FP04-3 Dax1 Functions As a Co-Activator of Lh▀ Transcription

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: FP04-GnRH & Gonadotroph Biology & Signaling
Saturday, June 15, 2013: 11:00 AM-11:30 AM
Presentation Start Time: 11:10 AM
Room 135 (Moscone Center)

Poster Board SAT-139
Debalina Bagchi*1, Carlos Jesus Santos2, Josefa Andrade3 and Margaret A Shupnik4
1Univ of Virginia, Charlottesville, VA, 2University of Puerto Rico, Cayey, PR, 3University of Virginia, Charlottesville, VA, 4Univ of VA Schl of Med, Charlottesville, VA
Luteinizing hormone (LH) is required for ovulation and steroidogenesis, and dysregulation can lead to infertility. Understanding LH transcriptional regulation may be beneficial in developing future therapies. LH consists of a shared α subunit with FSH and a unique β subunit. LHβ transcription is tightly regulated by GnRH, resulting in cyclic, co-ordinated binding of transcription factors EGR1 and SF1 on the promoter.  Co-activators or co-repressors are associated with Egr1 and SF1 on the promoter and ultimately regulate  transcription. One such cofactor is DAX1, and mutations in the gene and protein result in congenital adrenal hypoplasia and hypogonadism. DAX1 can physically interact with SF1, and previous studies depicted DAX1 as a repressor of SF1 target genes. However, a recent study showed that DAX1 acts as a co-activator for the SF1 target gene Mc2R in adrenal cells1. We examined the actions of DAX1 on the LHβ gene in LβT2 gonadotrope cells.  In chromatin immunoprecipitation assays, DAX1 associated with the LHβ promoter and GnRH stimulated association up to 7-fold; both the timing of binding and stimulation by GnRH are consistent with DAX1 acting as a co-activator. To investigate this possibility, we measured LHβ promoter activity and found DAX1 overexpression increased basal (2-fold) and GnRH-stimulated promoter activity up to 7-fold over GnRH alone. Decreasing expression of endogenous DAX1 by siRNA decreased GnRH-stimulated endogenous LHβ primary transcript mRNA. To understand how DAX1 functions to activate LHβ, we assessed DAX1 and GnRH stimulation of promoter constructs with distal and proximal GnRH response regions (-617 to +44bp) or only the proximal region (-245 to +44bp); both were stimulated by DAX1 plus GnRH over GnRH alone. The proximal region contains two SF1 binding sites; only mutation of the 3’SF1 site eliminated DAX1 stimulation. We previously showed that stimulation of LHβ transcription by the cAMP-PKA pathway also requires the 3’SF1 site2. Thus, we tested if DAX1 enhanced the transcriptional response to Forskolin, a PKA activator, and found DAX1 enhanced Forskolin induction of LHβ  2-fold. Our data indicate that DAX1 acts as a co-activator of GnRH-stimulated LHβ gene transcription, and that the proximal region of the promoter and the 3’SF1 site are critical for this activation. Overall, the data suggest that DAX1 may play a regulatory role in modulating transcription in the mature gonadotrope, in addition to its role in development.

(1) Xu B, Yang WH, Gerin I, Hu CD, Hammer GD, Koenig RJ. Mol Cell Biol. 2009 Apr;29(7):1719-34 (2) Ferris HA, Walsh HE, Stevens J, Fallest PC, Shupnik MA. Biol Reprod. 2007 Dec;77(6):1073-80.

Nothing to Disclose: DB, CJS, JA, MAS

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: This work was supported by the Eunice Kennedy Shriver National Institute of Child Health Development/National Institute of Health though cooperative agreement (UB54 HD28934 project to MAS) as a part of Specialized Cooperative Centers Program in Reproduction and Infertility Research.