SUN-194 Erasing the Methyl Mark: Mechanism of Androgen Action in the Ovary

Abstract

Androgen actions through androgen receptor (AR) are critical for follicle growth and female fertility. While excess androgen level leads to polycystic ovary syndrome (PCOS), a certain amount of direct androgen action is critical for normal ovarian function. Thus, with respect to androgen actions in the ovary, balance is key. However, how androgens regulate ovarian physiology or its underlying mechanism is still poorly understood. Androgen actions are mediated through ‘nuclear/genomic’ or ‘extra-nuclear/non-genomic’ actions of AR. Recently we reported that androgens through both the extra-nuclear and nuclear pathways inhibit the expression and activity of Ezh2, (a histone methyl-transferase involved in gene repression mark, H3K27me3) to induce different genes that are imperative for ovarian function. Here, we further extended our study to elucidate other histone modifications induced by androgens and its downstream physiological effect in follicular development. In addition to Ezh2, we have found that androgens also regulated the expression of a histone demethylase called Jumonji domain containing protein 3 (JMJD3/KDM6B), that is responsible of removing H3K27me3 mark. Results showed that DHT in GCs increased hypoxia-inducible factor 1 alpha (HIF1α) protein levels through the PI3K/Akt pathway, which in turn induced JMJD3 expression. We found that DHT had not affect HIF1α mRNA but increased HIF1α protein levels. This effect of DHT on HIF1α protein level was blocked by flutamide (AR inhibitor), LY294002 (PI3K inhibitor) as well as cyclohexamide (protein translational inhibitor). Interestingly the 5’ prime end of JMJD3 have HIF1α response elements (HRE) and it is well established that several JMJDs are induced by HIF1α under both normal conditions and hypoxic stress. Our studies showed that DHT treatment induced JMJD3 expression and decreased H3K27me3 levels. Inhibition of AR, PI3K/AKT pathway and siRNA-mediated knockdown of HIF1α attenuated androgen-induced expression of JMJD3. Moreover, ChIP studies showed increased binding of HIF1α on the JMJD3 promoter region with DHT treatment. In our efforts to understand the downstream effects of androgen-induced JMJD3 expression, we found Dicer1 was upregulated upon DHT treatment to mouse granulosa cells. Given the critical role of Dicer in follicular development, ovarian physiology and female fertility, in general, along with the importance of androgens in ovarian physiology, we propose that regulation of Dicer is one of the underlying mechanisms of androgen actions in the ovary. Androgens in a transcription-independent manner, through the PI3K/Akt signaling pathway, enhance HIF1α protein synthesis under normoxic conditions which in turn regulate the expression of Dicer through JMJD3. Altogether, this study provides a mechanistic insight of androgen actions in ovarian function by epigenetic modulation.