The hypothalamic Kiss1 neurons control gonadotropin-releasing hormone (GnRH) release through the secretion of kisspeptins. Kiss1 neurons serve as a nodal center to convey regulatory cues that determine the attainment and maintenance of reproductive function. Despite this critical role, little is known regarding the mechanisms that ultimately impinge kisspeptin synthesis and release. In our preliminary data using Drop-Seq of arcuate neurons from adult mice, we have identified Nhlh2 as a highly Kiss1 neuron-specific transcription factor (TF) of the basic helix-loop- helix family. Moreover, this TF is inhibited in conditions of negative energy balance, in line with the regulation of the Kiss1 gene. In addition, JASPAR analysis has revealed 12 potential binding sites of Nhlh2 within the 5?UTR of the Kiss1 promoter. Based on these findings, we hypothesize that Nhlh2 may play a crucial role in the development and function of Kiss1 neurons postnatally, as well as in the regulation of kisspeptin synthesis by metabolic factors. To test this hypothesis, we aim to characterize the role of Nhlh2 in Kiss1 neurons through: a) the study of the expression profile of Nhlh2 in the hypothalamus (especially in Kiss1 neurons) across postnatal development; b) ablation of Nhlh2 expression from Kiss1 neurons using Kiss1-cre:Nhlh2fl/fl mice to characterize the reproductive and metabolic role of Nhlh2 in Kiss1 neurons in vivo; and c) confirming the recruitment to the Kiss1 promoter and the enhancer action of Nhlh2 through chromatin- immunoprecipitation (ChIP) assays and in vitro luciferase assays. Altogether, the successful completion of this proposal would significantly contribute to our understanding of the mechanisms that control kisspeptin synthesis, which may lead to the development of new strategies to treat reproductive disorders, such as delayed puberty onset, polycystic ovarian syndrome or hormone- related cancers.
Reproductive function is tightly regulated by central and peripheral factors that affect the release of kisspeptins at the hypothalamic level. In turn, kisspeptins dictate the pattern of GnRH release, which determines the timing of puberty onset and successful reproduction. Understanding the mechanisms that regulate kisspeptin synthesis and release is paramount in understanding reproductive function. In this study, we aim to address the role of the transcription factor Nhlh2, (that is almost exclusive of Kiss1 neurons in the adult mouse brain and regulated by metabolic factors) in the development and metabolic regulation of Kiss1 neurons and, therefore, reproduction. This work will contribute significantly to unmasking the intracellular mechanisms that take place within Kiss1 neurons to control reproduction.
