Expression of human asparagine synthetase (ASNS), which catalyzes asparagine and glutamate biosynthesis, is transcriptionally induced following amino acid deprivation. Previous overexpression and electrophoresis mobility shift analysis showed the involvement of the transcription factors ATF4, C/EBPbeta, and ATF3-FL through the nutrient-sensing response element-1 (NSRE-1) within the ASNS promoter. Amino acid deprivation caused an elevated mRNA level for ATF4, C/EBPbeta, and ATF3-FL, and the present study established that the nuclear protein content for ATF4 and ATF3-FL were increased during amino acid limitation, whereas C/EBPbeta-LIP declined slightly. The total amount of C/EBPbeta-LAP protein was unchanged, but changes in the distribution among multiple C/EBPbeta-LAP forms were observed. Overexpression studies established that ATF4, ATF3-FL, and C/EBPbeta-LAP could coordinately modulate the transcription from the human ASNS promoter. Chromatin immunoprecipitation demonstrated that amino acid deprivation increased ATF3-FL, ATF4, and C/EBPbeta binding to the ASNS promoter and enhanced promoter association of RNA polymerase II, TATA-binding protein, and TFIIB of the general transcription machinery. A time course revealed a markedly different temporal order of interaction between these transcription factors and the ASNS promoter. During the initial 2 h, there was a 20-fold increase in ATF4 binding and a rapid increase in histone H3 and H4 acetylation, which closely paralleled the increased transcription rate of the ASNS gene, whereas the increase in ATF3-FL and C/EBPbeta binding was considerably slower and more closely correlated with the decline in transcription rate between 2 and 6 h. The data suggest that ATF3-FL and C/EBPbeta act as transcriptional suppressors for the ASNS gene to counterbalance the transcription rate activated by ATF4 following amino acid deprivation.