A Silencer Element in the Regulatory Region of Glutamine Synthetase Controls Cell Type-specific Repression of Gene Induction by Glucocorticoids

Abstract

Glutamine synthetase is a key enzyme in the recycling of the neurotransmitter glutamate. Expression of this enzyme is regulated by glucocorticoids, which induce a high level of glutamine synthetase in neural but not in various non-neural tissues. This is despite the fact that non-neural cells express functional glucocorticoid receptor molecules capable of inducing other target genes. Sequencing and functional analysis of the upstream region of the glutamine synthetase gene identified, 5 to the glucocorticoid response element (GRE), a 21-base pair glutamine synthetase silencer element (GSSE), which showed considerable homology with the neural restrictive silencer element NRSE. The GSSE was able to markedly repress the induction of gene transcription by glucocorticoids in non-neural cells and in embryonic neural retina. The repressive activity of the GSSE could be conferred on a heterologous GRE promoter and was orientation-and position-independent with respect to the transcriptional start site, but appeared to depend on a location proximal to the GRE. Gel-shift assays revealed that non-neural cells and cells of early embryonic retina contain a high level of GSSE binding activity and that this level declines progressively with age. Our results suggest that the GSSE might be involved in the restriction of glutamine synthetase induction by glucocorticoids to differentiated neural tissues.Glutamine synthetase (GS 1 ; L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2) is a "housekeeping" enzyme that is expressed at a particularly high level in neural tissues (1, 2). High levels of GS expression are restricted to glial cells (1,(3)(4)(5) and are essential for the recycling of the neurotransmitter glutamate. Synaptically released glutamate is taken up into glial cells, where it is converted by GS into glutamine, which re-enters the neurons and is hydrolyzed by glutaminase to form glutamate again (6, 7). In this way, the neurotransmitter pool is replenished and glutamate neurotoxicity is prevented.Studies in the chicken neural retina showed that GS expression is regulated by systemic glucocorticoids, which induce in this tissue a very high level of GS by directly stimulating the transcription of the gene (8 -10). The ability of glucocorticoids to induce GS expression is developmentally controlled. Glucocorticoids can induce a high level of GS expression in neural retina at late embryonic ages, but not in early embryonic retina (2, 11-13). The direct involvement of glucocorticoids in the control of GS gene transcription is evidenced by the nuclear run-on transcription assay, as well as by the finding that the upstream region of the GS gene contains a glucocorticoid response element (GRE) that can bind the glucocorticoid receptor protein and confer responsiveness to glucocorticoids on an attached reporter gene (8 -10, 14, 15). This mode of regulation, which is dependent on the presence of active glucocorticoid receptor molecules, explains the cell type specificity of GS expression in the neural retina; GS expression is ...

Dual Role of NRSF/REST in Activation and Repression of the Glucocorticoid Response

“…pUAS66GRE contains a fragment of 66 bp inserted between the UAS and GRE sequence in pUASGRE. The fragment was derived from a subclone of the previously described p2.4GS construct (1) and includes the sequence that spans between nucleotides 528 and 572 in the promoter of avian glutamine synthetase (GenBank TM accession number AF105022). pRepUASGRE was constructed by replacing the CMV promoter in the pCEP4 vector (Invitrogen) with the UAS-GRE-TK promoter and the adjacent luciferase gene derived from pUASGRE.…”

“…Expression of glucocorticoid inducible genes may also be determined by transcriptional repressors that block the hormonal response. This latter type of action is exerted, for example, by the neural restrictive silencing factor NRSF/REST, which is expressed in non-neural tissues and can restrict the hormonal induction of avian glutamine synthetase to neural tissues (1). The molecular mechanism that underlies the interplay between GR and negative or positive transacting factors is largely unknown.…”

“…This is despite the fact that non-neural cells express functional glucocorticoid receptor molecules capable of inducing other target genes. Analysis of the regulatory region of avian glutamine synthetase has revealed the presence of an NRSF/REST binding site (NRSE), upstream of the glucocorticoid response element (GRE) (1). This site represses the induction of gene transcription by glucocorticoids in non-neural cells and in embryonic neural retina, but not in neural cells.…”

Transcriptional Regulation of Metabolism

“…Two broad regions mediating this response have been identified in the upstream promoter and in the first intron of GS. Detailed studies of GS expression in chicken brains showed that glucocorticoids act by relieving the repression mediated by a silencer element located upstream of the glucocorticoid receptor binding site (5). This glucocorticoid-mediated induction of GS occurs particularly in conditions of trauma or high catabolic rate and results in increased glutamine synthesis at the expense of the AA that purvey the amino groups.…”

Expression of glutamine synthetase in Tegillarca granosa (Bivalvia, Arcidae) hemocytes stimulated by Vibrio parahaemolyticus and lipopolysaccharides

“…In rainbow trout, the highest level of GS expression occurs in the brain, with decreasing levels in intestine, liver, red muscle, gill/kidney, white muscle, and heart (Murray et al, 2003). GS expression is high in neural tissues and in post-blood-fed female A. aegypti mosquito midguts, in which it is involved in peritrophic matrix formation (Avisar et al, 1999;Smartt et al, 2001). In plants, the evidence reported herein indicates that the pine GS gene family contains at least two isoforms that have unique and precise patterns of spatial and temporal expression, suggesting that they play distinct functional roles in nitrogen metabolism of conifers (Avila et al, 2001).…”