UPSTREAM STIMULATORY FACTOR, A BASIC-HELIX-LOOP-HELIX-ZIPPER PROTEIN,REGULATES THE ACTIVITY OF THE ALPHA-GLYCOPROTEIN HORMONE SUBUNIT GENEIN PITUITARY-CELLS

In an effort to determine whether basic-helix-loop-helix (bHLH) protei ns are important in pituitary-specific expression of the alpha-glycopr otein hormone subunit gene, we examined the effect of the dominant neg ative HLH protein, Id, on the activity of the alpha-subunit gene promo ter in pituitary cells. Id overexpression reduces the expression of al pha-subunit reporter genes in either alpha T3-1 gonadotrope-derived or alpha TSH thyrotrope-derived cells. A deletion fragment containing nu cleotides from -131 to +44 of the human alpha-subunit promoter is inhi bited to a similar degree as a -244 to +44 fragment in alpha T3-1 cell s. Nuclear proteins in alpha T3-1 cells bind two potential bHLH protei n binding sites (E-boxes, alpha EB1 and alpha EB2) present in this fra gment but not to mutations that specifically alter only these sequence s. An antibody-specific for upstream stimulatory factor, a widely expr essed bHLH-leucine zipper protein, is able to inhibit factor binding t o the alpha EB2 sequences but not the alpha EB1 site. Mutating the alp ha EB1 element of the alpha-subunit promoter decreases basal activity of this promoter to about 42% of control levels in alpha T3-1 cells. A mutation that abolishes upstream stimulatory factor binding, either a lone or in combination with the alpha EB1 mutation, reduces basal acti vity of the promoter to approximately 21% of control levels in alpha T 3-1 cells and abolishes the decrease in promoter activity seen when Id is overexpressed. These results demonstrate that the bHLH family of p roteins are important regulators of alpha-subunit gene expression in p ituitary cells.