Divergent and Composite Gonadotropin-Releasing Hormone-Responsive Elements in the Rat Luteinizing Hormone Subunit Genes

Abstract

GnRH pulses regulate gonadotropin subunit gene transcription in a frequency-dependent, subunit-specific manner. The alpha-subunit gene is stimulated by constant GnRH and by rapid to intermediate pulse frequencies, while stimulation of LHbeta subunit gene transcription requires intermediate frequency pulses. We have defined the GnRH-responsive elements of the rat LH subunit gene promoters by deletion/mutation analysis and transfection studies in rat pituitary cells and two clonal gonadotrope cell lines. The alpha-subunit gene GnRH-responsive region lies between -411 and -375 bp. The region contains two Ets-domain protein binding sites, and mutating either site obliterates the response. DNA protein binding studies demonstrate the two sites are not equivalent, and that Ets-1 does not mediate this response. Studies of the LHbeta promoter reveal a major GnRH-responsive region between -456 and -342 bp. Within this region, two Sp1 binding sites contribute to the GnRH response, and the 3'Sp1 site is also critical for basal expression. The 5'Sp1 site partially overlaps a CArG box, and mutating the CArG element specifically eliminates the response to pulsatile GnRH. DNA containing this mutation cannot form intermediate mobility complexes with nuclear proteins, but retains Sp1 binding. Mutation of the 3'Sp1 site and either the 5'Sp1 or CArG element partially restores GnRH stimulation, suggesting a downstream element contributes to the full GnRH response. These studies demonstrate that unique composite elements and transcription factors are responsible for GnRH stimulation of the LH subunit genes and may contribute to their differential responses to GnRH pulses.

Gonadotropin-releasing Hormone Receptor-coupled Gene Network Organization

“…For example, Klf4 is a zinc-finger protein in the Sp/XKLF family (19). Sp1 sites in the LH␤ promoter have been implicated in the response to pulsatile GnRH (20). Therefore, our data raise the possibility that Klf4 may bind to the LH␤ promoter and contribute to its GnRH-mediated activation.…”

FOXO1 Transcription Factor Inhibits Luteinizing Hormone β Gene Expression in Pituitary Gonadotrope Cells

“…11). GnRH signaling via protein kinase C and mitogen-activated protein kinase pathways (12)(13)(14)(15)(16) results in increased synthesis of EGR1 (17,18). EGR1 binds to the promoter and interacts in a synergistic manner with SP1, SF1, and PTX1 to up-regulate Lhb gene expression (19,20).…”

Embryonic Expression of the Luteinizing Hormone β Gene Appears to Be Coupled to the Transient Appearance of p8, a High Mobility Group-related Transcription Factor

“…In this case, however, the elements consist of those that bind early growth response 1 (Egr1) (11)(12)(13)(14)(15)(16), steroidogenic factor 1 (SF-1) (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), Pitx1 (21)(22)(23), and an as yet unidentified Otx-related homeodomain protein (24), as well as proteins that bind elements in the distal domain, such as nuclear factor-Y (NF-Y) and Sp1 (16,(25)(26)(27). Unlike ␣GSU, which is a marker of early pituitary development, expression of the LH␤ gene is one of the last steps that defines a mature gonadotrope.…”