MULTIPLE CHARACTERISTICS OF A PENTAMERIC REGULATORY ARRAY ENDOW THE HUMAN ALPHA-SUBUNIT GLYCOPROTEIN HORMONE PROMOTER WITH TROPHOBLAST SPECIFICITY AND MAXIMAL ACTIVITY

Trophoblast-specific expression of the human alpha-subunit glycoprotei n hormone gene requires a tightly linked array of five different regul atory elements [trophoblast-specific element (TSE), alpha-activating e lement (alpha ACT), a tandem cAMP response element (CRE), junctional r egulatory element (JRE), and a CCAAT box]. We examined their contextua l contributions to trophoblast-specific expression by using transfecti on assays to evaluate activity of systematic block replacement mutatio ns made within the 1500-bp 5'-flanking region of the human alpha-subun it gene. While all five elements were required for full activity, only the TSE and JRE displayed trophoblast specificity. Interestingly, the TSE-binding protein has limited tissue distribution whereas a JRE-bin ding protein appears trophoblast specific. Likewise, replacement studi es with an AP-1 element that binds heterodimers of jun and fos indicat ed that this element was incapable of compensating for either the tand em CRE or JRE. This preference for both CRE- and JRE-binding proteins provides another avenue for configuring an alpha-subunit promoter with trophoblast specificity. Additional analysis with a cAMP response ele ment binding protein (CREB)-Gal4 fusion protein further underscored th e importance of CREB as well as suggested that transcriptional contrib utions come from both the DNA-binding domain and transactivation domai n of this protein. We also examined the interactive nature of the pent americ array by placing a 15-bp random sequence between each element. Remarkably, only the insertion 3' of the CCAAT box diminished promoter activity. This suggested the absence of direct interactions between t he transcriptional factors that bind each element in the array. It als o suggested that the CCAAT box is position-dependent relative to the T ATA box. This position dependence appeared cell-specific, as it was no t manifest in a gonadotrope cell line (alpha T3-1 cells). Thus, the CC AAT box also has tissue-specific characteristics that assist in target ing expression of the alpha-subunit gene to trophoblasts. Together, th ese data suggest that multiple characteristics of a complex pentameric array of regulatory elements endow the alpha-subunit promoter with tr ophoblast specificity and maximal activity.