Abstract Maximal Activity of the LHB promoter requires Beta-catenin Travis Salisbury, April Binder, Jean Grammer & John H Nilson School of Molecular Biosciences, Center for Reproductive Biology, Washington State University, Pullman WA Human pathological conditions indicate that too little or too much luteinizing hormone (LH) causes infertility, inappropriate gonad development, and generalized endocrine dysfunction. These clinical studies emphasize a need to understand mechanisms that insure appropriate release of this reproductive hormone. In this regard, gonadotropin releasing hormone (GnRH) is a critical component of this intricate system that stimulates the release of LH from gonadotropes. While this requirement of GnRH is clear, how this hormone stimulates transcription of genes that encode LH remains a fundamental question. In this regard, prior reports have reported a requirement of EGR1 and SF1 for regulated gene expression in gonadotropes. Data presented herein suggest that the activity of these two transcription factors requires β-catenin, a cofactor of transcription. For instance, targeted reduction of β-catenin attenuates the activity of a GnRH-primed LHB promoter. Additional gene reporter assays indicate that overexpression of β-catenin, or its targeted reduction by siRNA, modulates the activity of both SF1 and EGR1. Beta-catenin binds to SF1 and an SF1 mutant that lacks a β-catenin binding domain has compromised transcriptional activity. Finally, GnRH promotes β-catenin co-localization with SF1 and EGR1 on the endogenous mouse Lhb promoter-regulatory region. Taken together our results have exposed a new role for β-catenin as cofactor for SF1 capable of insuring that sufficient amounts of LH are available for sustained pulsatile secretion, a requirement for fertility in both males and females. Neurohormonal control of transcription in gonadotropes Sustained secretion of LH exerts a transcriptional demand on the genes that encode its subunits. This demand is met by synergistic interactions between transcription factors. Sf1Orphan nuclear receptor: Steroidogenic factor 1 Immediate early gene: Early Growth Response Protein 1 Egr1 Pitx1 Paired like homeodomain transcription factor 1 p300 Cofactor of Transcription GnRH Egr1 Primary GnRH Response Gene p300 Secondary GnRH response gene Levels of Transcription Gse Egr1 Pitx1 GseEgr1 Pol ІІ A E F B Sf1 Egr1 Pitx1 Sf1 p300 Tertiary GnRH response gene Lhb Beta-catenin enhances activity of EGR1 and SF1 Transient co-transfection assays were performed in LβT2 cells with -779/+10 bovine LHB promoter-luciferase and phRG-B-renilla reporter constructs. The activity of these reporter constructs was assayed upon their co-transfection with expression vectors encoding either ∆ 90 β-catenin (100 ng), SF1 (20 ng), or EGR1 (10 ng). Data shown are the means ± SEM from three experiments performed in triplicate. Significant differences (P<0.01) are indicated between groups that do not share any letters in the superscript. siRNA against β-catenin reduces the activity of EGR1 and SF1. LβT2 cells were transiently co-transfected with the -779/+10 bovine LHB promoter- luciferase reporter vector and either 20 nM of siRNAc or 20 nM of siRNAβ with expression vectors encoding either SF1 or EGR1. Data shown are the means ± SEM of four experiments performed in triplicate. *, P<0.01 SF1 physically associates with β-catenin Hypothalamic control of Pituitary Hormone synthesis Hypothalamic neurons release pulses of GnRH GnrhrCga Lhb Fshb LH FSH LH Gonadotrope GnRH The β-catenin binding site in SF1 is required for functional synergism between SF1 and EGR1. Transient co-transfection assays were performed in LβT2 cells with -779/+10 bovine LHB promoter- luciferase and phRG-B-renilla reporter constructs. The activity of these reporter constructs was assayed upon their cotransfection with expression vectors encoding either SF1 (20 ng), SF A (50 ng), or EGR1 (10 ng). Luciferase activities for each data point were normalized against renilla activities. Data shown are the means ± SEM of three experiments. Significant differences (P<0.05) are indicated between groups that do not share any letters in the superscript. Nuclear extracts isolated from LβT2 cells treated with either vehicle (lanes 1-3) or 10 nM GnRH (lanes 4-6) for 60 minutes were immunoprecipitated with either anti- β-catenin antibodies or control rabbit serum. Precipitates were then subjected to immunoblot blot (IB) analysis with SF1 and β-catenin antibodies. Results are representative of three separate experiments. β-catenin binds to the endogenous Lhb promoter. Gonads Maximal transcriptional response of the LHB promoter to GnRH requires β-catenin. + (A) LβT2 cells were transiently co-transfected with - 779/+10 bovine LHB promoter-luciferase (200 ng) and phRG-B-renilla reporter constructs (10 ng) and an expression vector encoding AXIN (100 ng) and then treated with either GnRH (10 nM) or vehicle. Data shown are the means ± SEM of three experiments. *, P<0.01 (B) LβT2 cells were transiently transfected with -779/+10 bovine LHB promoter-luciferase vector and 100 nM of non- targeting control interfering RNA (siRNAc) or β- catenin interfering RNA (siRNAβ) and subsequently treated with either vehicle or GnRH (10 nM). Data shown are the means ± SEM of three experiments. *, P<0.01 (C) Transfection lysates from experiments in Fig. 2, panel B were probed by immunoblot analysis of β-catenin and AKT, the latter as a loading control. DBD LBD N’ C’ hinge Helix 1 AF Mouse SF-1 Beta-catenin is a new mediator of GnRH regulated transcription p300 N C N C Levels of Transcription Gse Egr1 Pitx1 GseEgr1 Pol ІІ A E F B Sf1 Egr1 Pitx1 Sf1 Tertiary GnRH response gene Lhb N C Beta catenin: cofactor of Transcription Is β-catenin central to a GnRH regulated gene network? Chromatin immunoprecipitation assays were performed with chromatin prepared from LβT2 cells prior to and post a GnRH stimulus (15 and 60 minutes) and immunoprecipitated with anti-β-catenin antibodies (lanes 1-6), anti-EGR1 antibodies (lanes 7-12), anti-SF1 antibodies (lanes 13-18), or control rabbit serum (lanes 1-18). Precipitated chromatin was amplified with primers that span response elements for EGR1, SF1 and PITX1 (proximal primer set) and with primers that span a distal fragment of Lhb promoters that lack sites for these transcription factors (distal primer set).