DIFFERENT COMPOSITE REGULATORY ELEMENTS DIRECT EXPRESSION OF THE HUMAN ALPHA-SUBUNIT GENE TO PITUITARY AND PLACENTA

To identify elements of the human alpha subunit gene necessary for cel l-specific expression, we generated an array of block mutations spanni ng approximately 400 base pairs (bp) of promoter proximal region and e xamined them using transient transfection analysis in pituitary (alpha T3) and placental (BeWo) cell lines, Comparison of promoter activity in the two cell types revealed both common and unique elements require d for transcription in pituitary and placenta, Two strong elements, th e cyclic AMP response element (CRE) and the upstream regulatory elemen t (URE), regulate expression of the a subunit gene in BeWo cells, In c ontrast, promoter activity in alpha T3 cells requires an array of weak er elements, These include the CREs, the URE, as well as two previousl y described elements, pituitary glycoprotein hormore basal element (PG BE) and gonadotrope-specific element (GSE), and two new elements we de signated as the a basal elements 1 and 2 (alpha BE1 and alpha BE2). Th ese new elements reside between -316 and -302 bp (alpha BE1) and -296 and -285 bp (alpha BE2) of the human a subunit promoter and bind disti nct proteins designated alpha BP1 and alpha BP2, respectively, Southwe stern blot analysis revealed that alpha BE1 specifically binds 54- and 56-kDa proteins, Additional studies disclosed several potential inter actions between proteins that bind the CRE and proteins that occupy PG BE, alpha BE1, and alpha BE2, suggesting that gonadotrope-specific exp ression occurs through a unique composite regulatory element that incl udes components of the placenta-specific enhancer.