Proteasome regulation of dynamic transcription factor occupancy on the GnRH-stimulated luteinizing hormone beta-subunit promoter.
GnRH is the main modulator of LH secretion and transcription of the LH subunit genes in pituitary gonadotropes. The LHbeta 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 LHbeta transcription is also influenced by the ubiquitin-proteasome system. GnRH-stimulated activity of an LHbeta reporter gene was prevented by proteasome inhibitors MG-132 and lactacystin. Inhibition was not rescued by overexpression of two key transcription factors for LHbeta, early growth response-1 (Egr-1) and steroidogenic factor-1 (SF-1). Increased endogenous LHbeta transcription after GnRH treatment was also prevented by MG-132, as measured by primary transcript assays. To investigate possible mechanisms of LHbeta transcriptional inhibition by proteasome blockade, we employed chromatin immunoprecipitation to measure LHbeta 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 LHbeta 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 LHbeta promoter, suggesting their degradation may be key for LHbeta proteasome-dependent transcription. Together, these results demonstrate that degradation via the proteasome is vital to GnRH-stimulated LHbeta expression, and this occurs in part by allowing proper transcription factor associations with the LHbeta promoter.
Walsh
Heidi E
HE
University of Virginia, Charlottesville, Virginia 22908, USA.
Shupnik
Margaret A
MA
D000818:Animals; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D007976:Leupeptins / Q000378:metabolism; D037101:Luteinizing Hormone, beta Subunit / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011401:Promoter Regions, Genetic; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D054339:Steroidogenic Factor 1 / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D051766:Early Growth Response Protein 1; D007976:Leupeptins; D037101:Luteinizing Hormone, beta Subunit; D061988:Proteasome Inhibitors; D021122:Protein Subunits; D034741:RNA, Small Interfering; D054339:Steroidogenic Factor 1; D025801:Ubiquitin; D007987:Gonadotropin-Releasing Hormone; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde