Cis- and trans-acting elements involved in amino acid regulation of asparagine synthetase gene expression.

Abstract

We have previously shown that asparagine synthetase (AS) mRNA expression can be dramatically upregulated by asparagine deprivation in tsll cells, mutants of BHK hamster cells which encode a temperaturesensitive AS. The expression of AS mRNA was also induced upon starvation for one of several essential amino acids in HeLa cells. We also showed that regulation of AS mRNA expression by amino acid concentration has both transcriptional and posttranscriptional components. Here we report the analysis of the elements in the human AS promoter region important for its basal activity and activation by amino acid starvation. Our results indicate that a DNA fragment spanning from nucleotides -164 to +44 of the AS promoter is sufficient for uninduced and induced gene expression. Mutations in a region located 15 to 30 bp downstream from the major transcription start site that shows good homology to a sequence in the first exon of c-fos implicated as a negative regulatory element resulted in a significant increase in basal gene expression but did not affect regulation. Interestingly, this region binds single-stranded-DNA-binding proteins that are specific for the AS coding strand. Mutations in either one of two putative binding sites for transcription factor Spl, in a region of approximately 60 bp where many minor RNA start sites are located, or at the major transcription start site decreased promoter activity, but significant induction by amino acid starvation was still observed. Strikingly, mutations centered around nucleotide -68 not only decreased the basal promoter activity but also abolished amino acid regulation. This DNA region contains the sequence 5'-CATGATG-3', which we call the amino acid response element (AARE), that can bind a factor(s) present in HeLa cells nuclear extracts that is not capable of binding to an AS promoter with mutations or deletions of the AARE. This finding is in line with the hypothesis that transcriptional activation of AS gene expression is mediated through the binding of a positive regulatory element. We did not detect changes in the level of binding of this factor to the AARE by using nuclear extracts from HeLa cells grown under starved conditions, suggesting that activation of this factor(s) results from posttranslational modification or complexing with other proteins that do not affect its DNAbinding properties.The signals regulating gene expression in prokaryotes and lower eukaryotes in response to nutrient variation are well known and characterized; it is well established that these organisms are quickly able to adjust to variations in nutrient supply and to intracellular amino acid levels by altering their patterns of gene expression. Much less is known about the response of higher eukaryotes to nutrient deprivation (9).We have previously shown (13,15)

An update on renal peptide transporters

“…Promoter analysis studies employing a luciferase reporter assay identified a region that responded to all dipeptides tested and also to selected amino acids, such as Lys, Arg, and especially Phe. This region comprises 254 bp and contains an amino acid response-like element as found in the asparagine synthetase gene (32). Rat and mouse Pept1 and the murine Pept2 promoter regions contain these amino acid response-like elements with a bp substitution in the fifth bp (25,52).…”

Differences in the Molecular Mechanisms Involved in the Transcriptional Activation of the CHOP and Asparagine Synthetase Genes in Response to Amino Acid Deprivation or Activation of the Unfolded Protein Response

“…Indeed the AS promoter sequence on the bottom strand (nt Ϫ57 to Ϫ75) revealed a high identity to the CHOP AARE on the top strand (nt Ϫ313 to Ϫ295), not only in the AARE core, but also in both of the border sequences. By nucleotide substitutions, this AS sequence had been shown by independent laboratories to be important for the response to amino acid starvation (8,9). Furthermore, previous studies had shown that the CHOP AARE-like NSRE-1 sequence is not The minimum core consensus sequence is represented.…”

“…The level of AS mRNA increases in response not only to asparagine starvation, but also to deprivation of any individual essential amino acid (5-7). Guerrini et al (8) used promoter deletion and mutation to analyze the proximal promoter of the human AS gene. They identified the palindromic sequence 5Ј-CATGATG-3Ј at nucleotides Ϫ70 to Ϫ64 as necessary for the AS promoter regulation by amino acid availability.…”

ATF4 Is a Mediator of the Nutrient-sensing Response Pathway That Activates the Human Asparagine Synthetase Gene

“…Guerrini et al (9) identified a region from nt Ϫ70 to Ϫ62 within the human A.S. promoter that functioned as an amino acid response element (AARE). More recently, Barbosa-Tessmann et al (10 -12) have demonstrated that the human A.S. gene is also induced by glucose deprivation and that this activation is mediated by the endoplasmic reticulum stress response (ERSR), also known as the unfolded protein response pathway in yeast (13).…”

“…More recently, Barbosa-Tessmann et al (10 -12) have demonstrated that the human A.S. gene is also induced by glucose deprivation and that this activation is mediated by the endoplasmic reticulum stress response (ERSR), also known as the unfolded protein response pathway in yeast (13). In vivo footprinting and mutagenesis demonstrated that the promoter sequence 5Ј-TGATGAAAC-3Ј (nt Ϫ68 to Ϫ60), the region first identified by Guerrini et al (9), was also responsible for induction of the A.S. transcription following activation of the ERSR pathway (12). The ERSR activation demonstrates that this A.S. promoter sequence serves in a broader capacity than simply as an AARE and, to reflect this broader substrate-detecting capability, this sequence is referred to as the nutrient-sensing response element-1 (NSRE-1).…”