An Enhancer-Derived Noncoding RNA in GnRH Gene Regulation

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 389-413-Signaling and Transcriptional Control in Endocrine Systems
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-389
Polly P Huang*, Anita K Iyer and Pamela L Mellon*
University of California San Diego, La Jolla, CA
Gonadotropin-releasing hormone (GnRH) is the central mediator of the hypothalamic-pituitary-gonadal axis and is essential for normal reproductive function. Pulsatile GnRH is released from a small population of neurons in the hypothalamus. Conserved regulatory elements in the mouse, rat, and human GnRH gene include three enhancers and proximal promoter, which confer specific expression of GnRH in GnRH neurons. We used two immortalized mouse neuronal cell lines for studying the molecular aspects of GnRH regulation: 1) hypothalamic mature GnRH (GT1-7) neurons developed using a transgene carrying -3 kb regulatory region of the rat GnRH gene, and 2) immature GnRH (GN11) neurons carrying ~1 kb of the human GnRH promoter with undetectable GnRH secretion. Our recent studies identified transcription of a novel noncoding RNA at enhancer 1 in GT1-7 neurons and mouse hypothalamus, but its structure and function remained undefined. First, using rapid amplification of cDNA ends (RACE), we determined that the GnRH enhancer-derived long noncoding RNA (GnRH E1RNA) is a 5’-capped, polyadenylated RNA with alternative 5’ start and 3’ termination sites. Noncoding RNA molecules are identified upstream of the mouse GnRH transcription start site, transcribed in both sense and antisense direction. Rat GnRH E1RNA (rE1RNA) is transcribed from the rat transgene contained in GT1-7 neurons and from a transiently transfected reporter plasmid containing the rat GnRH regulatory region only in GT1-7 cells. RT-PCR and RNA sequencing data showed that GnRH E1RNA expression correlates with GnRH mRNA expression in GT1-7 neurons, but is absent in GN11 neurons and NIH3T3 fibroblasts. Second, to investigate the effect of GnRH E1RNA overexpression in GT1-7 and GN11 cells, we transiently co-transfected expression vectors expressing either mouse GnRH E1RNA (mE1RNA) or rE1RNA with reporter constructs carrying the upstream regulatory region of the rat GnRH gene. Overexpression of either mouse or rat GnRH E1RNA in GT1-7 neurons down-regulates activity of regulatory elements, while overexpression in GN11 cells showed a modest up-regulation of activity. These effects require the combination of enhancer 3 or enhancer 2 with enhancer 1. Finally, siRNA knockdown of the mE1RNA in GT1-7 neurons showed a modest decrease in GnRH mRNA expression measured using RT-qPCR. Together, the data demonstrate that GnRH E1RNA is expressed in GnRH neurons and implicate GnRH E1RNA in maintaining proper GnRH gene regulation.

Nothing to Disclose: PPH, AKI, PLM

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Sources of Research Support: NIH R01 HD020377; U54 HD012303; R01 DK044838; R01 HD072754; TF32 HD058427; T32 DK007494; T32 DK007541
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