GnRH 유전자의 시공간적 조절기작에 대한 연구

Abstract

Gonadotropin-releasing hormone (GnRH) is a pivotal hypothalamic neurohormone governing reproduction and sexual development. Extensive studies thus for have been undertaken to characterize GnRH neuron-specific enhancers in distal promoter regions of the mammalian GnRH genes since immortalized GnRH neuronal cell lines such as GT1 and Gn11 became available. However, these previous works still cannot fully account for spatio-temporal regulation of GnRH gene expression taking place in a highly restricted population of hypothalamic neurons. There are only few of trans-acting factors and their binding cis-elements on the GnRH promoter are characterized, suggesting that as yet undiscovered transcription factors and their evoked mechanisms may underlie the cell type specificity of GnRH gene expression. In terms of temporal regulation, GnRH is known to release in a pulsatile manner, however no evidence of whether oscillatory secretion is related to transcriptional regulation of GnRH is available thus far. In this regard, I examined the spatio-temporal regulation of GnRH gene expression. In Chapter I, I aimed to elucidate the novel transcriptional regulation mechanisms of neuron-specific GnRH gene expression. Up to now, a region approximately 3.0 kb upstream of the mammalian GnRH promoter has been extensive studied, but these results can not fully account for the GnRH neuron specificity. Here I demonstrated a transcription-enhancer located in the first intron (intron A) region of the GnRH gene. This transcriptional enhancer harbors putative Sry-related high-mobility-group box (SOX) family transcription factor-binding sites, which are well-conserved across mammalian species. The class-C SOX member proteins (SOX-C; SOX4 and SOX11) specifically augmented this transcriptional activation by binding to these SOX-binding sites. Accordingly, SOX11 was highly enriched in immortalized GnRH-producing GT1-1 cells, and suppression of its expression significantly decreases GnRH gene expression and subsequent GnRH secretion. Chromatin immunoprecipitation (ChIP) revealed that endogenous SOX-C factors recognize and bind to the intronic enhancer in GT1-1 cells and the hypothalamic tissue. Immunohistochemical analysis showed that SOX4 or SOX11 are highly expressed in the majority of hypothalamic GnRH neurons in adult mice. Consequently, these findings suggest that SOX-C transcription factors function as important transcriptional regulators of cell type-specific GnRH gene expression by acting on the intronic transcriptional enhancer. In Chapter II, I aims to examine whether the chromatin remodeling mechanism are involved in the ultradian rhythmicity of GnRH transcription. To determine whether the GnRH gene expression show rhythmic oscillations at the level of gene transcription, GnRH primary transcript levels were measured in GT1 cells after a serum treatment for synchronization. Primary transcript levels of GT1 cells exhibited rhythmic oscillation in an hour interval and this rhythm was not abolished by inhibition of translation. But the oscillation was profoundly disrupted by the changes of histone acetylation and methylation states. ChIP assay revealed that histone acetylation and methylation also showed ultradian kinetics in the GnRH genomic regions. Furthermore, histone methylations in the transcription start site of GnRH gene showed antiphasic oscillations between transcriptional active (H3K4-Me3) and repressive (H3K9-Me2) histone markers. This novel mechanism may underlie the ultradian regulation of GnRH gene transcription.