Regulation of Hormone Receptors and Gonadal Genes

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

The Luteinizing Hormone Receptor (LHR) is expressed primarily in the gonads where it mediates signals that regulate ovarian and testicular function. LHR is transcriptional regulated by diverse networks in which coordination and interactions between regulatory effectors are essential for silencing/activation of LHR expression. The proximal Sp1 site of the promoter recruit histone (H) deacetylases and the Sin3A corepressor complex that contributes to the silencing of LHR transcription. Site specific acetylation /methylation induced by TSA causes phosphatase release that serves as a switch for Sp1 phosphorylation, recruitment of TFIIB and Pol II and transcriptional activation. Positive coactivator 4(PC4) recruited by Sp1, acts as its coactivator and has an essential role in the formation/assembly of PIC and TFIIB and Pol II recruitment in TSA mediated LHR transcription. Further studies demonstrated association between PC4 and acetylated H3 in TSA induced LHR de-repression in MCF7 cells. The recruitment of PC4 to SP1 and formation of the PC4-SP1 complex was shown to be essential for LHR transcription. More recently, MS/MS analysis revealed that PC4 associates with histone variant H3.3 acetylated at several Lys residues. IP studies with Flag PC4 demonstrated interaction of PC4 with H3.3 induced by TSA using H3.3 specific antibody and the presence of the complex PC4-H3.3 at the LHR promoter was demonstrated by reChiP. Depletion of endogenous PC4 or H3.3A/B by siRNA caused marked reduction of TSA induced formation of the complex, its recruitment to the LHR promoter and transcriptional activation of the LHR gene. This resulted from a decrease of accessibility of the chromatin at the promoter region of the LHR. Pull-down studies demonstrated association of H3/H3.3 with GST-PC4 in MCF7-extracts, while no direct association with H4 was found. From these and other findings we concluded that PC4 associates with the tetramer via H3 or H3.3. PC4-H3.3 interaction favors acetylation of H3.3 which leads to chromatin accessibility and gene transcription. These findings indicate a critical role of PC4 association with acetylated H3.3 in TSA-induced Sp1 activated LHR transcription (1). Gonadotropin regulated Testicular RNA Helicase (GRTH/DDX25), is a testis-specific member of the DEAD-box family of RNA helicases discovered in our laboratory, which is essential for the completion of spermatogenesis. It is present in Leydig cells and meiotic (pachytene spermatocytes) and haploid germ cells (round and elongated spermatids). Males lacking GRTH are sterile due azoospermia resulting from failure of round spermatids to elongate. We demonstrated its participation on the nuclear export/transport of specific mRNAs, the structural integrity of the Chromatoid Body (CB) storage/ processing of relevant mRNAs and their transit/association to the actively translating polyribosomes where it may regulate translational initiation of genes. GRTH is the only family member regulated by hormones. GRTH transcription is stimulated in Leydig cells by LH/cAMP through direct actions of androgen (A)/A receptor (AR) (autocrine), and in germ cells in paracrine fashion through AR in Sertoli cells. The upstream region of the GRTH gene directs its expression in germ cells and downstream in LCs in the Leydig cell. Through these regions A/AR exerts its direct (endogenous) regulation of the GRTH gene in LC, and indirectly in germ cells. Functional binding sites for Germ Cell Nuclear Factor (GCNF) present in round spermatids (RS) and spermatocytes (SP) and its regulation by A/AR were identified in the distal region-of the GRTH gene, operative selectively in RS. Current knowledge indicates actions of A on GCNF cell specific regulation of GRTH expression in germ cells (RS). Also, GRTH exerts negative autocrine regulation of GCNF linking A actions to germ cells through GCNF as an A regulated trans-factor that controls transcription/expression of GRTH. These findings provide a connection of androgen action to two relevant germ cell genes (GRTH and GCNF) essential for the progress of spermatogenesis and established their regulatory interrelationship. Our early studies revealed that missense mutation of R to H at aa 242 of GRTH found in 5.8% of patients with complete loss of sperm causes loss of the 61 kDa phospho-species (pGRTH) with preservation of the 52 kDa non-phospho form. This finding provided an avenue to elucidate the function of pGRTH in spermatogenesis. We generated a humanized mutant GRTH knock-in (KI) mice. Mutant mice are sterile with reduction on testicular size, lack sperm with arrest at step 8 of round spermatids (RS) and complete loss of the pGRTH species with preservation of the nuclear 52 kDa form. This mice model permits to study the biological /biochemical functions of the cytoplasmic pGRTH. In KI mice the nuclear export transport and functions of GRTH are preserved (ie. mRNA export, miRNA regulation) while the cytoplasmic functions including shuttling of messages, storage in the CB and translational events all requiring pGRTH are absent. Marked reduction of the CB size in RS and lack pGRTH in the CBs are observed. Germ cell apoptosis was present in pachytene spermatocytes (PS) and RS. In contrast to KO, KI showed no changes in miRNA biosynthesis excluding participation of pGRTH as transcriptional regulator of the microprocessor complex (Drosha, DCGR) affecting primiRNAs formation and indicative of the participation of non-phospho GRTH in these processes. In KI mice there is loss of chromatin remodeling and related proteins including, TP2, PRM2 and TSSK6. Significant decreases of their mRNA and half-lives indicate that their association with pGRTH in the cytoplasm protect these mRNAs from degradation. Also, our work showed that pGRTH stimulates TP2 translation in a 3'UTR dependent manner (3). In recent studies we elucidated the GRTH phospho-site at a threonine (Tr239) structurally adjacent to the mutant site found in patients (R242H). Molecular modelling of the phospho-site based on the RecA domain 1 of the DDX9 crystal structure, pointed to the amino acids that formed the GRTH/PKA interface, solvent accessibility and H-bonding. These include in addition of the core residues T239 and R242 amino acids E165, K240, and D237 (4). The relevance of these residues were demonstrated by disruption of amino acids using site directed mutagenesis (single or double mutations) which caused reduction or abolition of the p-GRTH at Tr239. The phospho-Tr239 form is the cytoplasmic species demonstrated as essential for the progress of spermatogenesis beyond step 8 of round spermatids and for viable sperm formation. It is important to note that the deleterious effects on GRTH phosphorylation caused by the mutations did not result from changes of PKA alpha-catalytic binding affinity but rather to consequential structural changes that can affect PKA catalytic efficiency. Studies based on the abolition of the phospho-form provide the basis for drug design, virtual and throughput screening for discovery of a reversible chemical inhibitor for use as male contraceptive.