The Luteinizing Hormone Receptor (LHR): The LHR is expressed primarily in the gonads where it mediates LH signals that regulate ovarian and testicular function. The LHR gene transcription is regulated by complex and 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 recruits histone (H) deacetylases and the Sin3A corepressor complex that contributes to the silencing of LHR transcription. Site specific acetylation/methylation-induced phosphatase release serves as an on switch for Sp1 phosphorylation at Ser641 that causes p107 repressor release from Sp1, recruitment of TFIIB and Pol II and transcriptional activation. Maximal derepression of the gene is dependent on DNA demethylation of the promoter, H3/H4 acetylation and HDAC/Sin3A release. Positive Cofactor 4 (PC4) has an important role in the formation/assembly of PIC in TSA-mediated LHR transcription. It is recruited by Sp1 following TSA treatment and acts as its coactivator. However, PC4 does not participate in TSA release of phosphatases, Sp1 phosphorylation or release repressor/complexes. Although TFIIB recruitment is dependent on PC4 we have ruled out TFIIB as its direct target and acetylation of PC4 in the activation process. However, TSA induced acetylation of a PC4 interacting proteins (16 kDa), identified as Acetylated H3 by MS, and its presence in the complex in association to chromatin at the promoter was demonstrated by ChiP/reChiP. The role of these interactions on chromatin structure and their participation in the assembly of the PIC and transcriptional activation are under investigation. Gonadotropin regulated Testicular RNA Helicase (GRTH/DDX25): GRTH is a testis-specific member of the DEAD-box family of RNA helicases present in Leydig cells (LC) and meiotic germ cells. It is a multi-functional protein essential for the completion of spermatogenesis. Males lacking GRTH are sterile due to the absence of sperm resulting from failure of round spermatids to elongate. In addition, to its intrinsic RNA helicase activity, GRTH is a shuttling protein that exports specific mRNAs from the nucleus to cytoplasmic sites Our studies have demonstrated the essential participation of the GRTH export/transport of mRNAs in the structural integrity of the Chromatoid Body (storage/processing of mRNAs) and their transit/association to actively translating polyribosomes where it may regulate translational initiation of genes. We have identified mRNAs which are associated with GRTH and regulated at polysomal sites of cell populations of the mouse testis. The reduction in mRNAs associated at polysomal sites in the differential studies (KO vs WT) not detected at total cellular level but in the cytoplasm with abolition of protein expression are reflective of the importance of the transport function of GRTH to relevant sites and underscore its impact in protein synthesis. This differential study has revealed messages regulated in different testicular cells associated with GRTH at polysomes (Geo #GSE38860). Network analysis has provided information about regulatory pathways that link to GRTH function, and fertile ground for exploration of GRTH regulation in the progress of spermatogenesis. In other studies we determine GRTH regulation of miRNAs in round spermatids. Differential expression profiles from WT and GRTH KO mice revealed a panel of miRNAs and their primary miRNAs significantly increased in KO (Geo#GSE 33969). The testis specific upregulated miRNA-469 repressed the translation of Transition Protein 2 (TP2) and Protamine 2 (Prm2) through binding to their mRNA coding region. This is consistent with the preservation of TP2 and Prm2 mRNA expression and failure of their protein expression in KO. Thus, GRTH has an important role in microRNA regulation acting as a negative regulator miRNA-469 biogenesis and expression levels of DROSHA/DGCR8 (mRNA/protein). miRNA-469 silencing of TP2 and Prm2 (chromatin remodelers) is essential for their timely translation at later stages of spermiogenesis, which is critical to attain mature sperm. GRTH is regulated by LH through androgen (A) at the transcriptional level in LCs (direct) and germ cells (presumably indirect) of the testis where its expression is both cell- and stage specific. This helicase displays a novel negative autocrine control of androgen production in LCs by preventing overstimulation of the LH-induced androgen pathway through enhanced degradation of StAR protein. Our initial studies using transgenic mice carrying sequential deletions of 5'flanking sequences define an A responsive l.4 kb region adjacent to ATG codon that contains an ARE half-site at -827. The transcriptional regulation/expression of GRTH by androgen (A) was studied using in vitro and in vivo models where its expression induced by A or hCG (via A) was inhibited by AR antagonist. Deletion mapping and mutagenesis demonstrated that the ARE was critical for DHT induced activation and recruited AR in a DHT dependent manner. ChiP analysis revealed recruitment of AR, steroid coactivator 1 (SRC-1), mediator1 (Med-1), TFII and Pol II to both the ARE and a region around the initiator sites at the promoter. ChiP-3C revealed short-range chromosomal looping between the distal AR/ARE and the core transcriptional machinery at the promoter. Med-1 and SRC-1-were not require for looping but these were essential for the activation of the complex. These findings have provided new insights on the molecular mechanism of AR-regulated transcription of the GRTH gene in LCs. Prolactin receptor (PRLR): The PRLR is a member of the lactogen/cytokine receptor family which mediates the diverse cellular actions of PRL. PRL is a major factor in the proliferation and differentiation of breast epithelium and is essential for lactation. It has been also implicated in the development of breast cancer, tumoral growth and chemoresistance. hPRLR expression is controlled at the transcriptional level by multiple promoters (one generic, PIII, and five human specific hPN1-hPN5) that were defined and characterized in our laboratory. Each promoter directs transcription/expression of a specific non-coding Exon 1 (E1-3, hEN1-hEN5), a common non-coding exon 2 and coding exons (E3-E11). The transcription of PRLR in breast cancer cells by the preferentially utilized PIII which lacks an estrogen responsive element is directed by Estradiol (E2)/ERa through complex formation with SP1 and C/EBPb that associate with cognate elements induces TFIIB and Pol II recruitment. BRET revealed ERa constitutive homodimers. E2-enhanced complex formation of the ERa dimer with SP1 and C/EBPb dimers at the PIII promoter has an essential role in the transcriptional activation of the hPRLR gene in breast cancer cells. In further studies we investigated the effect of E2 and Testosterone on the expression of various E1 isoforms (hE1-3, hEN1-hEN5) transcribed under the control their specific promoters in MCF-7 cells. Real-time PCR of exons 1 showed that E2 stimulated the expression of all exon 1 isoforms except hE1N2. This effect was prevented by ER antagonist. Exogenous testosterone showed comparable stimulation that was prevented by an aromatase inhibitor confirming the endogenous estrogen action in PRLR expression. Changes on PRLR protein expression and cell proliferation displayed similar stimulatory patterns. Current studies are addressing homologous effect of PRL on PRLR through individual promoters. Investigations on PRLR promoter usage and their regulation under hormonal treatment may provide insights into resistance states of breast cancer patients under adjuvant therapies.

Project Start
Project End
Budget Start
Budget End
Support Year
37
Fiscal Year
2012
Total Cost
$1,520,321
Indirect Cost
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State
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Kavarthapu, Raghuveer; Dufau, Maria L (2016) Role of EGF/ERBB1 in the transcriptional regulation of the prolactin receptor independent of estrogen and prolactin in breast cancer cells. Oncotarget :
Kavarthapu, Raghuveer; Dufau, Maria L (2015) Germ Cell Nuclear Factor (GCNF/RTR) Regulates Transcription of Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in Testicular Germ Cells--The Androgen Connection. Mol Endocrinol 29:1792-804
Yang, Ruifeng; Tsai-Morris, Chon-Hwa; Kang, Jung Hoon et al. (2015) Elucidation of RNA binding regions of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) to transcripts of a chromatin remodeling protein essential for spermatogenesis. Horm Mol Biol Clin Investig 22:119-30
Chason, Rebecca J; Kang, Jung-Hoon; Gerkowicz, Sabrina A et al. (2015) GnRH agonist reduces estrogen receptor dimerization in GT1-7 cells: evidence for cross-talk between membrane-initiated estrogen and GnRH signaling. Mol Cell Endocrinol 404:67-74
Kavarthapu, Raghuveer; Tsai Morris, Chon-Hwa; Dufau, Maria L (2014) Prolactin induces up-regulation of its cognate receptor in breast cancer cells via transcriptional activation of its generic promoter by cross-talk between ERα and STAT5. Oncotarget 5:9079-91
Kavarthapu, Raghuveer; Tsai-Morris, Chon-Hwa; Fukushima, Masato et al. (2013) A 5'-flanking region of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) gene directs its cell-specific androgen-regulated gene expression in testicular germ cells. Endocrinology 154:2200-7
Tsai-Morris, Chon-Hwa; Sato, Hisashi; Gutti, Ravi et al. (2012) Role of gonadotropin regulated testicular RNA helicase (GRTH/Ddx25) on polysomal associated mRNAs in mouse testis. PLoS One 7:e32470
Villar, Joaquin; Tsai-Morris, Chon-Hwa; Dai, Lisheng et al. (2012) Androgen-induced activation of gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) transcription: essential role of a nonclassical androgen response element half-site. Mol Cell Biol 32:1566-80
Dufau, Maria L; Sato, Hisashi; Gutti, Ravi et al. (2011) Gonadotropin-regulated testicular helicase (GRTH/DDX25): a master post-transcriptional regulator of spermatogenesis. Adv Exp Med Biol 707:23-9
Kang, Jung-Hoon; Tsai-Morris, Chon-Hwa; Dufau, Maria L (2011) Complex formation and interactions between transcription factors essential for human prolactin receptor gene transcription. Mol Cell Biol 31:3208-22

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