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 glycoprotein hormone gene requires a tightly linked array of five different regulatory elements [trophoblast-specific element (TSE), alpha-activating element (alphaACT), a tandem cAMP response element (CRE), junctional regulatory element (JRE), and a CCAAT box]. We examined their contextual contributions to trophoblast-specific expression by using transfection assays to evaluate activity of systematic block replacement mutations made within the 1500-bp 5'-flanking region of the human alpha-subunit 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-binding protein appears trophoblast specific. Likewise, replacement studies with an AP-1 element that binds heterodimers of jun and fos indicated that this element was incapable of compensating for either the tandem 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 element binding protein (CREB)-Gal4 fusion protein further underscored the importance of CREB as well as suggested that transcriptional contributions come from both the DNA-binding domain and transactivation domain of this protein. We also examined the interactive nature of the pentameric 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 the transcriptional factors that bind each element in the array. It also suggested that the CCAAT box is position-dependent relative to the TATA box. This position dependence appeared cell-specific, as it was not manifest in a gonadotrope cell line (alphaT3-1 cells). Thus, the CCAAT box also has tissue-specific characteristics that assist in targeting expression of the alpha-subunit gene to trophoblasts. Together, these data suggest that multiple characteristics of a complex pentameric array of regulatory elements endow the alpha-subunit promoter with trophoblast specificity and maximal activity.
Budworth
P R
PR
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Quinn
P G
PG
Nilson
J H
JH
D017362:Cyclic AMP Response Element-Binding Protein / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D018507:Gene Expression Regulation, Developmental; D015972:Gene Expression Regulation, Neoplastic; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics*; D006801:Humans; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009928:Organ Specificity; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship; D016385:TATA Box; D018808:Transcription Factor AP-1 / Q000378:metabolism / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism* / Q000494:pharmacology; D015533:Transcriptional Activation; D014327:Trophoblasts / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D015292:Glycoprotein Hormones, alpha Subunit; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors