ß-catenin regulates FSHß induction by GnRH: next generation RNA-Sequencing identifies Brms1L as a mediator of beta-catenin regulation of FSHß gene expression

The regulation of gonadotropin synthesis by GnRH (Gonadotropin-releasing hormone) plays an essential role in the neuroendocrine control of reproduction. The known signaling mechanisms involved in gonadotropin synthesis have been expanding. For example, involvement of ß-catenin in LHß induction by GnRH has been discovered. We examined the role of ß-catenin in FSHß gene expression in LßT2 gonadotrope cells. GnRH caused a sustained increase in nuclear ß-catenin levels, which was significantly reduced by JNK inhibition. siRNA-mediated knockdown of ß-catenin mRNA demonstrated that induction of FSHß mRNA by GnRH depended on ß-catenin and that regulation of FSHß by ß-catenin occurred independently of the JNK-c-jun pathway. ß-catenin depletion had no impact on FSHß mRNA stability. In LßT2 cells transfected with FSHß promoter luciferase fusion constructs, GnRH responsiveness was conferred by the proximal promoter (-944/-1), and was markedly decreased by ß-catenin knockdown. However, none of the TCF/LEF binding sites in that region were required for promoter activation by GnRH. Chromatin immunoprecipitation further corroborated the absence of direct interaction between ß-catenin and the 1.8 kb FSHß promoter. To elucidate the mechanism for the ß-catenin effect, we analyzed ~1 billion reads of next generation RNA sequencing ß-catenin knockdown assays and selected the nuclear cofactor Brms1L as one candidate for further study. Subsequent experiments confirmed that Brms1L mRNA expression was decreased by ß-catenin knockdown as well as by JNK inhibition. Furthermore, knockdown of Brms1L significantly attenuated GnRH-induced FSHß expression. Thus, our findings indicate that the expression of Brms1L depends on ß-catenin activity and contributes to FSHß induction by GnRH. Overall design: A total of 24 samples were analyzed, namely 6 different experimental conditions, each comprised of 4 replicates. Control samples are included in the analysis. For the RNA-Seq assay, samples were multiplexed in the form of 3 samples per lanes, resulting in a total of 8 lanes.