Tet2 Enables Elevated GnRH Neuron Activity and Maintains Activating Histone Modifications within the GnRH Gene

Presentation Number: LBF-087
Date of Presentation: March 6th, 2015

Joe Kurian*
Southern Illinois University School of Medicine, Springfield, IL

Abstract

Late onset hypogonadism (LOH) is characterized by sexual dysfunction, muscle weakness, and/or depression in ageing men. Testosterone replacement has become an increasingly popular request of men suffering these symptoms; though the true impact and long-term safety of this treatment is not well defined. Defining the root cause of LOH is consequently critical to determining the most efficacious and safe treatment of the syndrome. LOH can be caused by a primary failure of testicular function or secondary disruptions of hypothalamic-pituitary function with reduced or abnormally stable plasma luteinizing hormone (LH) levels. Secondary LOH is related to lifestyle factors (e.g., diet and obesity) or coexisting conditions (e.g., diabetes, COPD, cardiovascular disease). We hypothesize that these environmental factors have an epigenetic impact on hypothalamic function that consequently interfere with the regulation of pituitary release of LH. We previously found that gonadotropin releasing hormone (GnRH) neuron activity coincides with the level of DNA methylation in the primate GnRH gene. We subsequently discovered that a mature GnRH cell line (GT1-7) expressed significantly higher levels an enzyme that regulates active DNA demethylation (Tet2) when compared to the immature GnRH cell line (GN11). Remarkably, GnRH neuron specific disruption of Tet2 led to an age dependent decrease of male plasma LH in mice. Whether disruption of Tet2 leads to epigenetic changes in mature GnRH neurons remained unclear. Using Chromatin immune-precipitation, we evaluated Tet2 binding and an activating histone modification (H3K4me3) across the mouse GnRH gene 5’ region in four conditions: 1) GN11, 2) GT1-7, 3) GN11 cells overexpressing Tet2, and 4) GT1-7 cells with Tet2 eliminated by CRISPR/cas9 mediated gene disruption. Tet2 binding was low in GN11 cells across the entire 5’ region, but significantly elevated in GT1-7 cells at the promoter and neuron specific enhancer region. The activating H3K4me3 modification largely mirrored Tet2 binding patterns, with significantly higher abundance in GT1-7 compared to GN11 cells. Overexpression of Tet2 in GN11 cells significantly increased Tet2 binding of the promoter and to a lesser extent the neuron specific enhancer; H3K4me3 levels were also significantly higher at the promoter compared to non-transfected GN11 cells. Disruption of Tet2 in GT1-7 cells decreased Tet2 binding across the entire region; remarkably, H3K4me3 levels significantly dropped at the neuron specific enhancer after Tet2 disruption in GT1-7 cells. They further suggest the novel concept that Tet2 mediated epigenetic patterns must be actively maintained, as opposed to established solely as a developmental process. Future studies will evaluate how the environment influences Tet2 activity in the hypothalamus and how this relates to the etiology of LOH.

 

Nothing to Disclose: JK