The Role of Citrullination in Gonadotrope Function

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Gonadotropin Releasing Hormone (GnRH) stimulates peptidylarginine deiminase (PAD) catalyzed citrullination of histones and cytoskeletal filaments in gonadotropes, yet the physiological consequence of this on reproductive function is lacking. This gap in knowledge is an important problem because proper gonadotrope function is critical for all vertebrate reproduction and a major therapeutic target for infertility and contraception. Our long-term goal is to develop a detailed understanding of the molecular targets of citrullination and its physiological implications in gonadotropes. The objective of this proposal is to identify citrullination as a novel regulator of gonadotrope specific gene programs and cytoskeletal function in vivo. Our published data demonstrates that GnRH induces PAD catalyzed citrullination of arginine residues on histones to regulate expression of the luteinizing hormone (LH) β subunit gene. Preliminary RNA-seq data suggests that citrullination also regulates the of expression endoplasmic reticulum processing and golgi vesicle trafficking gene networks not previously characterized in gonadotropes. In addition to histones, our data shows that PADs citrullinate β-tubulin and β-actin to regulate cytoskeletal reorganization following GnRH stimulation. Our central hypothesis is that GnRH utilizes PAD catalyzed citrullination of histones and cytoskeletal proteins to implement critical molecular changes necessary for LH synthesis, spatial repositioning of cells, and effective hormone secretion. The central hypothesis will be tested with the following specific aims: (1) To test whether genes governing gonadotropin synthesis and processing are regulated by GnRH induced PAD catalyzed histone citrullination in vitro and in vivo. (2) To identify citrullinated cytoskeletal filaments and determine how these modifications regulate gonadotrope function. In Aim 1, the expression of target genes identified by RNA-seq in LβT2 gonadotropes will be examined following GnRH and PAD inhibitor treatment using chromatin immunoprecipitation (ChIP). Target genes will next be examined in gonadotropes purified by fluorescence activated cell sorting (FACS) from GRIC-GFP mice. Primary gonadotropes will be treated with GnRH and PAD inhibitor and target genes examined by qPCR and ChIP. Proposed studies in Aim 2 will identify citrullinated proteins from LβT2 and primary gonadotropes using a proteomic approach. The physiologic importance of citrullination on cellular architecture and LH secretion will be examined in primary gonadotropes following treatment with GnRH and a PAD inhibitor. The proposed research is innovative because we plan to determine how GnRH acting through the unexplored mechanism of citrullination can temporally initiate an epigenetic event and cytoskeletal reorganization to control gonadotrope physiology, which represents a new and substantial departure from current studies in the gonadotrope field. The work is significant because it is an important step to characterize a completely novel, unexplored mechanism stimulated by GnRH that is critical for gonadotrope function.