The main research interest of my laboratory is the neuroendocrine regulation of pro-opiomelanocortin (POMC) gene expression in the pituitary. In this proposal we will focus on a single transcriptional activator that we have previously described, designated PO-B, which we have shown by mutational analysis contributes to 70% of POMC basal transcription. The PO-B DNA binding site is critically positioned between the POMC TATA box and CAP site which we believe is necessary for its efficient effects on transcriptional induction. PO-B is found in a number of cell types suggesting that PO-B regulates genes other than POMC. Indeed PO-B is highly inducible by differentiating agents in HL-60 pro-myelocytic cells. We have now purified PO-B to homogeneity. Interestingly dephosphorylation of the purified protein increases PO-B DNA binding affinity less than 30-fold. Our experiments will address the hypothesis that the efficient effects of PO-B on POMC and cellular gene transcription are regulated by its phosphorylation status and critical positioning of the PO-B DNA binding element between the TATA box and CAP site. The specific goals are: 1) To obtain a full length PO-B cDNA clone. 2) To develop antibodies to PO-B. 3) To identify the signal transduction pathways that control PO-B function in the pituitary. 4) To utilize oligonucleotide substitution mutational analysis to determine if the effects of the PO-B element are limited to the context of the POMC promoter.5)To determine if the specific interactions of PO-B with DNA that can be detected in vitro represent the interactions of PO-B with the integrated promoter in vivo. 6) To optimize the conditions of our in vitro transcriptions assays such that the effects of purified PO-B on POMC transcription can be reproducibly and sensitively detected.
