Identification of Hypothalamic Arcuate Nucleus-Specific Enhancer Region of Kiss1 Gene in Mice

Abstract

Pulsatile secretion of GnRH plays a pivotal role in follicular development via stimulating tonic gonadotropin secretion in mammals. Kisspeptin neurons, located in the arcuate nucleus (ARC), are considered to be an intrinsic source of the GnRH pulse generator. The present study aimed to determine ARC-specific enhancer(s) of the Kiss1 gene by an in vivo reporter assay. Three green fluorescent protein (GFP) reporter constructs (long, medium length, and short) were generated by insertion of GFP cDNA at the Kiss1 locus. Transgenic female mice bearing the long and medium-length constructs showed apparent GFP signals in kisspeptin-immunoreactive cells in both the ARC and anteroventral periventricular nucleus, in which another population of kisspeptin neurons are located. On the other hand, transgenic mice bearing 5'-truncated short construct showed few GFP signals in the ARC kisspeptin-immunoreactive cells, whereas they showed colocalization of GFP- and kisspeptin-immunoreactivities in the anteroventral periventricular nucleus. In addition, chromatin immunoprecipitation and chromosome conformation capture assays revealed recruitment of unoccupied estrogen receptor-α in the 5'-upstream region and intricate chromatin loop formation between the 5'-upstream and promoter regions of Kiss1 locus in the ARC. Taken together, the present results indicate that 5'-upstream region of Kiss1 locus plays a critical role in Kiss1 gene expression in an ARC-specific manner and that the recruitment of estrogen receptor-α and formation of a chromatin loop between the Kiss1 promoter and the 5' enhancer region may be required for the induction of ARC-specific Kiss1 gene expression. These results suggest that the 5'-upstream region of Kiss1 locus functions as an enhancer for ARC Kiss1 gene expression in mice.

The roles of kisspeptin revisited: inside and outside the hypothalamus

“…The bidirectional regulation of Kiss1 expression by estrogen might be mediated by ERα, because estrogen changes AVPV or ARC Kiss1 expression in ovariectomized ERβ knockout mice, but not in ERα knockout mice [60]. Recent advances in epigenetic research for the regulation of Kiss1 expression [68, 69] provide a clue as to how estrogen regulates Kiss1 expression in these two hypothalamic regions in a different manner. In the AVPV, estrogen-ERα complexes, which are highly recruited at the Kiss1 promoter, are likely involved in the histone acetylation of the Kiss1 promoter and the subsequent Kiss1 expression by a chromatin loop formation between the Kiss1 promoter and the 3′-downstream enhancer region [68].…”

“…In the AVPV, estrogen-ERα complexes, which are highly recruited at the Kiss1 promoter, are likely involved in the histone acetylation of the Kiss1 promoter and the subsequent Kiss1 expression by a chromatin loop formation between the Kiss1 promoter and the 3′-downstream enhancer region [68]. In the ARC, histone acetylation of the Kiss1 promoter and a chromatin loop formation between the Kiss1 promoter and the 5′-upstream enhancer region seems to be involved in Kiss1 expression in the absence of estrogen [68, 69]. Taken together, histone acetylation of the Kiss1 promoter is positively correlated with Kiss1 expression in both hypothalamic nuclei, and region-specific enhancers serve as switches for the bidirectional regulation of Kiss1 expression (Fig.…”

Molecular and Epigenetic Mechanism Regulating Hypothalamic <b><i>Kiss1</i></b> Gene Expression in Mammals

“…Goto et al [14] found ARC-specific chromatin loop formation between the promoter region and the 5′ upstream region of the Kiss1 gene, suggesting that the 5′ upstream region of the Kiss1 gene plays a key role in upregulating Kiss1 gene expression as an enhancer in the ARC. This study also showed a binding of unoccupied ERα and putative transcriptional factor binding sites in the ARC-specific putative enhancer region of the Kiss1 locus.…”

“…The assay revealed that the 3′ downstream region of the Kiss1 locus contributes to induction of estrogen-induced AVPV Kiss1 gene expression [11], while the 5′ upstream region of the Kiss1 locus is conducive to ARC Kiss1 gene expression [14]. More specifically, chromatin loop formation between the Kiss1 promoter region and the 3′ downstream region in the AVPV or the 5′ upstream region in the ARC, respectively, may act as a switch, which could turn on Kiss1 gene expression.…”

“…The epigenetic regulation of the Gnrh1 gene encoding gonadotropin-releasing hormone (GnRH) expression was reviewed elsewhere by Kurian and Terasawa [10], who demonstrated an inverse relationship between DNA methylation and Gnrh1 gene expression during the peripubertal period. Likewise, understanding of the epigenetic regulation of the Kiss1 gene encoding kisspeptin (initially named metastin) expression has become increasingly clear [11,12,13,14]. …”