Upstream stimulatory factor, a basic-helix-loop-helix-zipper protein, regulates the activity of the alpha-glycoprotein hormone subunit gene in pituitary cells.
In an effort to determine whether basic-helix-loop-helix (bHLH) proteins are important in pituitary-specific expression of the alpha-glycoprotein hormone subunit gene, we examined the effect of the dominant negative HLH protein, Id, on the activity of the alpha-subunit gene promoter in pituitary cells. Id over-expression reduces the expression of alpha-subunit reporter genes in either alpha T3-1 gonadotrope-derived or alpha TSH thyrotrope-derived cells. A deletion fragment containing nucleotides from -131 to +44 of the human alpha-subunit promoter is inhibited to a similar degree as a -244 to +44 fragment in alpha T3-1 cells. Nuclear proteins in alpha T3-1 cells bind two potential bHLH protein binding sites (E-boxes, alpha EB1 and alpha EB2) present in this fragment but not to mutations that specifically alter only these sequences. An antibody-specific for upstream stimulatory factor, a widely expressed bHLH-leucine zipper protein, is able to inhibit factor binding to the alpha EB2 sequences but not the alpha EB1 site. Mutating the alpha 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 alone or in combination with the alpha EB1 mutation, reduces basal activity of the promoter to approximately 21% of control levels in alpha T3-1 cells and abolishes the decrease in promoter activity seen when Id is overexpressed. These results demonstrate that the bHLH family of proteins are important regulators of alpha-subunit gene expression in pituitary cells.
Jackson
S M
SM
Department of Medicine, University of Colorado Health Science Center, Denver 80262, USA.
Gutierrez-Hartmann
A
A
Hoeffler
J P
JP
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics*; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1; D051379:Mice; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins; D014157:Transcription Factors / Q000378:metabolism / Q000494:pharmacology*; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D015292:Glycoprotein Hormones, alpha Subunit; C497453:ID1 protein, human; C497455:Idb1 protein, mouse; D051798:Inhibitor of Differentiation Protein 1; D012097:Repressor Proteins; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; D004247:DNA