Transcription of the LH subunit genes is stimulated by GnRH and may be modulated physiologically by steroids such as 17β-estradiol (E). We found that E treatment amplified GnRH stimulation of the rat LHβ and α-subunit promoters, and expression of the endogenous mRNA, in LβT2 gonadotrope cells 2- to 5-fold above GnRH alone. We examined gene expression in LβT2 cells after E and/or GnRH treatment, and found that E suppressed expression of transcription factor Zfhx1a, and enhanced GnRH stimulation of Egr-1 mRNA and protein. E effects were abolished in the presence of antiestrogen. Egr-1 is critical for LHβ expression; however, the role of Zfhx1a, which binds to E-box sequences, was untested. We found E-box motifs in both the rat LHβ (−381, −182, and −15 bp) and α-subunit (−292, −64, −58 bp) promoters. Zfhx1a overexpression suppressed basal and GnRH-stimulated activity of both promoters. Mutation of the α-subunit promoter E boxes at either −64 or −58 bp eliminated Zfhx1a suppression, whereas mutation of the −292 bp E box had no effect. Gel shift assays demonstrated that Zfhx1a bound to the −64 and −58, but not −292, bp E-box DNA. Similarly, mutation of LHβ promoter E boxes at either −381 or −182, but not −15, bp reduced Zfhx1a suppression, correlating with binding of Zfhx1a. The −381 bp LHβ E box overlaps with an Sp1 binding site in the distal GnRH-stimulatory region, and increased Sp1 expression overcame Zfhx1a suppression. Thus, one mechanism by which E may enhance GnRH-stimulated LH subunit promoter activity is through regulation of both activators and suppressors of transcription.
Regulation of the Human Secretin Gene Is Controlled by the Combined Effects of CpG Methylation, Sp1/Sp3 Ratio, and the E-Box Element
