Abstract
Transcription of the luteinizing hormone receptor gene is dependent on Sp1-induced promoter activation from two Sp1 binding domains (Sp1(2), and Sp1(4)) within the 173-base pair promoter. Of the two Sp1 binding domains, the canonical GC box (GGGCGG) was determined by mutation to be the binding element for only the Sp1(2) domain. The Sp1 binding element within the Sp1(4) domain was identified by mutation and immunological/competition studies as the 5'-GGG GTG GGG that conforms to a Zif-268 like three zinc finger binding domain, rather than the canonical 3' Sp1(4) GC box (GGGCGG). The guanines in the third trinucleotide (GGG GTG GGG) were not required for Sp1 binding, although they increased binding affinity. Non-Sp1 protein(s) bind the 3' Sp1(4) GC box, and by themselves exhibit transcriptional activity. Tissue specific differences were localized to this non-Sp1 binding domain, which functionally substituted for the downstream activating M1 regulatory domain in non-expressing but not in expressing cells. Mutations of both non-Sp1 and M1 domains were required for inhibition of promoter activity in constructs that retained the Sp1 binding elements in non-expressing cells, indicating that together these domains may play a role in regulation of luteinizing hormone receptor gene expression.
Highlights
The structure of the isolated LHR promoter domain that elicits constitutive promoter activity in both cell types has been characterized previously as a TATA-less region that carries multiple GC boxes [2]
The Sp l binding element in SpI2 was previously localized through mutation to the canonical GC box (GGGCGG) that conforms to those identified in the promoter regions of GC-rich housekeeping genes [5]
Identification ofthe Sp I Binding Element in Spi..-Previous mutational studies of the Sp1 2 domain have shown that the canonical GC box (GGGCGG) within Sp1 2 binds a nuclear factor that has the binding and immunological properties of the Spl protein [5]
Summary
The structure of the isolated LHR promoter domain that elicits constitutive promoter activity in both cell types has been characterized previously as a TATA-less region that carries multiple GC boxes [2]. Two potential Sp l binding domains, SpI2 ( -73 to -94 bp), and Sp l , (-135 to -154 bp), each contain a single GC box that competes for trans-binding factors with standard Spl elements. These LHR Spi domains were identified by deletion t To whom correspondence should be addressed. The 20-nuc1eotide domain that was determined to be responsible for this increase was defined as SpI4 and contained a canonical GC box These studies indicated that unlike the SpI2 domain, mutation of the entire 20-nucleotide G-rich Sp l , domain was necessary to abolish competition for Spl trans-factors, and SpI4-induced promoter activity [5]. Tandem Sp l/non-Sp l binding elements were identified on the Sp l , domain, and each element induced transcript activation in a differential expressing versus non-expressing cell mode of action
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