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Proteasome Regulation of Dynamic Transcription Factor Occupancy on the GnRH-Stimulated Luteinizing Hormone β-Subunit Promoter

Neuroscience Graduate Program (H.E.W.) and Department of Internal Medicine (M.A.S.), Division of Endocrinology and Metabolism, University of Virginia Medical School, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Margaret A. Shupnik, Ph.D., Box 800578 HSC, University of Virginia, Charlottesville, Virginia 22908. E-mail: mas3x{at}virginia.edu.

GnRH is the main modulator of LH secretion and transcription of the LH subunit genes in pituitary gonadotropes. The LHβ gene is preferentially transcribed during pulsatile GnRH stimuli of one pulse/30 min and is thus carefully controlled by specific signaling pathways and transcription factors. We now show that GnRH-stimulated LHβ transcription is also influenced by the ubiquitin-proteasome system. GnRH-stimulated activity of an LHβ reporter gene was prevented by proteasome inhibitors MG-132 and lactacystin. Inhibition was not rescued by overexpression of two key transcription factors for LHβ, early growth response-1 (Egr-1) and steroidogenic factor-1 (SF-1). Increased endogenous LHβ transcription after GnRH treatment was also prevented by MG-132, as measured by primary transcript assays. To investigate possible mechanisms of LHβ transcriptional inhibition by proteasome blockade, we employed chromatin immunoprecipitation to measure LHβ promoter occupancy by transcription factors. Without GnRH, binding was low and unorganized. With GnRH, Egr-1 and SF-1 associations were stimulated, cyclic, and coincidental, with a period of approximately 30 min. MG-132 disrupted GnRH-induced Egr-1 and SF-1 binding and prevented phosphorylated RNA polymerase II association with the LHβ promoter. Egr-1, but not SF-1, protein was induced by GnRH and accumulated with MG-132. Egr-1 and SF-1 were ubiquitinated in gonadotropes and ubiquitinated forms of these factors associated with the LHβ promoter, suggesting their degradation may be key for LHβ proteasome-dependent transcription. Together, these results demonstrate that degradation via the proteasome is vital to GnRH-stimulated LHβ expression, and this occurs in part by allowing proper transcription factor associations with the LHβ promoter.