Gilles Salbert

Epigenetic switch involved in activation of pioneer factor FOXA1-dependent enhancers

Transcription factors (TFs) bind specifically to discrete regions of mammalian genomes called cis-regulatory elements. Among those are enhancers, which play key roles in regulation of gene expression during development and differentiation. Despite the recognized central regulatory role exerted by chromatin in control of TF functions, much remains to be learned regarding the chromatin structure of enhancers and how it is established. Here, we have analyzed on a genomic-scale enhancers that recruit FOXA1, a pioneer transcription factor that triggers transcriptional competency of these cis-regulatory sites. Importantly, we found that FOXA1 binds to genomic regions showing local DNA hypomethylation and that its cell-type-specific recruitment to chromatin is linked to differential DNA methylation levels of its binding sites. Using neural differentiation as a model, we showed that induction of FOXA1 expression and its subsequent recruitment to enhancers is associated with DNA demethylation. Concomitantly, histone H3 lysine 4 methylation is induced at these enhancers. These epigenetic changes may both stabilize FOXA1 binding and allow for subsequent recruitment of transcriptional regulatory effectors. Interestingly, when cloned into reporter constructs, FOXA1-dependent enhancers were able to recapitulate their cell type specificity. However, their activities were inhibited by DNA methylation. Hence, these enhancers are intrinsic cell-type-specific regulatory regions of which activities have to be potentiated by FOXA1 through induction of an epigenetic switch that includes notably DNA demethylation.

Distribution of Estrogen Receptor-lmmunoreactive Cells in the Brain of the Rainbow Trout (Oncorhynchus mykiss)

Using antibodies against the hormone binding domain of the trout estrogen receptor (ER), the distribution of ER‐immunoreactive (ER‐IR) cells was studied in the brain of maturing diploid and triploid female rainbow trout using a streptavidin‐biotin‐peroxidase method followed by a nickel‐intensified diaminobenzidine reaction. This technique resulted in an excellent signal/background ratio allowing unambiguous identification of positive cells. In all animals, ER‐IR cells were consistently located in three brain regions, the ventral telencephalon, the anterior ventral preoptic region, and the mediobasal hypothalamus. About 250 ER‐IR cells were observed in the ventral and dorsal parts of the ventral telencephalon. In the anterior nucleus preopticus periventricularis, about 2400 ER‐IR cells were observed surrounding the preoptic recess. In the posterior hypothalamus, approximately 2700 ER‐IR cells were located in the anterior, posterior and inferior divisions of the nucleus lateralis tuberis and in the nucleus saccus vasculosus. In these regions, cell nuclei exhibiting different densities of staining were observed and absolutely no labeling of cytoplasmic processes was detected. These results are in partial agreement with those obtained either after injection of tritiated‐estradiol in other teleots species or in situ hybridization of ER mRNAs in trout. In particular, no immunoreactivity was observed in the thalamic region nor in the nucleus posterioris periventricularis. These data indicate that target cells for estradiol are essentially located in brain regions involved in the neuroendocrine control of pituitary functions and having direct connections with the hypophysis.

Serotonin and dopamine turnover in the female rainbow trout (Oncorhynchus mykiss) brain and pituitary: Changes during the annual reproductive cycle

Brain serotonin (5HT) and dopamine (DA) turnover were studied at various stages of the reproductive cycle of the female rainbow trout by simultaneous determination by HPLC of neurotransmitters and major related metabolites. An increase of 5HT turnover in telencephalon and hypothalamus and a decrease of DA turnover in pituitary and hypothalamus were observed during the periovulatory period. Some changes also occurred during vitellogenesis: decreased 5HT metabolite in telencephalon and preoptic area and increased DA content in preoptic area. These data suggest that physiological fluctuations of biogenic amines could be involved in both ovarian recrudescence and ovulation, with major effects on the hypothalamo-hypophysial complex during the periovulatory period.

Localization of the estradiol receptor mRNA in the forebrain of the rainbow trout.

In situ hybridization was used to localize the cells that express the estradiol receptor gene (ER) in the forebrain (hypothalamus, preoptic area, telencephalon) of the rainbow trout (Oncorhynchus mykiss). Both sense and anti-sense [35S]UTP-labeled single-stranded RNA probes were generated from the estradiol binding domain of the ER cDNA. The sense probe was used to evaluate the background of the hybridization reaction. In the forebrain, specific signal appeared in three areas: the posterior hypothalamus, the preoptic area, and the ventral telencephalon. Our localization correlates with [3H]estradiol binding studies in other teleost species. In the pituitary, we observed a weak signal when compared to the signal observed in the forebrain (about ten grains/cell in the pituitary against 35 grains/cell in the posterior hypothalamus). A significant difference was also observed between the intensity of labeling per cell when different forebrain nuclei were compared. We provide here evidence for a tissue-specific regulation of the ER mRNA levels in the trout hypothalamo-pituitary axis.

Effects of nonylphenol on estrogen receptor conformation, transcriptional activity and sexual reversion in rainbow trout (Oncorhynchus mykiss)

Estrogenic potency of 4-n-nonylphenol diethoxylate, 4-n-nonylphenol (NP) and metabolites were tested using two bioassays: rainbow trout hepatocyte culture and recombinant yeast stably expressing rainbow trout estrogen receptor (rtER) and containing estrogen-dependent reporter genes. Since NP was the only compound active in both systems, its interaction with rtER was studied in more detail. Qualitative and quantitative differences were observed in the presence of 17beta-estradiol (E2) or NP when estrogen-dependent promoters containing one to three estrogen-responsive elements were used in yeast. Moreover, limited proteolysis of rtER after E2 or NP binding presented different patterns after SDS-PAGE analysis suggesting that NP induces a differential conformation of rtER compare to E2. This finding may have important implications with respect to the biological activity of NP. Thus, the effects of NP on the activation of an E2-dependent gene and on sexual differentiation were assessed on all-male trout embryos exposed to NP for 1 h per day for 10 days. Although in situ hybridization demonstrated that E2, and to a lesser extend NP, were able to increase rtER mRNA level in the liver of embryos, no indication of total or partial sexual reversion was observed (even in E2 treated fishes) when the gonads were examined 8 months after hatching.

Estrogen receptors in the brain-pituitary complex and the neuroendocrine regulation of gonadotropin release in rainbow trout

In all vertebrates, steroid feedback is one of the mechanisms by which the brain is constantly kept informed on the physiological status of the periphery. It is, therefore, a very efficient system of synchronization between the different actors of the brain-pituitary-effector axis, particularly in the context of reproduction. The central effects of steroids are mediated by specific receptors belonging to the superfamily of intracellular receptors and acting as ligand dependant transcription factors to modulate the expression of certain target genes. In order to investigate the molecular events underlying steroid actions in the brain/pituitary complex, it is necessary, as a first step, to locate precisely steroid expressing cells in the brain/pituitary complex and to identify the phenotype of these cells on the basis of the neurohormone(s) or hormones they produce. This paper will review recent data obtained in our laboratory regarding the distribution and regulation of expression of estrogen receptors in the brain and pituitary gland of the rainbow trout (Oncorhynchus mykiss), using both in situ hybridization or immunohistochemistry. In addition, information concerning the phenotypic identification of cells expressing these receptors is presented, especially with respect to the main neuroendocrine systems controlling reproduction. The functional meaning of these data are discussed in the context of the reproductive physiology of trout.

Formation of an hERα–COUP-TFI complex enhances hERα AF-1 through Ser118 phosphorylation by MAPK

The enhancement of the human estrogen receptor α (hERα, NR3A1) activity by the orphan nuclear receptor COUP‐TFI is found to depend on the establishment of a tight hERα–COUP‐TFI complex. Formation of this complex seems to involve dynamic mechanisms different from those allowing hERα homodimerization. Although the hERα–COUP‐TFI complex is present in all cells tested, the transcriptional cooperation between the two nuclear receptors is restricted to cell lines permissive to hERα activation function 1 (AF‐1). In these cells, the physical interaction between COUP‐TFI and hERα increases the affinity of hERα for ERK2/p42MAPK, resulting in an enhanced phosphorylation state of the hERα Ser118. hERα thus acquires a strengthened AF‐1 activity due to its hyperphosphorylation. These data indicate an alternative interaction process between nuclear receptors and demonstrate a novel protein intercommunication pathway that modulates hERα AF‐1.

Regulation of gene expression and biological activity of rainbow trout estrogen receptor

Rainbow trout estrogen receptor (rtER) concentration was highly induced in the liver after in vivo estradiol (E2) treatment or in vitro, in hepatocyte aggregate culture. Determination of transcription rate and mRNA half-life demonstrated that E2-induction of hepatic rtER level is caused essentially by an increase in the transcriptional and post-transcriptional activity of rtER gene. However, the expression of rtER gene in the liver seems to be down-regulated by glucocorticoids. We have used transient transfection assays with reporter plasmids linked to 5′ flanking regions of the rtER gene promoter, to identify cis-elements responsible for E2 inducibility. Deletion analysis localized a functional estrogen-responsive-element (ERE), near the transcription start site, with one mutation on the first base compared to the consensus sequence. This element and 200 bp fragment of the rtER promoter encompassing the ERE appear to be the major cis-acting element involved in the regulation of the gene. Data obtained from transfection experiments and footprinting analysis, suggested that the receptor is one of the major trans-factors implicated in the regulation of its own gene. However, interaction of ER with other transcription factors is required for maximal E2-stimulation.

Differential regulation of the estrogen receptor mRNA by estradiol in the trout hypothalamus and pituitary

In an attempt to understand the molecular mechanisms by which steroids can modulate brain functions in fish, we first localized the cells which produce estrogen receptor mRNA in the rainbow trout forebrain (Salbert et al., 1991). We now report how estradiol itself can alter the estrogen receptor mRNA content of these cells in a sterile strain of female rainbow trout. We also examined liver and pituitary levels of the estrogen receptor mRNA under the same estrogenic treatment. As revealed by slot blot and in situ hybridisations, a single injection (1.5 mg/kg) of estradiol can induce a strong increase (about five-fold) in the estrogen receptor mRNA levels in the liver, as well as a moderate increase (about two-fold) in two nuclei of the hypothalamus/preoptic area: the nucleus lateralis tuberis and the nucleus preopticus periventricularis. Conversely, no modifications of these levels were observed in the pars intermedia and the proximal pars distalis of the pituitary. Moreover, a comparison between estrogen receptor mRNA levels in the brain of sexually active female trout and in the brain of these sterile animals revealed that, in these latter, estrogen receptor mRNA levels are lower but can be increased by a single estradiol injection and reached the levels observed in mature females.

Effects of estradiol on brain aminergic turnover of the female rainbow trout (Oncorhynchus mykiss) at the beginning of vitellogenesis

Brain serotonin and dopamine (DA) turnovers in the female rainbow trout were studied at the beginning of the vitellogenesis and related to blood estradiol (E2) levels; pituitary and plasma gonadotropin (GtH) were also assayed. Ovariectomy did not modify brain aminergic turnover. E2 replacement on ovariectomized fish increased hypothalamic DA turnover (increased DA and increased DA metabolites). E2 stimulated GtH synthesis (positive feedback) but did not enhance GtH release; hypothalamic E2-mediated aminergic inhibition upon release was suspected. Individual relations between blood E2 levels and catecholaminergic neurotransmitters were determined. A linear positive correlation (r = 0.82) was found for the hypothalamus, but not for the pituitary, the preoptic area, or the telencephalon. These data suggest that an activation of hypothalamic tyrosine hydroxylase (the limiting step of catecholamines synthesis) by E2 could develop as vitellogenesis proceeds.