Abstract
Several cis-acting elements interact through their cognate trans-acting factors to confer placenta-specific expression to the alpha subunit gene of glycoprotein hormones. These elements include two tandemly arranged cAMP response elements (CREs, located between base pairs (bp) -146 and -111 relative to the transcription start site), an upstream regulatory element (URE, located between -180 and -150), and a CCAAT box (located between bp -100 and -80). Here, we identify a new regulatory region, junctional regulatory element (JRE), with critical nucleotides located between the CREs and CCAAT box (bp -120 to -100). Although the binding site of the JRE abuts the 3' CRE, methylation interference assays indicate that no overlap occurs between the contact points of the proteins that bind to the JRE and CRE. This new regulatory element is highly conserved across species and contains a palindromic binding site for a 50-kDa nuclear factor(s) which is distinct from the factors that bind the URE, CRE, and CCAAT box of the alpha subunit gene. Finally, gene transfer studies suggest that, although JRE binding activity is found in several cell types, this element acts relatively cell-specifically. We conclude that this element and its cognate trans-acting factor act in conjunction with the complexes formed over the URE, CREs, and CCAAT box to enhance the placenta-specific activity of the alpha subunit promoter.
Highlights
A C&-acting Element Located between the CAMP Response Elements and CCAAT Box Augments Cell-specific Expression of the G~ycoprotein Hormone cySubunit Gene*
We identify a new regulatory region, junctional reefulatory element (JRE), with critical nucleotides located between the CREs and CCAAT box
The binding site of the junctional regulatory element (JRE) abuts the 3’ CRE, methylation interference assays indicate that no overlap occurs between the contact points of the proteins that bind to the JRE and CRE
Summary
Construction of Vectors-Construction of the wild-type and mutant a subunit promoter vectors has been described previously (Kennedy et al, 1990) These vectors were constructed by ligating together a series of overlapping 20-bp oligonucleotides that span the region -170 to +44 of the Conditions of binding reactions and the gel system have been described previously (Kennedy et al, 1990). The binding reaction w&s subjected tb electrophoresis through 1% low melting agarose gels prior to UV irradiation as previously described (Kennedy et al, 1990). The oligonucleotides used were (only sequence of the coding strand is shown; linker sequences are shown in lowercase letters) 5’-ctagAAA These oligonucleotides were end labeled, partially methylated, and standard gel retardation assays were performed. Because activitv of all vectors was expressed relative to activity of the wild-type vector, this vector was included in all transcriptions to correct for variation in transcription efficiency
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