Divergent and composite gonadotropin-releasing hormone-responsive elementsin the rat luteinizing hormone subunit genes

GnRH pulses regulate gonadotropin subunit gene transcription in a frequency -dependent, subunit-specific manner. The a-subunit gene is stimulated by co nstant GnRH and by rapid to intermediate pulse frequencies, while stimulati on of LH beta subunit gene transcription requires intermediate frequency pu lses, We have defined the GnRH-responsive elements of the rat LH subunit ge ne promoters by deletion/mutation analysis and transfection studies in rat pituitary cells and two clonal gonadotrope cell lines, The alpha-subunit ge ne GnRH-responsive region lies between -411 and -375 bp. The region contain s two Ets-domain protein binding sites, and mutating either site obliterate s the response, DNA protein binding studies demonstrate the two sites are n ot equivalent, and that Ets-l does not mediate this response. Studies of th e LH beta promoter reveal a major GnRH-responsive region between -456 and - 342 bp, Within this region, two Spl binding sites contribute to the GnRH re sponse, 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 specific ally eliminates the response to pulsatile GnRH. DNA containing this mutatio n cannot form intermediate mobility complexes with nuclear proteins, but re tains Spl binding. Mutation of the 3'Sp1 site and either the 5'Sp1 or CArG element partially restores GnRH stimulation, suggesting a downstream elemen t contributes to the full GnRH response, These studies demonstrate that uni que composite elements and transcription factors are responsible for GnRH s timulation of the LH subunit genes and may contribute to their differential responses to GnRH pulses.