We investigate the molecular basis of peptide hormone control of gonadal function, with particular emphasis on the structure and regulation of the luteinizing hormone receptor (LHR) and prolactin receptor (PRLR) genes and the regulatory mechanism(s) involved in the control of steroidogenesis and spermatogenesis. Our studies focus on regulation of human LHR gene transcription (nuclear orphan receptors, epigenetic, DNA methylation, second messenger) as well as on the multiple promoter control of hPRLR gene transcription. We are elucidating the function of two inhibitory short forms of the prolactin receptors and their relevance to physiological regulation and breast cancer. We also investigate hormone-regulated membrane coupling and intracellular events involved in the modulation of steroid biosynthesis in the testis as well as novel gonadotropin-regulated genes of relevance to the progression of testicular gametogenesis, ovarian function, and other reproductive processes.
Epigenetic control of luteinizing hormone receptor transcription
Zhang, Fatima,1 Liao, Dufau
Characterization of the regulatory mechanisms for the LHR gene has been advanced by identification of the gene’s promoter in different species and the various transcription factors that are involved in its basal transcription. LHR gene promoter activity is controlled by two activating Sp1-/Sp3-binding domains and an inhibitory direct repeat motif recognized by nuclear orphan receptors EAR2 and EAR3/COUP-TF1. Repression results from a direct interaction between EAR3/COUP and Sp1 bound to the proximal Sp1 site, which perturbs the interaction of Sp1 and TFIIB and the recruitment of RNA polymerase II. We previously demonstrated that, independent of this mechanism, transcription of the LHR gene is subject to repression by histone deacetylation at its promoter region, where a histone deacetylase (HDAC)/mSin3A complex is anchored at a proximal Sp1 site (Zhang and Dufau, J Biol Chem 2002;277:33431). Our more recent studies have shown that epigenetic silencing and activation of the LHR is achieved through coordinated regulation at both histone and DNA levels.
We began our studies by characterizing the LHR gene promoter methylation status in (1) JAR and MCF-7 cells, where the transcription of the gene is markedly silenced, and in (2) simian virus 40–transformed normal placenta PLC cells, where LHR expression is an active state. The HDAC inhibitor trichostatin A (TSA) evoked robust but significantly lower activation of the LHR gene in JAR than in MCF7 cells. The effect was localized to the 176bp promoter region, which is highly methylated in JAR and lightly methylated in MCF7 cells. Consequently, TSA and the DNA-demethylating reagent 5-Aza C caused marked synergistic activation of the LHR gene in JAR cells but not in MCF7 cells. Multiple site-specific lysine acetylation of H3/H4 is associated with such LHR gene activation. Methylation or acetylation of H3 at K9 is present at the silenced and derepressed LHR promoter, respectively. While DNA methylation did not affect the histone code of the LHR promoter, demethylation of the LHR CpG sites was necessary for maximal stimulation of the gene. Mechanistically, the combined actions of TSA and 5-AzaC resulted in complete demethylation of the promoter in JAR cells. Release of the repressive HDAC/mSin3A complex from the LHR gene promoter in both cell types required both TSA-induced changes of histone modifications and, concurrently, a demethylated promoter. In addition, Dnmt1 was largely dissociated from the LHR gene promoter in the presence of TSA or TSA plus 5-AzaC while binding to MDB2 in JAR cells was diminished upon conversion of the promoter to a demethylated state. Release of MBD2 from the demethylated LHR gene promoter in JAR cells could contribute to elimination of the HDAC complex from the promoter as a consequence of the recognized interaction between MBD2 and HDAC.
All changes described above induced a more permissive chromatin, in which recruitment of polymerase II and TFIIB to the promoter significantly increased. In contrast to activation of the LHR gene by TSA and 5-AzaC in JAR and MCF7-cells, we observed basal expression of the LHR gene in LHR-expressing PLC cells, in which the promoter is unmethylated and associated with hyperacetylated histones. Consequently, PLC cells are unresponsive to drug treatment. In summary, we defined the combinatorial requirements of histone modifications and DNA methylation/demethylation affecting the repression/derepression modalities of LHR transcription. The findings elucidated a regulatory mechanism whereby concurrent dissociation of repressors and association of activators and basal transcriptional components, resulting from coordinated histone hyperacetylation and DNA methylation, lead to derepression of LHR gene expression. Our results thus uncovered a novel silencing/derepression mechanism that pertains to a class distinct from that derived from epigenetic studies of tumor suppressor genes.
Tsai-Morris C-H, Dufau ML. The luteinizing hormone receptor. In: Huret JL, ed. Atlas of Genetics and Cytogenetics in Oncology and Haematology URL. 2005.
Zhang Y, Dufau ML. Gene silencing by nuclear orphan receptors. Vitam Horm 2004;68:1-48.
Zhang Y, Dufau ML. Repression of the luteinizing hormone receptor gene promoter by cross-talk among EAR3/COUP-TFI, Sp1/Sp3 and TFIIB. Mol Cell Biol 2003;23:6958-6972.
Zhang Y, Fatima N, Dufau ML. Coordinated changes in DNA methylation and histone modifications regulate silencing/derepression of luteinizing hormone receptor gene transcription. Mol Cell Biol 2005;25:7929-7939.
Gonadotropin-regulated testicular genes
Tsai-Morris, Sheng, Gutti, Maeda, Dufau; in collaboration with Lei
Gonadotropin-regulated acyl CoA synthetase (GR-LACS) is a 79 kD protein that we cloned from a rat cDNA library (Tang et al., Proc Natl Acad Sci USA 2001;98:6581). The protein, which is transcriptionally downregulated by gonadotropin and can activate long-chain fatty acids, belongs to the long-chain fatty acyl-CoA synthetase family. GR-LACS has sequence identity with two conserved regions of the LACS family (ATP/AMP binding domain and fatty acid acyl-CoA synthetase [FACS] signature motif) but shares low overall amino acid sequence similarity with all other known members of the family. GR-LACS is abundantly and constitutively expressed in steroid-producing rat Leydig cells (but minimally in germinal cells) and is downregulated during desensitization by gonadotropin. We thus hypothesize that GR-LACS contributes to the provision of energy requirements and biosynthesis of steroid precursors and participates through acyl-CoA’s several functions in the regulation of the male gonad.
After determining GR-LACS’s cell-, tissue-, and species-specific expression in rats and mice, we found that GR-LACS protein is expressed in the rodent brain and gonads but, in the mouse, in the adrenal cortex only. In both rats and mice, the protein is found in most regions of the brain, is highly expressed in the hippocampal region, and is associated with ovarian follicles undergoing atresia (i.e., transition from pre-antral to antral stages and subordinate antral follicles not selected for ovulation), thus potentially serving as a marker for atresia. GR-LACS is present in newborn and immature testis tubules but in the Leydig cells only after puberty. Its presence at pre-meiotic stages, at a time of intense proliferation of Sertoli cells and spermatogonia, could contribute to the development of normal adult spermatogenesis. We observed a distinct GR-LACS protein species of 64 kDa, which was more abundant than the 79 kDa long form, as well as a minor form of 73 kDa species in the rat brain and mouse ovary, which we are currently investigating.
To understand the transcriptional regulation of GR-LACS in gonadal cells, we determined the structural requirements of the mouse gene. The minimal promoter domain of this TATAless gene resides within -254/-217 bp 5´ of the ATG codon, and we identified four transcriptional start sites within the region (-266/-216 bp). Upstream sequences to -404 contribute to the inhibition of transcription in mouse Leydig tumor cells. Removal of sequences downstream of the transcription start site (TSS) (-271/-1 bp) reduced transcriptional activity to basal levels. Functional analyses indicate that transcription of GR-LACs requires an Sp1-/Sp3-binding element located downstream of the TSS within exon 1, which is essential for basal promoter activity.
We previously identified a novel gonadotropin-regulated testicular helicase (GRTH/Ddx25) that is present in the nucleus and cytoplasm of pachytene spermatocytes and round spermatids and binds to mRNA species as an integral component of messenger ribonucleoprotein particles, with storage in chromatoid bodies located in the cytoplasm of spermatids (Tsai-Morris et al., 2004). GRTH-targeted null male mice are sterile as a result of spermatid arrest at step 8 of spermatogenesis, demonstrating failure to elongate and markedly fewer chromatoid bodies. In spermatids, transcription steps 1 through 8 did not undergo alteration, but translation was selectively abrogated. GRTH is cell-specific and hormonally regulated in the testis, which contains three species of GRTH resulting from alternative utilization of translation initiation codons. In the rat, germ cells (round spermatids and spermatocytes) primarily use the first ATG codon (+) and contain a major protein of 61/56 kDa, whereas Leydig cells preferentially use the second ATG codon with expression of 48/43 kDa species; in the mouse, we observed only the 61/56 kDa species. Our current studies are defining the function of this helicase as an RNA-binding protein and its storage and translational function during sperm progression.
We recently determined the subcellular distribution of GRTH protein in cytoplasm and nucleus of mouse Leydig and germinal cells. N-terminal and C-terminal antibodies recognize both a cytoplasmic species of 61 kDa and a nuclear species of 56 kDa, with both species generated from utilization of the first ATG codon. The observed differences in molecular weight species are attributable to phosphorylation of the 61 kDa cytoplasmic form. In addition to its storage function, the cytoplasmic species of GRTH associates with polyribosomes to regulate the translational activity of specific subsets of expressed genes.
Sheng Y, Li J, Dufau ML, Tsai-Morris C-H. The gonadotropin-regulated long chain acyl CoA synthetase gene: a novel downstream Sp1/Sp3 binding element critical for transcriptional promoter activity. Gene 2005;360:20-26.
Sheng Y, Tsai-Morris C-H, Dufau ML. Cell-specific and hormonally regulated expression of gonadotropin regulated testicular RNA helicase gene (GRTH/Dsx25) resulting from alternative utilization of translation initiation codons in the rat testis. J Biol Chem 2003;278:27796-27803.
Tsai-Morris C-H, Lei CH, Jiang Q, Sheng Y, Dufau ML. Genomic organization and transcriptional analysis of gonadotropin-regulated testicular RNA helicase. Gene 2004;331:83-94.
Tsai-Morris C-H, Sheng Y, Lee E, Lei KJ, Dufau ML. Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) is essential for spermatid development and completion of spermatogenesis. Proc Natl Acad Sci USA 2004;101:6373-6378.
Prolactin receptor
Qazi, Dong, Tsai-Morris, Dufau
Prolactin is a polypeptide hormone of the growth/cytokine family that is produced by the lactotrophs of the anterior pituitary gland. It displays diverse biological actions in all vertebrates through high-affinity membrane receptors present in its several target tissues. It acts through the long form of the receptor (LF) to cause differentiation of mammary epithelium and to initiate and maintain lactation through activation of the Jak2/Stat5 pathway and subsequent transcriptional events. The hormone plays an essential role in the development of rodent mammary tumors and is a potent mitogen in human normal and cancerous breast tissues/cells. The available evidence strongly suggests that prolactin plays a role in the development of human breast tumors. We identified two novel short forms (SFs) with abbreviated cytoplasmic domain (S1a and S1b) that are products of alternative splicing and inhibit the activation induced by PRL through the LF. We observed a significantly lower ratio of SF in tumor tissue and breast cancer cell lines than in normal breast and control mammary cells. Therefore, the lower expression of SFs in cancer could cause gradations of unopposed, prolactin-mediated, long form–stimulatory function and contribute to breast tumor development/progression.
In light of the dimerization requirement for initiation of signal transduction by prolactin through the long form and the possibility that the inhibitory action of the SF (S1a and S1b) could result from the heterodimerization, we studied the association among LF and short forms S1a and S1b of hPRLR with various techniques, including in vitro co-immunoprecipitation, in vivo BRET analyses, biotinylation studies, and mutagenesis. We used 3´-tagged fusion constructs, which exhibited activities comparable to those of the wild type, to investigate homodimer and heterodimer formation. We detected the formation of homodimers of a fraction of the total receptors in Western blots only under reducing conditions. Initial co-immunoprecipitation studies using combinations of specific anti-tag antibodies demonstrated the presence of heterodimerized complexes between LF and the individual SFs. Neither homodimer nor heterodimer formation required the presence of hormone. Using BRET analysis, we further demonstrated a physical association of hPRLR variants in HEK293 cells and in vivo. Our results revealed the interaction between RL and Y moieties of the fusion proteins in intact cells and supported the existence of constitutive homodimers and heterodimers. Using surface biotinylation/avidin immunoprecipitation followed by Western analysis under non-reducing and reducing conditions, respectively, we further established that dimerization (homodimers LF:LF, S1a:S1a, and S1b:S1b) and heterodimers (LF:S1a and LF:S1b) of PRLR short and long forms occurs at the cell membrane. Mutation of unpaired cysteine residues at amino acids 208, 249, and 266, which are shared by LF and SFs, indicated that the residues do not contribute to hPRLR dimerization through covalent bonding. Therefore, other domains could contribute to this process, including receptor-receptor hydrogen bonding interactions within the ligand binding domain.
In summary, we have demonstrated significant hPRLR homodimer/heterodimer formation that is independent of the hormone, which does not induce formation of additional dimeric forms. Furthermore, the inhibitory effect of SFs on LF stimulatory activity induced by prolactin can be attributed to the formation of heterodimers. Although the heterodimeric forms are competent to bind hormone and may permit JAK-2 association to the cytoplasmic Box 1 (present in LF and SF), further signaling events probably cannot proceed, presumably because of the absence of downstream relevant sequences (i.e., Y587) in the SF heterodimer partner. Consequently, signaling via the JAK/STAT pathway is not operative, and the activation of transcription of relevant genes is reduced or abolished.
Meng J, Tsai-Morris C-H, Dufau ML. Human prolactin receptor variants in breast cancer: low ratio of short forms to the long-form human prolactin receptor associated with mammary carcinoma. Cancer Res 2004;64:5677-5682.
Tsai-Morris C-H, Dufau ML. Human prolactin receptor. In: Huret JL, ed. Atlas of Genetics and Cytogenetics in Oncology and Haematology URL. 2005.
1Naheed Fatima, PhD, former Adjunct Investigator
Collaborators
Ke-jian G. Lei, PhD, Oral Infection and Immunity Branch, NIDCR, Bethesda, MD
For further information, contact dufaum@mail.nih.gov.