists following the loss of testicular androgens through castration. Therefore, we aim to identify and characterize the regulatory elements that confer this unique expression pattern by employing a transgenic reporter gene approach in mice. Transgenes were created by homologous recombination of a lacZ reporter gene into the Hoxb13 locus within the context of a 218 kb mouse genomic BAC. This construct recapitulated the reported expression pattern of Hoxb13, including persistent expression after castration. Reporter gene expression was also maintained in the diverse pathologies of the Transgenic Adenocarcinoma of the Mouse Prostate model. Deletion analyses identified a 7,675 region downstream of Hoxb13 required for lacZ expression. A bioinformatic analysis of transcription factor binding sites predicted a 37 bp enhancer in that region that is conserved between mice and humans. This enhancer contained two binding sites for Foxa1, which is known to regulate several prostate-specific genes. FOXA1 binding in vivo was confirmed in the LNCaP cancer cell line by chromatin immunoprecipitation. The enhancer was replaced by a loxP site in the context of the BAC transgene, and lacZ expression diminished specifically in the prostate while remaining robust in the colon. These elements can be utilized as tools to achieve androgen-independent manipulation of genes in the prostate through diverse transgenic applications. Furthermore, it defines a region required for prostate-specific expression and establishes a framework to begin a fine-scale identification of distinct factors regulating the unique expression of Hoxb13