Differential Functional Activities of Rainbow Trout and Human Estrogen Receptors Expressed in the Yeast Saccharomyces cerevisiae

Abstract

The cDNA of rainbow trout estrogen receptor (rtER), highly and stably expressed in yeast, Sacchat-om j c e s c,erevisirie, was used to analyse the biological activity of the receptor. The rtER niRNA encoded a 65-kDa protein which was iininunorevealed by a specific antibody and migrated with the authentic rtER major protein form detected in trout liver. Yeast rtER bound estradiol with high affinity and the dissociation constant (KCI = 1.35 nM) was very similar to the value measured from trout liver extracts but 3-5-fold higher than the KtI found for human estrogen receptor (hER). This indicates therefore that the rtER has a lower estradiol affinity compared to the human receptor. While the hER K,, remained unchanged at both 4°C or 22"C, it was slightly modified at 30°C. The K,I measured for rtER at 22°C and 30°C were about 2-fold, and 12-fold higher, respectively, than the K,( obtained at 4°C suggesting an alteration of the rtER affinity for its ligand at elevated temperature. To examine the estrogen-receptor-mediated activation of transcription in yeast, reporter plasmids integrated or not in the yeast genome were used. The reporter genes consist of one, two, or three copies of estrogen-responsive elements (ERE) upstream of the yeast proximal CYCI or UKA3 promoters fused to the lacZ gene of Excherichia coli coding for p-galactosidase.The induction of /I-galactosidase activity for all reporter genes was strictly dependent on the presence of rtER and estrogens. The activation of transcription mediated by rtER responded in an estradiol-dosedependent manner as in animal cells. However, compared to hER, the estradiol concentration necessary to achieve maximal activation was 10-fold higher. This is probably a consequence of the lower estradiolaffinity for rtER compared to hER. The levels of induction of the reporter genes containing two or three ERE were \trongly enhanced compared to the one ERE construct. This is in agreement with the synergistic effect previously described for multiple ERE. The magnitudes of transcriptional induction mediated by rtER and hER were similar when the reporter gene containing three ERE was used but changed when the one ERE construct was used. In this case transcriptional activation mediated by rtER was 10-20-fold lower. This suggests that rtER requires protein/protein interaction for i t s stabilization on DNA. Antiestrogens were able to bind rtER and promote gene transcription. However, to produce effects comparable to those obtained with estrogens, much higher concentrations were required. This may imply nonetheless that antihormones were capable of provoking efficient interactions of rtER with the transcriptional machinery Keywonl.~; steroid action ; estrogen receptor; yeast expression; transcriptional activity.

Control of vitellogenin genes expression by sequences derived from transposable elements in rainbow trout

“…In the absence of E2, a promoter construct containing an ERE cs separated by 20 bp is sufficient to drive the transcription of the reporter gene with rtER S . This strong E2-independent basal activity has already been observed in various cell systems (including yeast) and is assigned to the absence of the N-terminal domain [8,10,42]. In the presence of E2, transcription is further increased by 2.4 fold with construct containing one ERE cs .…”

“…Following E2-stimulation, β-galactosidase activity was strongly increased in hERα-expressing cells whereas rtER S -expressing cells exhibited an activity increased by twice in comparison to without E2. This control experiment clearly indicates that hERα transcriptional activity is strictly E2-dependent, whereas rtER S exhibits a marked E2-independent activity when acting towards ERE cs , as previously described [8,10].…”

“…Human ERα (hERα) and rtER S are well-conserved (92% and 60% similarities for the DNA-binding domain and the ligand-dependent transactivation domain, respectively), except for the ligand-independent transactivation domain, which is poorly conserved (20% similarity). hERα and rtER S also exhibit important functional differences [10]: (i)-rtER S has a marked transcriptional activity in the absence of estrogens; (ii)-rtER S needs a 10-fold higher estradiol (E2) concentration to achieve maximal transactivation compared to hERα; (iii)-rtER S displays a weaker transactivation activity compared to hERα, in yeast assays with a reporter gene containing one, two or three copies of ERE cs . It is important to note that the binding of E2 to rtER S, but not to hERα, was shown to be temperature sensitive [11].…”

Reconstitution d'une unité transcriptionnelle à partir du promoteur du gène du récepteur aux oestrogènes de truite arc-en-ciel dans la levure Saccharomyces cerevisiae

“…L'ADNc du rtER a été introduit (PETIT et al, 1995) dans le site BamHI du vecteur d'expression de levure YEpucG (don du Dr. B.S. Katzenellenbogen), qui est un plasmide mixte levure/bactérie portant un gène de résistance à l'Ampicilline, et un gène de sélection pour le tryptophane (gène TRP1 codant pour une enzyme de la voie de biosynthèse du tryptophane).…”

“…METZGER et al (1988) ont en effet reconstitué un système oestrogéno-dépendant en utilisant l'ER humain et un gène rapporteur artificiel. Le même type d'expérience a été réalisé avec le récepteur aux glucocorticoides (SCHENA et YAMAMOTO, 1988), celui aux hormones thyroïdiennes (OHASHI et al, 1991), mais également le rtER (PETIT et al, 1995). De surcroît, les facteurs de levure TFIID ou Gal4 sont capables d'activer la transcription de gènes cibles dans différentes cellules de mammifères (WEBSTER et al, 1988 ;KAKIDANI et PTASHNE, 1988).…”