The propiomelanocortin (POMC) gene produces a complex precursor polypeptide that is posttranslationally cleaved into a series of peptide hormones. In response to stress, one of these hormones, ACTH, induces the production of glucocorticoids in the adrenal gland, which in turn act to suppress POMC transcription in the pituitary in a classical feedback loop regulatory mechanism. This locus manifests two regulatory features of interest in our laboratory, negative regulation of gene expression by steroids and tissue-specific gene expression. The overall goals of the project are to identify and characterize tissue-specific and positive transacting factors that regulate POMC promoter activity, and to investigate mechanisms by which glucocorticoids mediate transcriptional repression. During characterization of potential regulatory regions in the POMC promoter by the classical mutagenesis and transfection approach, an unusual transcription factor (designated PO-B) was discovered. This protein activates POMC expression via a high-affinity DNA-binding site that is located between the TATA box and the cap site for initiation of RNA synthesis. The DNA recognition site for this factor has been characterized by the DNase I footprinting and DMS methylation interference techniques. In both in vitro transcription extracts and in vivo transfection studies, the TATA binding region is required, suggesting the factor acts cooperatively with TFIID. The factor has been extensively purified, and protein sequencing is underway.