Abstract
Placenta-specific expression of the gene encoding the alpha subunit of glycoprotein hormones involves the interaction of at least two different cis-acting elements: the upstream regulatory element (URE), which binds a placenta-specific trans-acting factor; and two tandem cAMP-response elements (CREs), which bind a ubiquitous protein (CRE-binding protein). To identify additional elements required for promoter activity, we used block replacement mutagenesis to produce a series of 10-base pair transversion mutations throughout the proximal promoter-regulatory region of the human alpha subunit gene. Transient expression of these constructs in choriocarcinoma cells enabled the identification of several closely spaced transcriptional control elements in addition to the previously identified URE and CREs. Gel mobility shift assays, methylation interference analyses, and UV cross-linking permitted identification of a factor that binds specifically to a canonical CCAAT box contained within the proximal promoter-regulatory of the human alpha subunit gene. This factor, which we refer to as alpha subunit CCAAT-binding factor (alpha CBF), is found in a variety of cell types and appears to be distinct from the previously characterized CCAAT-binding factors CTF/NF1, C/EBP, CP1, and NF-Y. We suggest that interaction of alpha CBF with factors binding to the URE and CREs may be required for maximal activity of the alpha subunit promoter in placental cells.
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