PARENT SESSION
NEUROENDOCRINOLOGY

Monday, August 2, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(119) MUTATION OF GATA BINDING SITES IN THE GNRH PROMOTER ALTERS PULSATILE GNRH GENE EXPRESSION.

Leclerc, Gilles¹, Boockfor, Fredric¹, ¹ Medical University of South Carolina, Charleston, SC, USA

ABSTRACT- Release of GnRH from the hypothalamus in pulses is necessary for proper reproductive function. Despite its importance, very little information is available on the cellular nature of this pulsatile phenomenon. Recent evidence obtained with GT1-7 clonal GnRH neurons indicates that a region of the GnRH promoter, termed the neuron specific enhancer (NSE), is important in generating GnRH gene expression pulses, especially the AT-a site that binds Oct-1. The purpose of the following study was to determine whether GATA-binding sites (GATA-A, position -1734 to -1753 and GATA-B, position -1704 to -1729) also located in this NSE region, are needed for pulsatile gene expression. Using the QuikChange site-directed mutagenesis kit, we mutated the core sequence "TGATAG" of GATA-A to "TCCATG" and GATA-B to "CTCGAG" of the GnRH promoter (-3026/+116). These mutated promoters were each fused to the luciferase gene reporter, and the resulting constructs microinjected into GT1-7 neurons. Photonic activity was monitored over time in individual cells and the episodic profiles determined. Initial analyses revealed that episodes of photonic activity (normally observed when unmodified promoter was used) were undetected in cells injected with plasmid containing the GATA-A mutation (n=12). In addition, cells containing the GATA-B mutated promoter construct exhibited a reduction in frequency of episodes of luciferase activity (0.10 pulses/hr vs. 0.23 pulses/hr, mutated vs. unmodified promoter), but this effect was not as marked as that observed for the GATA-A mutant. Taken together, it appears that GATA binding sites in the NSE portion of the GnRH promoter play an important role in the generation of GnRH gene expression pulses. These findings, when viewed in light of the importance of Oct-1 in episodic activity, reveal that multiple regulatory elements are necessary for pulse generation and suggest that a complex interaction of these elements must occur to yield a specific temporal event such as a pulse. (Supported by the NIH Grant HD-37657 to F.R.B.).

KEY WORDS: Neuron-specific enhancer, GATA, Gonadotropin-Releasing Hormone (GnRH), Gene expression