1. Post-transcriptional regulation of Egr1 in the gonadotrope is influenced by CSDA Theodore R. Chauvin & John H. Nilson School of Molecular Biosciences, Washington State University, Pullman, WA, USA; Center for Reproductive Biology, Pullman WA, USA The gonadotropin Luteinizing Hormone (LH) is essential for reproductive function in mammals. LH is a heterodimeric glycoprotein secreted from the pituitary and is composed of two subunits: the common alpha subunit (α- GSU) and the unique beta subunit (LHβ). The genes that encode these subunits are tightly regulated by inputs from the hypothalamic-pituitary- gonadal axis. GnRH from the hypothalamus acts upon cells in the pituitary called gonadotropes to stimulate the expression of Lhb This stimulation is dependent upon proper induction and accumulation of the transcription factor Early growth response gene 1 (Egr1) by GnRH. The controlled expression and regulation of Egr1 is critical for LH production. Using the gonadotrope-derived L  T2 cell line, we have uncovered that a gene, Cold shock domain protein A (Csda) may be important for regulating Egr1 mRNA stability. When endogenous Csda expression is lowered by siRNA techniques there is a reduction in the amount of Egr1 and Lhb mRNA after treatment with GnRH. We hypothesize that CSDA may be acting through the untranslated region (UTR) of Egr1 to regulate its stability. Using a luciferase reporter vector, which contains different elements of the Egr1 3’- UTR, we have started to investigate the mechanisms that control Egr1 stability in L  T2 cells along with how CSDA may contribute to this stability. These studies will allow us to better understand the mechanisms of mRNA stability and to further our knowledge of how Egr1 and Lhb are controlled. Abstract Egr1 is essential for fertility Early Growth response protein 1 (Egr1) is a zinc-finger phosphoprotein which binds DNA in GC-rich sequences on the promoters of target genes. Egr1 is considered to be an immediate early response gene Egr1 is induced by GnRH in gonadotropes Egr1 is necessary for Lhb expression and essential for fertility GnRH regulates the transcription of Lhb and Egr1 Pulses of GnRH control expression of Egr1 and Lhb Proper expression of Lhb depends upon multiple transcription factors including SF1, EGR1, and PITX1 The limiting transcription factor appears to be Egr1 which is induced by GnRH Hpg Axis Hypothalamus – releases GnRH Pituitary – composed of 5 cell types Gonadotropes – secrete LH thyrotropes – secrete TRH Lactotropes – secrete Prolactin Somatotropes – secrete GH Corticotropes – secrete ACTH Pulses of GnRH from the hypothalamus act upon gonadotrope cells in the pituitary control expression of and release of LH LH acts on the testis and ovary and is necessary for reproduction Gonadotropes GnRH Hypothalamus Pituitary Ovary Testis LH Cold Shock Domain protein A (aka MSY 3/4) Binds RNA and DNA Contains a highly conserved 70 amino acid sequence (CSD) Binds UCCAUCA Csd elements Highly expressed in testes - controls protamine mRNA stability Controls Vegf mRNA stability Csda S2 L  T2 Northern blot CSDA is an RNA binding protein expressed in L  T2 cells Conclusions GnRH appears to have no effect on the half life of Egr1 mRNA The 3’ UTR of Egr1 enhances luciferase activity in transfected L  T2 cells The 3’ Csd sequence in the proximal region of the Egr1 UTR appears to modulate luciferase activity in transfected L  T2 cells Reduction of CSDA by siRNA can also modulate activity of luciferase in transfected L  T2 cells Reduction of CSDA by siRNA reduced GnRH stimulation of both a transfected LHB-luciferase construct and endogenous Lhb Egr1 mRNA Polypyrimidine consensus polyadenylation signal AU rich regions and U rich regions Csd consensus Egr1 mRNA (NCBI RefSeq NM_007913) 3111 1896 1295 5’ UTR 3’ UTR coding region The 3’ untranslated region (UTR) of Egr1 is large and contains many elements that have the potential to regulate mRNA stability The 3’ end of Egr1 enhances luciferase activity 5.630.2 ± 2.555.4 ± 0.46Egr1 3’ distal UTR 6.837.0 ± 2.255.3 ± 0.32Egr1 3’ proximal UTR 9.117.5 ± 0.951.9 ± 0.02Egr1 3’ UTR 6.76.7 ± 1.521.0 ± 0.07pGL3 Fold Change+ GnRH-GnRH Relative activity in L  T2 cells Egr1 3’ UTR luciferaseSV40 Egr1 3’ distal luciferaseSV40 luciferaseSV40 Egr1 3’ proximal luciferaseSV40 pGL3 The effect of GnRH on Egr1 mRNA half-life L  T2 cells were treated with 10 nM GnRH Time (hours): 0 0.5 1, 2, 3, 4, 5, 6, 8, 12 AMD AMD + 100nM GnRH S2 Egr1 AMD + Vehicle AMD + GnRH Hours 0 1 2 3 4 5 6 8 12 1 2 3 4 5 6 8 12 AMD + GnRH y = 1.72 -.196x AMD + vehicle y = 1.99 -.225x Time (hours) Relative Egr1 expression 02468101214 0.1 1 10 100 Studying gonadotropes using a cell line G S/L C Tr M e17.5 LβT2 LβT2 cells Represent early developing gonadotropes Derived from a mouse pituitary at embryonic day 17.5 Express all necessary transcription factors for Lhb expression Can express Lhb Due to the complexity of the pituitary gland studying primary gonadotropes is very difficult To study gonadotropes we utilize a cell line that has been developed The 3’ Csd consensus and CSDA protein modulate luciferase activity Egr1 3’ UTR luciferaseSV40 luciferaseSV40 Egr1 3’ proximal luciferaseSV40 pGL3 Egr1 3’  proximal luciferaseSV40 384.71 ± 1.96Csda 7X7.67 ± 2.55Control Egr1  proximal 3’ 726.39 ± 1.36Csda 23X23.10 ± 7.19Control Egr1 proximal 3’ 390.69 ± 0.18Csda 1X1.14 ± 0.14Control pGL3 RNAi % Loss UTR fold- change Relative Activity RNAi The proximal Csd sequence was mutated and used in transient transfections along with and 100 nM of non-targeting control interfering RNA (Control RNAi) or Csda interfering RNA (Csda RNAi) Csda siRNA reduces GnRH stimulation of LHB-luciferase 0 2 4 6 8 10 12 14 16 Con siRNA + GnRH Csda siRNA + GnRH Fold change LβT2 cells were transiently transfected with -779/+10 bovine LHB promoter-luciferase vector and 100 nM of non-targeting control interfering RNA (Con siRNA) or Csda interfering RNA (Csda siRNA) and subsequently treated with either vehicle or GnRH (100 nM). Data shown are the means ± SEM of three experiments. Csda siRNA reduces GnRH stimulation of endogenous Lhb Lhb S2 Time 0 1 2 4 8 24 0 1 2 4 8 24 0 1 2 4 8 24 Sham Control siRNA Csda siRNA 0 1 2 3 4 5 6 0240 0 Sham Control siRNA Csda siRNA GnRH - + - + - + (100nM) Fold increase (Lhb/S2) LβT2 cells were transiently sham transfected or transfected with 100 nM of non-targeting control interfering RNA (Con siRNA) or Csda interfering RNA (Csda siRNA) and subsequently treated with either vehicle or GnRH (100 nM). RNA was isolated and Northern blot analysis was performed. RNA was isolated and Northern blot analysis performed The 3’ UTR of Egr1 was inserted into a luciferase reporter vector and used in transient transfection assays in order to identify regions of the UTR which may be important for mRNA stability