Transcription from the asparagine synthetase (A.S.) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the A.S. promoter referred to as nutrientsensing response elements (NSRE) 1 and 2, both of which are necessary for gene activation. The NSRE-1 sequence was used to screen ATF/CREB family members by electrophoresis mobility shift assays and supershift by specific antibodies. The results indicated that ATF4 binds to the NSRE-1 sequence and that the amount of the ATF4 complex was increased when extracts from amino acid-deprived or glucose-deprived cells were tested. Using electrophoresis mobility shift assay experiments and a probe that contained both NSRE-1 and NSRE-2, mutation of the NSRE-1 sequence completely prevented formation of the ATF4-containing complexes, whereas mutation of the NSRE-2 sequence did not. Overexpression of ATF4 increased A.S. promoter-driven transcription, whereas an inhibitory dominant negative ATF4 mutant blocked both basal and starvation-enhanced transcription. Collectively, the results provide both in vitro and in vivo evidence for a role of ATF4 in the transcriptional activation of the A.S. gene in response to nutrient deprivation.

After amino acid deprivation, the mRNA content for both asparagine synthetase (AS) and the system A transporter ATA2 is increased. The purpose of the reported experiments was to characterize the molecular mechanism for the ATA2 gene and to contrast the ATA2 regulatory characteristics with those of AS. Amino acid limitation was initiated by incubation of HepG2 human hepatoma cells in either amino acid-free Krebs-Ringer bicarbonate buffer or culture medium lacking the single amino acid histidine. For ATA2, like AS, the elevated mRNA content was due to increased transcription. However, there were fundamental differences between the mechanisms for nutrient regulation of the AS and ATA2 genes. When cells were deprived of amino acids, there was a lag period of approximately 4 h before an increase in AS mRNA occurred, whereas the elevation of ATA2 mRNA was readily detectable at 2-4 h. Consistent with these observations, de novo protein synthesis was absolutely required for the activation of the AS gene, but the increase in ATA2 mRNA was largely independent of protein synthesis. Furthermore, in contrast to AS, transcription from the ATA2 gene was not increased by glucose deprivation. Given this lack of ATA2 transcriptional activation by glucose starvation and that the induction of the AS gene by glucose or amino acid starvation is mediated by common genomic elements, it is likely that the ATA2 gene does not contain the same genomic amino acid-responsive cis-elements as the AS gene.