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

Abstract

In most of oviparous animals, vitellogenins (VTG) are the major egg yolk precursors. They are produced in the liver under the control of estrogens. In rainbow trout (Oncorhynchus mykiss), the vtg genes cluster contains an unusually large number of almost identical gene copies. In order to identify the regulatory elements in their promoters, we used a combination of reporter plasmids containing genomic sequences including putative estrogen response elements (EREs) and we performed transient transfection assays in MCF-7 and yeast cells.We found a functional ERE corresponding to the sequence GGGGCAnnnTAACCT (rtvtgERE), which differs from the consensus ERE (ERE cs ) by three base pairs. This nonpalindromic ERE is located in the env gene of a retrotransposon relic, 180 base pairs upstream of the transcriptional start site. Fluorescence anisotropy experiments confirmed that the purified human estrogen receptor α (hERα) can specifically bind to rtvtgERE. Furthermore, we observe that the stability of hERα-ERE cs and hERα-rtvtgERE complexes is similar with equilibrium dissociation constants of 3.0 nM and 6.2nM respectively, under our experimental conditions. Additionally, this rtvtgERE sequence displays a high E2-responsiveness through ER activation in cellulo.In the rainbow trout, the functional ERE (rtvtgERE) lies within promoter sequences which are mostly composed of sequences derived from transposable elements (TEs), which therefore may have acted as an evolutionary buffer to secure the proper expression of these genes.4

Investigation of the functional properties and subcellular localization of alpha human and rainbow trout estrogen receptors within a unique yeast cellular context

“…The ligands used here are agonists which promote the ERa binding to coactivators and antagonists which are able to inhibit these intermolecular interactions and/or promote interactions with corepressors [42][43][44]. The main results of this in cellulo study are: (i) in the absence of ligand, rtERa S is able to activate gene transcription whereas hERa does not activate, or very poorly, as previously observed in other yeast strains as well as in mammalian cells [22,[24][25][26][27]45,46]; (ii) rtERa S is less efficient in response to ligands than hERa. In our unique cellular system, E2 and DES are equally efficient and potent agonists of hERa and rtERa S .…”

“…This expression system has been chosen as a unique cellular context for at least two reasons: (i) no expression of endogenous ER and (ii) conserved activity of exogenously expressed hERa and rtERa S [22,[24][25][26][27]. This lower eukaryotic model therefore ensures that the observed cellular response to hormone stimulation was on the sole control of produced recombinant receptors which is an essential prerequisite for the present purpose.…”

“…b-galactosidase reporter vector, YRPE2, contains 2 consensus ERE sequences (ERE cs ) located upstream of a minimal cyc promoter controlling the lacZ gene expression [24]. This plasmid was kindly provided by Pr.…”

“…The construction of expression plasmids of hERa and rtERa S has been previously described [24]. To construct expression plasmids of recombinant chimeric GFP-hERa and GFP-rtERa S proteins, the hERa and rtERa S cDNA were PCR-amplified from YEPE15 [28] and pCMV5/rtera S [29] respectively, and subcloned into a modified pBluescript SK phagemid (Stratagene).…”

“…Thus, to improve our understanding of the mechanisms involved in ER signaling pathways, we compare, within a unique cellular context, i.e., Saccharomyces cerevisiae, and a unique ERE, the binding and functional properties, namely cellular activity and subcellular localization, of the hERa and rtERa S receptors, in response to agonists, estradiol and diethylstilbestrol, and antagonists, 4-OH tamoxifen, raloxifen (2 known partial antagonists in human cells) and ICI 182-780 (pure antagonist in human cells). This cellular system is widely used for expressing recombinant ERs and quantifying their ability to enhance gene transcription from yeast promoters linked to consensus estrogen-responsive elements (ERE) in response to estradiol [22][23][24][25][26][27].…”