Teleost FTZ-F1 homolog and its splicing variant determine the expression of the salmon gonadotropin IIbeta subunit gene.

Steroidogenic factor 1, a member of the fushi tarazu factor 1 (FTZ-F1) subfamily of nuclear receptors, is a key regulator in mammalian reproduction. From an embryonic complementary DNA library, the zebrafish homolog of FTZ-F1 (zFF1A) and an alternatively spliced variant (zFF1B) were isolated. zFF1B represented a C-terminally truncated version of zFF1A. Whole mount in situ hybridization and reverse transcriptase-PCR analysis revealed that both zFF1A and B transcripts were present in the developing pituitaries, adult fish brain, gonads, and liver, albeit zFF1B messenger RNA was absent in testis. Comparison of the primary sequences of zFF1 with those of other FTZ-F1 subfamily members showed a close structural relationship between the mouse liver receptor homolog, which activated the alpha1-fetoprotein gene in rodent liver. However, similar to mouse steroidogenic factor 1, zFF1A regulated chinook salmon gonadotropin IIbeta subunit gene expression. On the contrary, zFF1B, which could bind a consensus gonadotrope-specific element with an affinity similar to that of zFF1A, lacked both the trans-activation function and synergistic interaction with the estrogen receptor. Furthermore, cotransfection studies in HeLa cells showed that zFF1B was a strong competitor for the action of zFF1A on the chinook salmon gonadotropin IIbeta subunit gene promoter. Our investigation suggests that 1) zFF1 represents an ancestor protein of the vertebrate FTZ-F1 homologs; 2) the antagonistic relationship between zFF1A and -B may dictate the expression of the FTZ-F1 target genes in a variety of tissues, including the pituitary; and 3) the naturally occurring zFF1B provides evidence that the C-terminal portion of zFF1A (80 amino acid residues) contains a major trans-activation function and a protein-protein interface.

Differential recruitment of steroid hormone response elements may dictate the expression of the pituitary gonadotropin II beta subunit gene during salmon maturation.

The role of testosterone (T) and 17 beta-estradiol (E2) in the control of chinook salmon gonadotropin II beta subunit (sGTHII beta) gene was examined. Both E2 and T specifically stimulated GTHII beta gene expression in cultured juvenile rainbow trout pituitary cells. 5'-Flanking regions of the sGTHII beta gene linked to the chloramphenicol acetyltransferase (CAT) expression vector were transfected into these pituitary cells, and cultures were treated with steroid hormones. Estrogen-stimulated CAT activity occurred with constructs containing a 13-base pair estrogen responsive element (ERE) sequence [proximal ERE (pERE)] located at -273 to -260 upstream of its transcriptional start site. Binding specificity of pERE was confirmed by mobility shift and DNA methylation interference assays using the DNA binding domain of the human estrogen receptor. Interestingly, the pERE functioned to derepress the activity of the proximal silencer (pSil) only in the pituitary cells of juvenile trout but not in cells derived from maturing and sexually matured fish. Another potential ERE sequence comprised of three tandemly linked half-ERE palindromes was located from -2736 to -2659 [distal ERE (dERE)] of the sGTHII beta gene. Distal ERE might be responsible for the steroid responsiveness of the longest sGTHII beta/CAT construct (-3500CAT) observed in the pituitary cells of maturing fish. The function of pERE and dERE were further examined in the heterologous HeLa cells by mutagenesis and cotransfection with a rainbow trout estrogen receptor expression vector. Disruption of the palindromic structure of pERE severely impaired its function. When the sequences between pERE and dERE were deleted, a 200-fold increase in CAT activity was observed in response to E2. A model is proposed to describe the regulation of GTHII beta gene expression at different reproductive stages.

Induction of chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1.

In this study, the functional role of two cAMP-response elements (CRE) in the promoter of the chinook salmon GH gene and their interactions with the transcription factor Pit-1 in regulating GH gene expression were examined. A chimeric construct of the chloramphenicol acetyltransferase (CAT) reporter gene with the CRE-containing GH promoter (pGH.CAT) was transiently transfected into primary cultures of rainbow trout pituitary cells. The expression of CAT activity was stimulated by an adenylate cyclase activator forskolin as well as a membrane-permeant cAMP analog 8-bromo-cAMP. Furthermore, these stimulatory responses were inhibited by a protein kinase A inhibitor H89, suggesting that these CREs are functionally coupled to the adenylate cyclase-cAMP-protein kinase A cascade. This hypothesis is supported by parallel studies using GH4ZR7 cells, a rat pituitary cell line stably transfected with dopamine D2 receptors. In this cell line, D2 receptor activation is known to inhibit adenylate cyclase activity and cAMP synthesis. Stimulation with a nonselective dopamine agonist, apomorphine, or a D2-specific agonist, Ly171555, suppressed the expression of pGH.CAT in GH4ZR7 cells, and this inhibition was blocked by simultaneous treatment with forskolin. These results indicate that inhibition of the cAMP-dependent pathway reduces the basal promoter activity of the CRE-containing pGH.CAT. The functionality of these CREs was further confirmed by deletion analysis and site-specific mutagenesis. In trout pituitary cells, the cAMP inducibility of pGH.CAT was inhibited after deleting the CRE-containing sequence from the GH promoter. When the CRE-containing sequence was cloned into a CAT construct with a viral thymidine kinase promoter, a significant elevation of cAMP inducibility was observed. This stimulatory response, however, was abolished by mutating the core sequence, CGTCA, in these CREs, suggesting that these cis-acting elements confer cAMP inducibility to the salmon GH gene. The interactions between CREs and the transcription factor Pit-1 in mediating GH gene expression were also examined. In HeLa cells, a human cervical cancer cell line deficient in Pit-1, both basal and cAMP-induced expression of pGH.CAT were apparent only with the cotransfection of a Pit-1 expression vector. These results taken together indicate that the two CREs in the chinook salmon GH gene are functionally associated with the cAMP-dependent pathway and that their promoter activity is dependent on the presence of Pit-1

Characterization of an estrogen-responsive element implicated in regulation of the rainbow trout estrogen receptor gene.

We previously reported that the expression of the rainbow trout estrogen receptor (rtER) gene is markedly increased by estradiol (E2). In this paper, we have used transient transfection assays with reporter plasmids expressing chloramphenicol acetyl transferase (CAT), linked to 5' flanking regions of the rtER gene promoter, to identify cis-elements responsible for E2 inducibility. Deletion analysis localized an estrogen-responsive element (ERE), at position +242, with one mutation on the first base compared with the consensus sequence. This element confers estrogen responsiveness to CAT reporter linked to both the herpes simplex virus thymidine kinase promoter and the homologous rtER promoter. Moreover, using a 0.2 kb fragment of the rtER promoter encompassing the ERE and the rtER DNA binding domain obtained from a bacterial expression system, DNase I footprinting experiments demonstrated a specific protection covering 20 bp (+240/+260) containing the ERE sequence. Based on these studies, we believe that this ERE sequence, identified in the rtER gene promoter, may be a major cis-acting element involved in the regulation of the gene by estrogen.

Eukaryotic conditional expression system.

Existing conditional expression systems can be classified in two major categories that are based either on the induction or on the de-repression of transcription. The system described here combines both mechanisms, since a unique transcription factor can be shifted from a repression to a stimulation activity by simply changing its ligand. The resulting advantage of this system is the complete absence of basal expression before active induction. The principle of this method is based on the unexpected ability of the chimeric protein containing the DNA-binding domain of the yeast Gal4 transcription factor fused to the COOH half of the estradiol receptor (GalER), to act as a repressor when bound to the drug 4OH-tamoxifen, in the context of a previously described optimized Gal4-responsive promoter. The efficacy of this system has been assessed in transient expression assays using the chloramphenicol acetyl transferase (CAT), and in situ, through the activity of a Gal4 responsive beta-galactosidase gene.

Rainbow trout estrogen receptor presents an equal specificity but a differential sensitivity for estrogens than human estrogen receptor.

The estrogen receptor is a transcription factor that mediates the actions of estrogens which plays a major role in salmonid vitellogenesis. Previously, we cloned and described the estradiol receptor in rainbow trout. To study the transactivation characteristics, the full length cDNA was inserted in an expression vector and tested by transfection on an estrogen-dependent promoter (pERE-TK-CAT). For the first time, direct comparison between the trout receptor (rtER) and human receptor (hER) in an analogous system has been possible. We demonstrate that rtER can, like hER, transactivate transcription in a strictly hormone-dependent manner. The specificity and sensitivity of the rtER response to different steroids have been studied. With rtER, the E2 concentration needed for half maximal activation is 10 times higher than with hER. In addition, we show that rtER has a weak affinity for androgens and transactivation could be induced using high testosterone concentration. Experiments show that both rtER and hER have an equal specificity for estrogens but that the rtER binds its ligand with a lower sensitivity than hER. Several hypotheses concerning the role of main amino acids within the receptor protein are proposed based on the different properties of the receptors and comparison of sequences.

Presence of distinct cis-acting elements on gonadotropin gene promoters in diverse species dictates the selective recruitment of different transcription factors by steroidogenic factor-1.

The nuclear receptor steroidogenic factor-1 (SF-1) regulates the cell-specific expression of the pituitary gonadotropin subunit genes. Several potential DNA-binding sites for SF-1, estrogen receptor (ER) and the immediate-early transcription factor NGFI-A are found in LHbeta genes from many species. In this study, we have examined the action and interaction of these transcription factors on LHbeta gene promoters from two representative vertebrate species, i.e. rat and salmon. Cotransfection studies in COS-1 cells have shown that the action of SF-1 on salmon gonadotropin IIbeta (sGTHIIbeta) gene promoter was dramatically enhanced when combined with ER. The rat LHbeta promoter was activated by SF-I or ER individually, but these two factors, however, were unable to act in synergism on this promoter. In contrast, NGFI-A, specifically in cooperation with SF-1, transactivated the rat LHbeta gene expression but was ineffective on the sGTHIIbeta gene. Gel shift experiments showed that this lack of activation was due to the low affinity of the salmon NGFI-A-responsive element for its binding protein. In conclusion, our studies demonstrate that differential recruitment of distinct transcription factors by SF-1 might be a common mechanism to activate the cell-specific gonadotropin gene expression in different species.

Salmon gonadotropin IIbeta subunit promoter contains multiple DNA elements responsible for stimulation by gonadotropin-releasing hormone through protein kinase C-dependent and -independent pathways.

Gonadotropin-releasing hormone (GnRH) stimulates gonadotropin (GTH) production by activating GTH subunit gene transcription. In salmonid fish, the expression of the beta subunit gene of GTH II (sGTH IIbeta) is stimulated by GnRH at the final stages of reproduction. DNA elements required for the GnRH stimulation were examined by analyzing sGTH IIbeta promoter activity by transfection studies in a gonadotrope-derived cell line, alphaT3-1. A GnRH analog (GnRHa) specifically stimulated the sGTH IIbeta promoter (3358 bp) expression 3.6-fold, while phorbol myristate acid (PMA) stimulated it 6.2-9-fold. Analysis of a series of 5'-deletion mutants has revealed that a proximal region (-258 to -199) was important in GnRHa stimulation through protein kinase C (PKC)-independent signal transduction pathways, because an internal deletion mutant (delta(246 - 217)/3358) showed a significant decrease in the level of GnRHa stimulation, but showed no change in stimulation by PMA. A large upstream region (-3358 to -1260) showed an enhancing activity of the GnRHa stimulation, and a far upstream 530 bp segment in this region (-3358 to -2829) may be responsible for this activity. The present results suggest that sGTH IIbeta gene may be controlled by GnRH through multiple DNA elements including those responsive to PKC-dependent and -independent signal transduction pathways.

Roles of heat shock factor 1 and 2 in response to proteasome inhibition: consequence on p53 stability.

A single heat shock factor (HSF), mediating the heat shock response, exists from yeast to Drosophila, whereas several related HSFs have been found in mammals. This raises the question of the specific or redundant functions of the different members of the HSF family and in particular of HSF1 and HSF2, which are both ubiquitously expressed. Using immortalized mouse embryonic fibroblasts (iMEFs) derived from wild-type, Hsf1(-/-), Hsf2(-/-) or double-mutant mice, we observed the distinctive behaviors of these mutants with respect to proteasome inhibition. This proteotoxic stress reduces to the same extent the viability of Hsf1(-/-)- and Hsf2(-/-)-deficient cells, but through different underlying mechanisms. Contrary to Hsf2(-/-) cells, Hsf1(-/-) cells are unable to induce pro-survival heat shock protein expression. Conversely, proteasome activity is lower in Hsf2(-/-) cells and the expression of some proteasome subunits, such as Psmb5 and gankyrin, is decreased. As gankyrin is an oncoprotein involved in p53 degradation, we analyzed the status of p53 in HSF-deficient iMEFs and observed that it was strongly stabilized in Hsf2(-/-) cells. This study points a new role for HSF2 in the regulation of protein degradation and suggests that pan-HSF inhibitors could be valuable tools to reduce chemoresistance to proteasome inhibition observed in cancer therapy.

Low-power millimeter wave radiations do not alter stress-sensitive gene expression of chaperone proteins.

This article reports experimental results on the influence of low-power millimeter wave (MMW) radiation at 60 GHz on a set of stress-sensitive gene expression of molecular chaperones, namely clusterin (CLU) and HSP70, in a human brain cell line. Selection of the exposure frequency is determined by its near-future applications for the new broadband civil wireless communication systems including wireless local area networks (WLAN) for domestic and professional uses. Frequencies around 60 GHz are strongly attenuated in the earth's atmosphere and such radiations represent a new environmental factor. An exposure system operating in V-band (50-75 GHz) was developed for cell exposure. U-251 MG glial cell line was sham-exposed or exposed to MMW radiation for different durations (1-33 h) and two different power densities (5.4 microW/cm(2) or 0.54 mW/cm(2)). As gene expression is a multiple-step process, we analyzed chaperone proteins induction at different levels. First, using luciferase reporter gene, we investigated potential effect of MMWs on the activation of transcription factors (TFs) and gene promoter activity. Next, using RT-PCR and Western blot assays, we verified whether MMW exposure could alter RNA accumulation, translation, or protein stability. Experimental data demonstrated the absence of significant modifications in gene transcription, mRNA, and protein amount for the considered stress-sensitive genes for the exposure durations and power densities investigated. The main results of this study suggest that low-power 60 GHz radiation does not modify stress-sensitive gene expression of chaperone proteins.

Interplay between liganded and orphan nuclear receptors controls reproductive pathways.

Nuclear receptors are transcription factors that belong to an evolutionary ancient superfamily. These proteins, which are even present in primitive metazoans, are implicated in all levels of cell fate: proliferation, differentiation, and apoptosis. Some of these nuclear receptors behave as ligand-inducible transcription factors, as they have acquired during evolution the ability to bind ligands. This is the case for some proteins that recognize small hydrophobic signaling molecules, and particularly the estrogen receptor (ER or NR3A1), which regulates the target gene's transcription rate under estrogen binding. It is now known that the ER alone regulates the transcription of many genes, such as those implicated in reproductive functions. However, this ER-mediated signaling pathway could be modulated by other transcription factors. Our work has established that two other orphan nuclear receptors (SF-1 or NR5A1 and the COUP-TFs, NR2F1 and NR2F2) can enhance two ER-regulated genes implicated in salmonid reproductive functions: the ER gene itself, and the sGTHIIbeta gene. Moreover, some xenoestrogens could disturb these regulations. Therefore, our data contribute to the concept that interplay between nuclear receptors is an important event for the transcriptional regulation of genes controlling cellular functions.

A zebrafish ftz-F1 (Fushi tarazu factor 1) homologue requires multiple subdomains in the D and E regions for its transcriptional activity.

A zebrafish Ftz-F1 homologue, zFF1A (zebrafish Ff1a or Nr5a2, a member of nuclear receptor superfamily) and its C-terminally truncated variant (zFF1B) were previously identified. Due to lack of the identity box (I-box) and activation function 2 (AF-2) domain, zFF1B lacks transactivation function and fails to synergize with estrogen receptor (ER) in regulating promoters. It was speculated that the I-box might be involved in the zFF1A/ER interaction. In the present study, the function of the I-box was examined. In the absence of the I-box or with an altered heptad 9, the AF-2 of zFF1A was not functional, either in the presence or absence of ER. The GST pull-down assay showed that zFF1A and its mutants exerted similar physical contacts with ER-LBD, suggesting that the "dimerization" domain (I-box) is essential for the transcriptional activity of zFF1A. Moreover, nuclear receptor coactivator selectively activated zFF1 with the I-box but exerted no effect on zFF1B, indicating that the I-box is able to interact with the coactivators. By deletion study and analysis of the identified domains in GAL4-DNA binding domain, other regions of zFF1A critical for its AF were also delineated. Consistent with the mutation analysis, AF-2 was active only in the presence of the I-box. We also identified a novel AF domain (AF-3) located in the hinge region (amino acids 155-267), although the activity of AF-3 was inhibited by its flanking region. We suggest that the D and E regions of zFF1A possess both positive and negative transactivation functions, and interdomain "cross-talk" may confer the full transcriptional activity of the protein.