SUN-470 The Homeodomain Transcription Factors Six3 and OTX2 Act on Mouse and Human Kisspeptin Promoters to Repress Transcription

Abstract

Idiopathic hypogonadotropic hypogonadism (IHH) is a gonadotropin-releasing hormone (GnRH) deficiency disorder that, in a subset of individuals, can occur alongside anosmia (Kallmann Syndrome). Known genetic causes for these disorders account for only 45% of affected individuals, leaving the majority of unknown origin. In mice, the homeodomain transcription factors Six3 and OTX2 are necessary for a normal number of GnRH neurons, GnRH neuron migration, and/or GnRH mRNA levels. The loss of just a single allele results in subfertility, hypogonadism, and/or anosmia, making them promising candidates for dysregulation in IHH and Kallman Syndrome. Previous work has shown the importance of these factors within GnRH neurons, but little is known about their roles in upstream cell types, such as kisspeptin neurons. Using qPCR and RNA-seq we have verified that Six3 and OTX2 are present in both an arcuate population (KTaR) and AVPV population (KTaV) of immortalized kisspeptin neurons. This led us to hypothesize that Six3 and OTX2 are critical regulators of kisspeptin gene transcription. To test this hypothesis we performed transient transfections in KTaR and KTaV cell lines with Six3 and OTX2 expression vectors along with either a mouse or human kisspeptin luciferase reporter. We found that in both populations, Six3 repressed both mouse (p < 0.05) and human (p < 0.01) kisspeptin transcription. We also found that OTX2 repressed both mouse and human kisspeptin transcription in the KTaV cell line (p < 0.05), but did not alter transcription in the KTaR cell line. These results suggest that Six3 and OTX2 can regulate the kisspeptin promoter in vitro and produce a response that is consistent between both mouse and human promoters. Effects of Six3 are similar between kisspeptin populations, while OTX2 may function in a population-specific manner.